Lakshmi A Devi, Ph.D.

7,8-dihydroxyflavone, a small-molecule TrkB agonist, reverses memory deficits and BACE1 elevation in a mouse model of Alzheimer's disease
Devi, Latha; Ohno, Masuo
2012 Jan;37(2):434-444, Biological psychiatry
Increasing evidence suggests that reductions in brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) may have a role in the pathogenesis of Alzheimer's disease (AD). However, the efficacy and safety profile of BDNF therapy (eg, gene delivery) remains to be established toward clinical trials. Here, we evaluated the effects of 7,8-dihydroxyflavone (7,8-DHF), a recently identified small-molecule TrkB agonist that can pass the blood-brain barrier, in the 5XFAD transgenic mouse model of AD. 5XFAD mice at 12-15 months of age and non-transgenic littermate controls received systemic administration of 7,8-DHF (5 mg/kg, i.p.) once daily for 10 consecutive days. We found that 7,8-DHF rescued memory deficits of 5XFAD mice in the spontaneous alternation Y-maze task. 5XFAD mice showed impairments in the hippocampal BDNF-TrkB pathway, as evidenced by significant reductions in BDNF, TrkB receptors, and phosphorylated TrkB. 7,8-DHF restored deficient TrkB signaling in 5XFAD mice without affecting endogenous BDNF levels. Meanwhile, 5XFAD mice exhibited elevations in the beta-secretase enzyme (BACE1) that initiates amyloid-beta (Abeta) generation, as observed in sporadic AD. Interestingly, 7,8-DHF blocked BACE1 elevations and lowered levels of the beta-secretase-cleaved C-terminal fragment of amyloid precursor protein (C99), Abeta40, and Abeta42 in 5XFAD mouse brains. Furthermore, BACE1 expression was decreased by 7,8-DHF in wild-type mice, suggesting that BDNF-TrkB signaling is also important for downregulating baseline levels of BACE1. Together, our findings indicate that TrkB activation with systemic 7,8-DHF can ameliorate AD-associated memory deficits, which may be, at least in part, attributable to reductions in BACE1 expression and beta-amyloidogenesis.
— id: 155559, year: 2012, vol: 37, page: 434, stat: Journal Article,

Mitochondrial dysfunction and accumulation of the beta-secretase-cleaved C-terminal fragment of APP in Alzheimer's disease transgenic mice
Devi, Latha; Ohno, Masuo
2012 Jan;45(1):417-424, Neurobiology of disease
Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-beta (Abeta) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the beta-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the beta-site APP-cleaving enzyme 1 (BACE1(+/-)) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1(+/-).5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1(+/-) deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1(+/-) deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.
— id: 155558, year: 2012, vol: 45, page: 417, stat: Journal Article,

G protein-coupled receptor heterodimerization: A role in allosteric modulation of ligand-mediated receptor binding
Gomes I; Ijzerman AP; Ye K; Maillet EL; Devi LA
2011 Jun;79(6):1044-1052, Molecular pharmacology
It is becoming increasingly recognized that G protein-coupled receptors physically interact. These interactions may provide a mechanism for allosteric modulation of receptor function. In this study we examined this possibility by using an established model system of a receptor heteromer consisting of mu and delta opioid receptors. We examined the effect of a number of mu receptor ligands on the binding equilibrium, association and dissociation kinetics of a radiolabeled delta receptor agonist, [(3)H]Deltorphin II. Reciprocally, we also examined the effect of delta receptor ligands on the binding equilibrium, association and dissociation kinetics of a radiolabeled mu receptor agonist, [(3)H]DAMGO. We show that mu receptor ligands are capable of allosterically enhancing delta receptor radioligand binding, and vice versa. Thus, there is strong positive cooperativity between the two receptor units with remarkable consequences for ligand pharmacology. We find that the data can be simulated by adapting an allosteric receptor model previously developed for small molecules, suggesting that the ligand-occupied protomers function as allosteric modulators of the partner receptor's activity
— id: 131933, year: 2011, vol: 79, page: 1044, stat: Journal Article,

Sex- and brain region-specific acceleration of beta-amyloidogenesis following behavioral stress in a mouse model of Alzheimer's disease
Devi, Latha; Alldred, Melissa J; Ginsberg, Stephen D; Ohno, Masuo
2010 ;3:34-34, Mol Brain
BACKGROUND: It is hypothesized that complex interactions between multiple environmental factors and genetic factors are implicated in sporadic Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. Importantly, recent evidence reveals that expression and activity levels of the beta-site APP cleaving enzyme 1 (BACE1), which initiates amyloid-beta (Abeta) production, are elevated in AD brains. In this study, we investigated a molecular mechanism by which sex and stress interactions may accelerate beta-amyloidogenesis and contribute to sporadic AD. RESULTS: We applied 5-day restraint stress (6 h/day) to the male and female 5XFAD transgenic mouse model of AD at the pre-pathological stage of disease, which showed little amyloid deposition under non-stressed control conditions. Exposure to the relatively brief behavioral stress increased levels of neurotoxic Abeta42 peptides, the beta-secretase-cleaved C-terminal fragment (C99) and plaque burden in the hippocampus of female 5XFAD mice but not in that of male 5XFAD mice. In contrast, significant changes in the parameters of beta-amyloidosis were not observed in the cerebral cortex of stressed male or female 5XFAD mice. We found that this sex- and brain region-specific acceleration of beta-amyloidosis was accounted for by elevations in BACE1 and APP levels in response to adverse stress. Furthermore, not only BACE1 mRNA but also phosphorylation of the translation initiation factor eIF2alpha (a proposed mediator of the post-transcriptional upregulation of BACE1) was elevated in the hippocampus of stressed female 5XFAD mice. CONCLUSIONS: Our results suggest that the higher prevalence of sporadic AD in women may be attributable to the vulnerability of female brains (especially, the hippocampus) to stressful events, which alter APP processing to favor the beta-amyloidogenesis through the transcriptional and translational upregulation of BACE1 combined with elevations in its substrate APP.
— id: 155560, year: 2010, vol: 3, page: 34, stat: Journal Article,

Increased abundance of opioid receptor heteromers after chronic morphine administration
Gupta, Achla; Mulder, Jan; Gomes, Ivone; Rozenfeld, Raphael; Bushlin, Ittai; Ong, Edmund; Lim, Maribel; Maillet, Emeline; Junek, Mats; Cahill, Catherine M; Harkany, Tibor; Devi, Lakshmi A
2010 ;3(131):ra54-ra54, Science signaling
The mu and delta types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the mu-delta heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of mu-delta heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the mu-delta heteromer could be a therapeutic target in the treatment of chronic pain and addiction
— id: 122723, year: 2010, vol: 3, page: ra54, stat: Journal Article,

Modulation of Opiate-Related Signaling Molecules in Morphine-Dependent Conditioned Behavior: Conditioned Place Preference to Morphine Induces CREB Phosphorylation
Moron, Jose A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A
2010 Mar;35(4):955-966, Neuropsychopharmacology
Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.Neuropsychopharmacology advance online publication, 2 December 2009; doi:10.1038/npp.2009.199
— id: 105488, year: 2010, vol: 35, page: 955, stat: Journal Article,

Modulation of opioid receptor function by protein-protein interactions
Alfaras-Melainis, Konstantinos; Gomes, Ivone; Rozenfeld, Raphael; Zachariou, Venetia; Devi, Lakshmi
2009 ;14:3594-3607, Frontiers in biosciences
Opioid receptors, MORP, DORP and KORP, belong to the family A of G protein coupled receptors (GPCR), and have been found to modulate a large number of physiological functions, including mood, stress, appetite, nociception and immune responses. Exogenously applied opioid alkaloids produce analgesia, hedonia and addiction. Addiction is linked to alterations in function and responsiveness of all three opioid receptors in the brain. Over the last few years, a large number of studies identified protein-protein interactions that play an essential role in opioid receptor function and responsiveness. Here, we summarize interactions shown to affect receptor biogenesis and trafficking, as well as those affecting signal transduction events following receptor activation. This article also examines protein interactions modulating the rate of receptor endocytosis and degradation, events that play a major role in opiate analgesia. Like several other GPCRs, opioid receptors may form homo or heterodimers. The last part of this review summarizes recent knowledge on proteins known to affect opioid receptor dimerization
— id: 105490, year: 2009, vol: 14, page: 3594, stat: Journal Article,

Building a new conceptual framework for receptor heteromers
Ferre, Sergi; Baler, Ruben; Bouvier, Michel; Caron, Marc G; Devi, Lakshmi A; Durroux, Thierry; Fuxe, Kjell; George, Susan R; Javitch, Jonathan A; Lohse, Martin J; Mackie, Ken; Milligan, Graeme; Pfleger, Kevin D G; Pin, Jean-Philippe; Volkow, Nora D; Waldhoer, Maria; Woods, Amina S; Franco, Rafael
2009 Mar;5(3):131-134, Nature Chemical Biology
Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, we recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. We also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development
— id: 96333, year: 2009, vol: 5, page: 131, stat: Journal Article,

Novel endogenous peptide agonists of cannabinoid receptors
Gomes, Ivone; Grushko, Julia S; Golebiewska, Urszula; Hoogendoorn, Sascha; Gupta, Achla; Heimann, Andrea S; Ferro, Emer S; Scarlata, Suzanne; Fricker, Lloyd D; Devi, Lakshmi A
2009 Sep;23(9):3020-3029, FASEB journal
Hemopressin (Hp), a 9-residue alpha-hemoglobin-derived peptide, was previously reported to function as a CB(1) cannabinoid receptor antagonist (1) . In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hpalpha) or two (VD-Hpalpha) additional amino acids, as well as a beta-hemoglobin-derived peptide with sequence similarity to that of hemopressin (VD-Hpbeta). Characterization of the alpha-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB(1) cannabinoid receptor antagonist, both RVD-Hpalpha and VD-Hpalpha function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca(2+) indicate that these peptides activate a signal transduction pathway distinct from that activated by the endocannabinoid, 2-arachidonoylglycerol, or the classic CB(1) agonist, Hu-210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB(1) receptor is involved in the integration of signals from both lipid- and peptide-derived signaling molecules
— id: 105489, year: 2009, vol: 23, page: 3020, stat: Journal Article,

Multiple actions of spinophilin regulate mu opioid receptor function
Charlton, Joanna J; Allen, Patrick B; Psifogeorgou, Kassi; Chakravarty, Sumana; Gomes, Ivone; Neve, Rachael L; Devi, Lakshmi A; Greengard, Paul; Nestler, Eric J; Zachariou, Venetia
2008 Apr 24;58(2):238-247, Neuron
Spinophilin, a dendritic spine-enriched scaffold protein, modulates synaptic transmission via multiple functions mediated by distinct domains of the protein. Here, we show that spinophilin is a key modulator of opiate action. Knockout of the spinophilin gene causes reduced sensitivity to the analgesic effects of morphine and early development of tolerance but a higher degree of physical dependence and increased sensitivity to the rewarding actions of the drug. At the cellular level, spinophilin associates with the mu opioid receptor (MOR) in striatum and modulates MOR signaling and endocytosis. Activation of MOR by opiate agonists such as fentanyl and morphine promotes these events, which feedback to suppress MOR responsiveness. Our findings support a potent physiological role of spinophilin in regulating MOR function and provide a potential new target for the treatment of opiate addiction
— id: 96337, year: 2008, vol: 58, page: 238, stat: Journal Article,

Cell surface targeting of mu-delta opioid receptor heterodimers by RTP4
Decaillot, Fabien M; Rozenfeld, Raphael; Gupta, Achla; Devi, Lakshmi A
2008 Oct 14;105(41):16045-16050, Proceedings of the National Academy of Sciences of the United States of America
Mu opioid receptors are G protein-coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that mu-delta opioid heterodimers have a pharmacologic profile distinct from those of the mu or delta homodimers. Because the heterodimers exhibit distinct signaling properties, the protein and mechanism regulating their levels have significant effects on morphine-mediated physiology. We report the characterization of RTP4, a Golgi chaperone, as a regulator of the levels of heterodimers at the cell surface. We show that the association with RTP4 protects mu-delta receptors from ubiquitination and degradation. This leads to increases in surface heterodimer levels, thereby affecting signaling. Thus, the oligomeric organization of opioid receptors is controlled by RTP4, and this governs their membrane targeting and functional activity. This work is the first report of the identification of a chaperone involved in the regulation of the biogenesis of a family A GPCR heterodimer. The identification of such factors as RTP4 controlling dimerization will provide insight into the regulation of heterodimers in vivo. This has implications in the modulation of pharmacology of their endogenous ligands, and in the development of drugs with specific therapeutic effects
— id: 96334, year: 2008, vol: 105, page: 16045, stat: Journal Article,

Homology modeling and site-directed mutagenesis to identify selective inhibitors of endothelin-converting enzyme-2
Gagnidze, Khatuna; Rozenfeld, Raphael; Mezei, Mihaly; Zhou, Ming-Ming; Devi, Lakshmi A
2008 Jun 26;51(12):3378-3387, Journal of medicinal chemistry
Endothelin-converting enzyme-2 (ECE-2), a member of M13 family of zinc metallopeptidases, has previously been shown to process a number of neuropeptides including those derived from prodynorphin, proenkephalin, proSAAS, and amyloid precursor protein. ECE-2, unlike ECE-1, exhibits restricted neuroendocrine distribution and acidic pH optimum; it is consistent with a role in the regulation of neuropeptide levels in vivo. Here, we report the generation of a three-dimensional (3D) molecular model of ECE-2 using the crystal structure of neprilysin (EC 3.4.24.11) as a template. On the basis of the predictions made from the molecular model, we mutated and tested two residues, Trp 148 and Tyr 563, in the catalytic site. The mutation of Tyr 563 was found to significantly affect the catalytic activity and inhibitor binding. The molecular model was used to virtually screen a small molecule library of 13 000 compounds. Among the top-scoring compounds three were found to inhibit ECE-2 with high affinity and exhibited specificity for ECE-2 compared to neprilysin. Thus, the model provides a new useful tool to probe the active site of ECE-2 and design additional selective inhibitors of this enzyme
— id: 96336, year: 2008, vol: 51, page: 3378, stat: Journal Article,

Antibodies against G-protein coupled receptors: novel uses in screening and drug development
Gupta, Achla; Heimann, Andrea S; Gomes, Ivone; Devi, Lakshmi A
2008 Jul;11(6):463-467, Combinatorial chemistry & high throughput screening
Antibodies are components of the body's humoral immune system that are generated in response to foreign pathogens. Modern biomedical research has employed these very specific and efficient molecules designed by nature in the diagnosis of diseases, localization of gene products as well as in the rapid screening of targets for drug discovery and testing. In addition, the introduction of antibodies with fluorescent or enzymatic tags has significantly contributed to advances in imaging and microarray technology, which are revolutionizing disease research and the search for effective therapeutics. More recently antibodies have been used in the isolation of dimeric G protein-coupled receptor (GPCR) complexes. In this review, we discuss antibodies as powerful research tools for studying GPCRs, and their potential to be developed as drugs themselves
— id: 96335, year: 2008, vol: 11, page: 463, stat: Journal Article,

Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies
Gupta, Achla; Rozenfeld, Raphael; Gomes, Ivone; Raehal, Kirsten M; Decaillot, Fabien M; Bohn, Laura M; Devi, Lakshmi A
2008 Apr 18;283(16):10735-10744, Journal of biological chemistry
The majority of studies examining activity-induced conformational changes in G protein-coupled receptors have focused on transmembrane helices or intracellular regions. Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process. To begin to address this, we generated a series of antibodies to the N-terminal region of opioid receptors. Characterization of these antibodies revealed that they differentially recognize activated receptors. Recently, we generated monoclonal antibodies that recognize regions proximal to glycosylation sites in the receptor N terminus. Characterization of these antibodies revealed that agonist treatment leads to a decrease in epitope recognition by the antibody presumably because of a movement of the region of the N terminus proximal to glycosylation sites. The time course of the decrease in antibody recognition suggested that it could be due to a post-activation-mediated event. Examination of the involvement of receptor residues in the C-tail and beta-arrestin binding using site-directed mutagenesis and cells or tissues lacking beta-arrestin 2 suggests a role for these desensitization-related mechanisms in governing antibody binding to the receptor. Thus, these N-terminally directed antibodies can differentially recognize post-activation-mediated changes in the C-terminal (intracellular) region of the receptor. Therefore, these conformation-sensitive antibodies represent powerful reagents to probe receptor activation states and provide a potential tool for identifying and characterizing new compounds of therapeutic interest
— id: 96341, year: 2008, vol: 283, page: 10735, stat: Journal Article,

Inquiry learning. Integrating content detail and critical reasoning by peer review
Iyengar, Ravi; Diverse-Pierluissi, Maria A; Jenkins, Sherry L; Chan, Andrew M; Devi, Lakshmi A; Sobie, Eric A; Ting, Adrian T; Weinstein, Daniel C
2008 Feb 29;319(5867):1189-1190, Science
— id: 96339, year: 2008, vol: 319, page: 1189, stat: Journal Article,

Teaching resources. Using web-based discussion forums as a model of the peer-review process and a tool for assessment
Jenkins, Sherry L; Iyengar, Ravi; Diverse-Pierluissi, Maria A; Chan, Andrew M; Devi, Lakshmi A; Sobie, Eric A; Ting, Adrian T; Weinstein, Daniel C
2008 ;1(9):tr2-tr2, Science signaling
This Teaching Resource describes how to use an online asynchronous discussion as a mechanism to introduce students to the peer-review process, as well as to assess student performance and understanding. This method was applied to a graduate course on signal transduction and the Teaching Resource includes a syllabus, detailed plan for incorporating the online discussion, sample journal club questions, and sample student responses to the discussion forum, faculty responses, and student revisions
— id: 96338, year: 2008, vol: 1, page: tr2, stat: Journal Article,

Regulation of CB1 cannabinoid receptor trafficking by the adaptor protein AP-3
Rozenfeld, Raphael; Devi, Lakshmi A
2008 Jul;22(7):2311-2322, FASEB journal
Cannabinoid receptor 1 (CB(1)) is an abundant G protein-coupled receptor, involved in a number of physiological processes. This receptor is localized at the plasma membrane, as well as in intracellular vesicles. The trafficking events leading to this intracellular localization remain controversial. In this study, we examine the differential trafficking of CB(1) receptors and its implication on signaling. We find that the transfected tagged receptors are predominantly at the plasma membrane, whereas endogenous receptors exhibit an intracellular localization. We also find that intracellular endogenous CB(1) receptors do not have an endocytic origin. Instead, these receptors associate with the adaptor protein AP-3 and traffic to the lysosomes. siRNA-mediated AP-3delta knockdown leads to enhanced cell surface localization of CB(1) receptors. Finally, we show that CB(1) receptors in the late endosomal/lysosomal compartment are associated with heterotrimeric G proteins and mediate signal transduction. These results suggest that intracellular CB(1) receptors are functional and that their spatial segregation is likely to significantly affect receptor function
— id: 96340, year: 2008, vol: 22, page: 2311, stat: Journal Article,

D2 receptors regulate dopamine transporter function via an extracellular signal-regulated kinases 1 and 2-dependent and phosphoinositide 3 kinase-independent mechanism
Bolan, Elizabeth A; Kivell, Bronwyn; Jaligam, Vanaja; Oz, Murat; Jayanthi, Lankupalle D; Han, Yang; Sen, Namita; Urizar, Eneki; Gomes, Ivone; Devi, Lakshmi A; Ramamoorthy, Sammanda; Javitch, Jonathan A; Zapata, Agustin; Shippenberg, Toni S
2007 May;71(5):1222-1232, Molecular pharmacology
The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D(2) dopamine receptors (D(2)R) has been reported. The high affinity of DA and other DAT substrates for the D(2)R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP(+)) with live cell imaging techniques to identify the role of two D(2)R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D(2)R regulation of DAT. Addition of the D(2)/D(3) receptor agonist quinpirole (0.1-10 muM) to human embryonic kidney cells coexpressing human DAT and D(2) receptor (short splice variant, D(2S)R) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP(+) accumulation. The D(2)/D(3) agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP(+) accumulation. D(2S)R activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP(+) accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D(2)R stimulation. These experiments demonstrate that D(2S)R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D(2)R. Together, these results suggest a novel mechanism by which D(2S)R autoreceptors may regulate DAT in the central nervous system
— id: 96346, year: 2007, vol: 71, page: 1222, stat: Journal Article,

Conformation state-sensitive antibodies to G-protein-coupled receptors
Gupta, Achla; Decaillot, Fabien M; Gomes, Ivone; Tkalych, Oleg; Heimann, Andrea S; Ferro, Emer S; Devi, Lakshmi A
2007 Feb 23;282(8):5116-5124, Journal of biological chemistry
A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest
— id: 96348, year: 2007, vol: 282, page: 5116, stat: Journal Article,

The emerging functions of endocannabinoid signaling during CNS development
Harkany, Tibor; Guzman, Manuel; Galve-Roperh, Ismael; Berghuis, Paul; Devi, Lakshmi A; Mackie, Ken
2007 Feb;28(2):83-92, Trends in pharmacological science
In the postnatal brain, endocannabinoids acting as retrograde messengers regulate the function of many synapses. By contrast, the understanding of endocannabinoid functions that regulate fundamental developmental processes such as cell proliferation, migration, differentiation and survival during patterning of the CNS is just beginning to unfold. Increasing the knowledge of basic developmental and signaling principles that are controlled by endocannabinoids will provide important insights into the molecular mechanisms that establish functional neuronal circuits in the brain. Moreover, determining the molecular basis of permanent modifications to cellular structure and intercellular communication imposed by cannabis smoking during pregnancy will provide novel therapeutic targets for alleviating pathogenic changes in affected offspring. Here, we summarize recent findings regarding the ontogeny of the endocannabinoid system in neurons that sculpt the temporal and spatial diversity of cellular functions during CNS development
— id: 96347, year: 2007, vol: 28, page: 83, stat: Journal Article,

Hemopressin is an inverse agonist of CB1 cannabinoid receptors
Heimann, Andrea S; Gomes, Ivone; Dale, Camila S; Pagano, Rosana L; Gupta, Achla; de Souza, Laura L; Luchessi, Augusto D; Castro, Leandro M; Giorgi, Renata; Rioli, Vanessa; Ferro, Emer S; Devi, Lakshmi A
2007 Dec 18;104(51):20588-20593, Proceedings of the National Academy of Sciences of the United States of America
To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB(1) cannabinoid receptors, we used the recently described conformation-state sensitive antibodies and screened a panel of endogenous peptides from rodent brain or adipose tissue. This led to the identification of hemopressin (PVNFKFLSH) as a peptide ligand that selectively binds CB(1) cannabinoid receptors. We find that hemopressin is a CB(1) receptor-selective antagonist, because it is able to efficiently block signaling by CB(1) receptors but not by other members of family A G protein-coupled receptors (including the closely related CB(2) receptors). Hemopressin also behaves as an inverse agonist of CB(1) receptors, because it is able to block the constitutive activity of these receptors to the same extent as its well characterized antagonist, rimonabant. Finally, we examine the activity of hemopressin in vivo using different models of pain and find that it exhibits antinociceptive effects when administered by either intrathecal, intraplantar, or oral routes, underscoring hemopressin's therapeutic potential. These results represent a demonstration of a peptide ligand for CB(1) cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB(1) receptors
— id: 96342, year: 2007, vol: 104, page: 20588, stat: Journal Article,

Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins: a proteomics study focusing on endocytic proteins
Moron, Jose A; Abul-Husn, Noura S; Rozenfeld, Raphael; Dolios, Georgia; Wang, Rong; Devi, Lakshmi A
2007 Jan;6(1):29-42, Molecular & cellular proteomics
Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses
— id: 96349, year: 2007, vol: 6, page: 29, stat: Journal Article,

Use of proteomics for the identification of novel drug targets in brain diseases
Moron, Jose A; Devi, Lakshmi A
2007 Jul;102(2):306-315, Journal of neurochemistry
In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases
— id: 96344, year: 2007, vol: 102, page: 306, stat: Journal Article,

An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function
Rozenfeld, Raphael; Abul-Husn, Noura S; Gomez, Ivone; Devi, Lakshmi A
2007 ;7:64-73, ScientificWorldJournal
Morphine and related opiates are commonly used in the clinical management of various types of pain. However, the antinociceptive properties of morphine are often overshadowed by the development of tolerance and dependence following its chronic use. The mechanisms underlying opiate tolerance are not fully understood, but appear to involve numerous and complex physiological adaptations. Recently, a role for the heterodimerization of mu and delta opioid receptors in the development of morphine tolerance has been proposed. This novel mechanism could help us to understand several observations, such as the critical role of delta opioid receptor regulation, the impact of delta opioid receptor binding site occupancy, and the participation of beta-arrestin2, in the development of morphine tolerance
— id: 96343, year: 2007, vol: 7, page: 64, stat: Journal Article,

Receptor heterodimerization leads to a switch in signaling: beta-arrestin2-mediated ERK activation by mu-delta opioid receptor heterodimers
Rozenfeld, Raphael; Devi, Lakshmi A
2007 Aug;21(10):2455-2465, FASEB journal
Opiates are analgesics of choice in the treatment of chronic pain, but their long-term use leads to the development of physiological tolerance. Thus, understanding the mechanisms modulating the response of their receptor, the mu opioid receptor (muOR), is of great clinical relevance. Here we show that heterodimerization of muOR with delta opioid receptors (deltaOR) leads to a constitutive recruitment of beta-arrestin2 to the receptor complex resulting in changes in the spatio-temporal regulation of ERK1/2 signaling. The involvement of beta-arrestin2 is further supported by studies using beta-arrestin2 siRNA in cells endogenously expressing the heterodimers. The association of beta-arrestin2 with the heterodimer can be altered by treatment with a combination of muOR agonist (DAMGO) and deltaOR antagonist (Tipp(psi)), and this leads to a shift in the pattern of ERK1/2 phosphorylation to the pattern observed with muOR alone. These data indicate that, in the naive state, muOR-deltaOR heterodimers are in a conformation conducive to beta-arrestin-mediated signaling. Destabilization of this conformation by cotreatment with muOR and deltaOR ligands leads to a switch to a non-beta-arrestin-mediated signaling. Taken together, these results show for the first time that muOR-deltaOR heterodimers, by differentially recruiting beta-arrestin, modulate the spatio-temporal dynamics of opioid receptor signaling
— id: 96345, year: 2007, vol: 21, page: 2455, stat: Journal Article,

Peptidomics of Cpefat/fat mouse hypothalamus and striatum: effect of chronic morphine administration
Decaillot, Fabien M; Che, Fa-Yun; Fricker, Lloyd D; Devi, Lakshmi A
2006 ;28(3):277-284, Journal of molecular neuroscience
Chronic morphine administration is known to affect several neuropeptide systems, and this could contribute to the behavioral effects of opiates. To quantitate global changes in neuropeptide levels upon chronic morphine administration, we took advantage of a method that allows selective isolation of neuropeptides from brains of mice lacking carboxypeptidase E (Cpefat/fat mice), a critical enzyme in the generation of many neuroendocrine peptides. We used a differential labeling procedure with stable isotopic tags and mass spectrometry to quantitate the relative changes in a number of hypothalamic and striatal peptides in Cpefat/fat mice chronically treated with morphine. A total of 27 distinct peptides were detected in hypothalamus and striatum. Of these, 27 were identified by mass spectrometry-based sequencing, 1 was tentatively identified by the mass and charge, and 9 were not identified. The identified peptides included fragments of proenkephalin, prothyrotropin-releasing hormone, secretogranin II, chromogranin Aand B, protachykinin B, provasopressin, promelanin concentrating hormone, and pro-SAAS. Upon morphine administration, although the levels of most of the peptides were unaltered (within a factor of 1.3 to 0.7 compared with saline control), the levels of a small number of peptides did show consistent changes (increased or decreased by 1.3-fold or more) in hypothalamus and/or striatum. Taken together, these results provide interesting insights into endogenous neuropeptide systems that are modulated by morphine and suggest further experiments to link candidate peptides with long-term effects of morphine
— id: 96351, year: 2006, vol: 28, page: 277, stat: Journal Article,

The use of receptor-specific antibodies to study G-protein-coupled receptors
Gupta, Achla; Devi, Lakshmi A
2006 Jul;73(4):673-681, Mount Sinai journal of medicine
The identification of G-protein-coupled receptor (GPCR) cDNAs has facilitated a number of studies characterizing the biochemical properties of the receptor protein. Most of these studies have used antibodies directed against the epitope-tagged receptor expressed in heterologous cells, because of the lack of sensitive and selective antibodies capable of recognizing endogenous receptors in their native state. In order to facilitate studies with endogenous receptors, efforts have been made to generate receptor-type selective, sensitive antibodies that are able to recognize endogenous receptors. In this review, we discuss the strategies as well as the details of the techniques used for the generation of monoclonal and polyclonal antibodies with a focus on family A GPCRs
— id: 96350, year: 2006, vol: 73, page: 673, stat: Journal Article,

mu opioid and CB1 cannabinoid receptor interactions: reciprocal inhibition of receptor signaling and neuritogenesis
Rios, Carl; Gomes, Ivone; Devi, Lakshmi A
2006 Jun;148(4):387-395, British journal of pharmacology
Several studies have described functional interactions between opioid and cannabinoid receptors; the underlying mechanism(s) have not been well explored. One possible mechanism is direct receptor-receptor interactions, as has been demonstrated for a number of G-protein-coupled receptors. In order to investigate interactions between opioid and cannabinoid receptors, we epitope tagged mu, delta and kappa opioid receptors with Renilla luciferase and CB1 cannabinoid or CCR5 chemokine receptors with yellow fluorescent protein and examined the extent of substrate hydrolysis induced bioluminescence resonance energy transfer (BRET) signal. We find that coexpression of opioid receptors with cannabinoid receptors, but not with chemokine receptors, leads to a significant increase in the level of BRET signal, suggesting that the opioid-cannabinoid interactions are receptor specific. In order to examine the implications of these interactions to signaling, we used GTPgammaS binding and mitogen-activated protein kinase (MAPK) phosphorylation assays and examined the effect of receptor activation on signaling. We find that the mu receptor-mediated signaling is attenuated by the CB1 receptor agonist; this effect is reciprocal and is seen in heterologous cells and endogenous tissue expressing both receptors. In order to explore the physiological consequences of this interaction, we examined the effect of receptor activation on the extent of Src and STAT3 phosphorylation and neuritogenesis in Neuro-2A cells. We find that the simultaneous activation of mu opioid and CB1 cannabinoid receptors leads to a significant attenuation of the response seen upon activation of individual receptors, implicating a role for receptor-receptor interactions in modulating neuritogenesis
— id: 96352, year: 2006, vol: 148, page: 387, stat: Journal Article,

Serotonin receptor activation leads to neurite outgrowth and neuronal survival
Fricker, Ashwana D; Rios, Carl; Devi, Lakshmi A; Gomes, Ivone
2005 Aug 18;138(2):228-235, Brain research. Molecular brain research
Serotonin 5-HT1 receptors are implicated in anxiety and depression. These receptors belong to the family A of G-protein-coupled receptors and couple to inhibitory G-proteins. Recent studies show that chronic activation of 5-HT1A receptors leads to proliferation of hippocampal neurons suggesting that neurogenesis contributes to the effects of antidepressants. However, the molecular mechanisms and pathways involved are not understood. We used Neuro 2A cells transfected with 5-HT1A receptors and SK-N-SH cells endogenously expressing the receptor to examine the effect of receptor activation on neuronal survival and neurite outgrowth. We find that receptor activation leads to increased neurite outgrowth that can be blocked by the receptor selective antagonist and by treatment with pertussis toxin or lactacystin implicating inhibitory G-proteins and proteasomal degradation in this process. Interestingly, the small G-protein Rap and the transcription factor STAT-3 are also involved since reducing the levels of Rap1 (using small interfering RNA) or STAT-3 (using dominant negative STAT3) significantly blocks 5-HT1A-receptor-mediated neurite outgrowth. The observed increase in the phosphorylation of Src and STAT-3, at sites leading to their activation, further supports a crucial role for these proteins in neurite outgrowth. We also find that prolonged activation of endogenous 5-HT1A receptors leads to increased cell survival even under starving conditions; this is completely blocked by co-treatment with the antagonist. Taken together, these findings indicate that activation of the 5-HT1A receptor leads to a number of neurotropic events by activating a series of signal transduction molecules leading to long-term changes required for neurogenesis
— id: 128989, year: 2005, vol: 138, page: 228, stat: Journal Article,

The G alpha(o/i)-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3
He, John Cijiang; Gomes, Ivone; Nguyen, Tracy; Jayaram, Gomathi; Ram, Prahlad T; Devi, Lakshmi A; Iyengar, Ravi
2005 Sep 30;280(39):33426-33434, Journal of biological chemistry
The study of the signaling pathways regulating neurite outgrowth in culture is important because of their potential role in neuronal differentiation in vivo. We have previously shown that the G alpha(o/i)-coupled CB1 cannabinoid receptor (CB1R) activates Rap1 to induce neurite outgrowth. G alpha(o/i) also activates the Src-Stat3 pathway. Here, we studied the relationship between the G alpha(o/i)-Rap1 and Src-Stat3 pathways and the role of these signaling pathways in CB1R-mediated neurite outgrowth in Neuro-2A cells. The CB1 agonist HU-210 induced pertussis toxin-sensitive Src and Stat3 phosphorylation. Dominant negative (DN) mutants of Src and Stat3 blocked CB1R-induced neurite outgrowth. Constitutively active Rap 1B and Ral-activated Src and CB1R-induced Src phosphorylation was inhibited by Rap1-DN and Ral-DN, indicating that both Rap1 and Ral mediate downstream signaling from G alpha(o/i) for Src activation. Rap1-activated Ral and Ral-DN blocked Rap-induced Src phosphorylation. G alpha(o)-induced Stat3 activation was blocked by Ral-DN, whereas v-Src-induced Stat3 activation was not inhibited by Ral-DN, indicating that the CB1R, through G alpha(o), mediates the sequential activation of Rap1 to Ral to Src to Stat3 in Neuro-2A cells. Downstream of Src, the CB1R also activated Rac1 and JNK, which enhanced CBR1-mediated Stat3 activation. Rac-DN blocked CB1R-induced activation of JNK. Pharmacological inhibition of JNK blocked Src and CB1R activation of Stat3, indicating that Rac and JNK are also involved in CB1R-mediated neurite outgrowth. Overall, this study demonstrated that G alpha(o/i)-coupled CB1R triggers neurite outgrowth in Neuro-2A through the activation of a signaling network containing two pathways that bifurcate at Src and converge at Stat3
— id: 128990, year: 2005, vol: 280, page: 33426, stat: Journal Article,

Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G(alpha)o/i-triggered proteasomal degradation of Rap1GAPII
Jordan, J Dedrick; He, John Cijiang; Eungdamrong, Narat J; Gomes, Ivone; Ali, Wasif; Nguyen, Tracy; Bivona, Trever G; Philips, Mark R; Devi, Lakshmi A; Iyengar, Ravi
2005 Mar 25;280(12):11413-11421, Journal of biological chemistry
The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth
— id: 64122, year: 2005, vol: 280, page: 11413, stat: Journal Article,

Mechanisms of action of antidepressants: from neurotransmitter systems to signaling pathways
Taylor, Chirisse; Fricker, Ashwana D; Devi, Lakshmi A; Gomes, Ivone
2005 May;17(5):549-557, Cellular signalling
Antidepressants are commonly used in the treatment of anxiety and depression, medical conditions that affect approximately 17-20% of the population. The clinical effects of antidepressants take several weeks to manifest, suggesting that these drugs induce adaptive changes in brain structures affected by anxiety and depression. In order to develop shorter-acting and more effective drugs for the treatment of anxiety and depression, it is important to understand how antidepressants bring about their beneficial effects. Recent reports suggest that antidepressants can induce neurogenesis in the adult brain, although the mechanisms involved are not clearly understood. In this review, we describe the different neurotransmitter systems that are affected by anxiety and depression and how they are modulated by antidepressant treatment with a focus on signaling molecules and pathways that are activated during neurotransmitter receptor induced neurogenesis
— id: 128992, year: 2005, vol: 17, page: 549, stat: Journal Article,

Interactions between delta opioid receptors and alpha-adrenoceptors
Rios, Carl; Gomes, Ivone; Devi, Lakshmi A
2004 Nov;31(11):833-836, Clinical & experimental pharmacology & physiology
1. Several studies have reported functional interactions between different subtypes of opioid and alpha2A-adrenoceptors in the induction of spinal cord analgesia. The mechanisms underlying this phenomenon are not well characterized. We propose that direct receptor-receptor associations could account for some of the observed functional interactions. In the present study, we examined the presence of delta opioid receptors and alpha2A-adrenoceptors in interacting complexes and the functional implications of such interactions on receptor activity. 2. Using the proximity based bioluminescence resonance energy transfer (BRET) assay, we found that the delta opioid receptors and alpha2A-adrenoceptors are in close enough proximity (< 100 A) in live cells that can foster physical interactions. 3. Using coimmunoprecipitation of differentially epitope-tagged receptors, we found that delta opiate receptors exist in interacting complexes with alpha2A-adrenoceptors in heterologous cells. 4. Finally, using receptor activity mediated neurite outgrowth in Neuro 2A cells as a physiological readout, we found that interactions between delta opiate receptors and alpha2A-adrenoceptors have functional consequences. The expression of alpha2A-adrenoceptors is sufficient to promote delta opiate receptor-mediated neurite outgrowth, suggesting that the presence of inactive alpha2A-adrenoceptors can enhance delta opiate receptor-mediated signalling. 5. Taken together, these findings suggest that modulation of receptor function as a result of physical associations between delta opiate receptors and alpha2A-adrenoceptors may account for the observed synergy between opiate and adrenergic agonists in spinal analgesia
— id: 128993, year: 2004, vol: 31, page: 833, stat: Journal Article,

Inhibitory specificity and potency of proSAAS-derived peptides toward proprotein convertase 1
Basak A; Koch P; Dupelle M; Fricker LD; Devi LA; Chretien M; Seidah NG
2001 Aug 31;276(35):32720-32728, Journal of biological chemistry
Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors
— id: 23499, year: 2001, vol: 276, page: 32720, stat: Journal Article,

Identification of peptides from brain and pituitary of Cpe(fat)/Cpe(fat) mice
Che FY; Yan L; Li H; Mzhavia N; Devi LA; Fricker LD
2001 Aug 14;98(17):9971-9976, Proceedings of the National Academy of Sciences of the United States of America
Cpe(fat)/Cpe(fat) mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumulation of many neuroendocrine peptides containing C-terminal basic residues. These processing intermediates can be readily purified on an anhydrotrypsin affinity resin. Using MS to obtain molecular mass and partial sequence information, more than 100 peptides have been identified. These peptides represent fragments of 16 known secretory pathway proteins, including proenkephalin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, and secretogranin II. Many of the identified peptides represent previously uncharacterized fragments of the precursors. For example, 12 of the 13 chromogranin B-derived peptides found in the present study have not been previously reported. Of these 13 chromogranin B-derived peptides, only five contain consensus cleavage sites for prohormone convertases at both the C and N termini. Two distinct chromogranin B-derived peptides result from cleavage at Trp-Trp bonds, a site not typically associated with neuropeptide processing. An RIA was used to confirm that one of these peptides, designated WE-15, exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpe(fat)/Cpe(fat) mice. These 'orphan' peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general technique of using affinity chromatography to isolate endogenous substrates from a mutant organism lacking an enzyme should be applicable to a wide range of enzyme-substrate systems
— id: 23498, year: 2001, vol: 98, page: 9971, stat: Journal Article,

Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking
Devi LA
2001 Oct;22(10):532-537, Trends in pharmacological science
Although classical models predict that G-protein-coupled receptors (GPCRs) function as monomers, several recent studies acknowledge that GPCRs exist as dimeric or oligomeric complexes. In addition to homodimers, heterodimers between members of the GPCR family (both closely and distantly related) have been reported. In some cases heterodimerization is required for efficient agonist binding and signaling, and in others heterodimerization appears to lead to the generation of novel binding sites. In this article, the techniques used to study GPCR heterodimers, and the 'novel pharmacology' and functional implications resulting from heterodimerization will be discussed
— id: 23497, year: 2001, vol: 22, page: 532, stat: Journal Article,

ProSAAS processing in mouse brain and pituitary
Mzhavia N; Berman Y; Che FY; Fricker LD; Devi LA
2001 Mar 2;276(9):6207-6213, Journal of biological chemistry
ProSAAS is a newly discovered protein with a neuroendocrine distribution generally similar to that of prohormone convertase 1 (PC1), a peptide-processing endopeptidase. Several proSAAS-derived peptides were previously identified in the brain and pituitary of the Cpe(fat)/Cpe(fat) mouse based on the accumulation of C-terminally extended peptides due to the absence of enzymatically active carboxypeptidase E, a peptide-processing exopeptidase. In the present study, antisera against different regions of proSAAS were used to develop radioimmunoassays and examine the processing profile of proSAAS in wild type and Cpe(fat)/Cpe(fat) mouse tissues following gel filtration and reverse phase high performance liquid chromatography. In wild type mouse brain and pituitary, the majority of proSAAS is processed into smaller peptides. These proSAAS-derived peptides elute from the reverse-phase column in the same positions as synthetic peptides that correspond to little SAAS, PEN, and big LEN. Mass spectrometry revealed the presence of peptides with the expected molecular masses of little SAAS and big LEN in the fractions containing immunoreactive peptides. The processing of proSAAS is slightly impaired in Cpe(fat)/Cpe(fat) mice, relative to wild-type mice, leading to the accumulation of partially processed peptides. One of these peptides, the C-terminally extended form of PEN, is known to inhibit PC1 activity and this could account for the reduction in enzymatically active PC1 seen in Cpe(fat)/Cpe(fat) mice. The observation that little SAAS and big LEN are the major forms of these peptides produced in mouse brain and pituitary raises the possibility that these peptides function as neurotransmitters or hormones
— id: 18585, year: 2001, vol: 276, page: 6207, stat: Journal Article,

Defective prodynorphin processing in mice lacking prohormone convertase PC2 [In Process Citation]
Berman Y; Mzhavia N; Polonskaia A; Furuta M; Steiner DF; Pintar JE; Devi LA
2000 Oct;75(4):1763-1770, Journal of neurochemistry
Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides
— id: 11498, year: 2000, vol: 75, page: 1763, stat: Journal Article,

G-protein-coupled receptor dimers in the lime light [In Process Citation]
Devi LA
2000 Sep;21(9):324-326, Trends in pharmacological science
— id: 11513, year: 2000, vol: 21, page: 324, stat: Journal Article,

Dimerization of G-Protein Coupled Receptors
Devi LA; Brady LS
2000 Oct 1;23(4 Suppl 1):S3-S4, Neuropsychopharmacology
— id: 11485, year: 2000, vol: 23, page: S3, stat: Journal Article,

Analysis of opioid peptide processing in mice lacking the various peptide processing enzymes
Devi, Lakshmi A
2000 Jan 29-Feb 01;86(1-3):15-15, Regulatory peptides
— id: 15858, year: 2000, vol: 86, page: 15, stat: Journal Article,

Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing
Fricker LD; McKinzie AA; Sun J; Curran E; Qian Y; Yan L; Patterson SD; Courchesne PL; Richards B; Levin N; Mzhavia N; Devi LA; Douglass J
2000 Jan 15;20(2):639-648, Journal of neuroscience
Five novel peptides were identified in the brains of mice lacking active carboxypeptidase E, a neuropeptide-processing enzyme. These peptides are produced from a single precursor, termed proSAAS, which is present in human, mouse, and rat. ProSAAS mRNA is expressed primarily in brain and other neuroendocrine tissues (pituitary, adrenal, pancreas); within brain, the mRNA is broadly distributed among neurons. When expressed in AtT-20 cells, proSAAS is secreted via the regulated pathway and is also processed at paired-basic cleavage sites into smaller peptides. Overexpression of proSAAS in the AtT-20 cells substantially reduces the rate of processing of the endogenous prohormone proopiomelanocortin. Purified proSAAS inhibits prohormone convertase 1 activity with an IC(50) of 590 nM but does not inhibit prohormone convertase 2. Taken together, proSAAS may represent an endogenous inhibitor of prohormone convertase 1
— id: 18587, year: 2000, vol: 20, page: 639, stat: Journal Article,

Acute ethanol treatment modulates delta opioid receptors in N18TG2 cells
Gomes I; Trapaidze N; Turndorf H; Devi LA; Bansinath M
2000 Jun;92(6):1789-1798, Anesthesiology
BACKGROUND: The in vitro adaptive responses of delta opiate receptors (DOR) to chronic ethanol treatment have been well documented. The acute effects of ethanol on these receptors are not well characterized beyond its effect on ligand binding. The aim of this study was to evaluate the acute effects of clinically relevant concentrations of ethanol (50-200 mm) on the saturation binding kinetics, receptor/ligand internalization, and agonist stimulation of G-protein coupling in N18TG2 cells expressing the Flag epitope-tagged mouse DOR. METHODS: Confocal microscopy was used to localize Flag epitope-tagged DOR in N18TG2 cells. Saturation binding assays at 4 degrees C and 37 degrees C were conducted in the absence or presence of ethanol on cells not pretreated or pretreated with ethanol for 30 min at 37 degrees C. Highly specific delta agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), was used in these studies. The effect of ethanol on agonist stimulation of G-protein coupling was examined using [35S]GTPgammaS (guanosine-5'-O-(3-thio)triphosphate) binding to membranes. Agonist-mediated receptor internalization was examined using flow cytometry of cells labeled with the antiserum directed against the Flag epitope, and the ligand internalization was examined using [3H]DPDPE. RESULTS: Ethanol decreased the binding of the agonist [3H]DPDPE, and not the antagonist [3H]diprenorphine, in a dose-dependent manner. These effects were temperature-dependent. Ethanol reversibly inhibited agonist stimulation of [35S]GTPgammaS binding. In non-pretreated cells, ethanol decreased the rate of receptor/ligand internalization, but this effect was not seen in ethanol pretreated cells. Taken together, these results suggest that pretreatment of N18TG2 cells with ethanol induces compensatory mechanisms that allow the receptor to function efficiently in its presence. CONCLUSION: Acute ethanol decreased the binding, agonist-mediated functional coupling and receptor/ligand internalization in N18TG2 cells expressing epitope-tagged DOR. In these cells, 30-min pretreatment with ethanol was sufficient to reverse these effects
— id: 11667, year: 2000, vol: 92, page: 1789, stat: Journal Article,

Kappa opioid receptor endocytosis by dynorphin peptides
Jordan BA; Cvejic S; Devi LA
2000 Jan;19(1):19-27, DNA & cell biology
Internalization and downregulation are important steps in the modulation of receptor function. Recent work with the beta2 adrenergic and opioid receptors have implicated these processes in receptor-mediated activation of mitogen-activated protein kinase (MAPK). We have used CHO cells expressing epitope-tagged rat kappa opioid receptors (rKORs) and prodynorphin-derived peptides to characterize the agonist-mediated endocytosis of rKORs and activation of MAPK. Kappa receptor-selective peptides induced receptor internalization and downregulation whereas nonpeptide agonists did not. An examination of the ability of dynorphin A-17-related peptides (lacking C-terminal amino acids) to promote KOR internalization, inhibition of adenylyl cyclase, and MAPK phosphorylation revealed that the N-terminal seven residues play an important role in eliciting these responses. Both dynorphin peptides and nonpeptide agonists induced rapid and robust phosphorylation of MAPKs. Taken together, these results point to a difference in the ability of dynorphin peptides and nonpeptide ligands to promote rKOR endocytosis and support the view that rKOR internalization is not required for MAPK activation
— id: 8558, year: 2000, vol: 19, page: 19, stat: Journal Article,

Opioids and Their Complicated Receptor Complexes
Jordan BA; Cvejic S; Devi LA
2000 Oct 1;23(4 Suppl 1):S5-S18, Neuropsychopharmacology
No field more eagerly awaits a molecular clarification for G-protein coupled receptor (GPCR) dimerization than the opioid receptor field. Extensive evidence of pharmacological and functional interactions between opioid receptor types has primed this field for such a resolution. In retrospect, much of the data collected on synergy between different opioid receptor types may represent the functional correlate for the newly found opioid receptor dimerization. While previous reports of functional synergy have been, for the most part, consistent in demonstrating cross-regulation between two receptor types, the lack of highly receptor-selective ligands allowed skeptics to remain doubtful over the interpretations of these results. Today, two important developments in the opioid receptor field help reinvigorate the hypothesis of functional, cross-modulating opioid receptor complexes: (1) The existence of highly selective ligands which eliminate any possibility of cross-reactivity between receptor types, and (2) the discovery that opioid receptors and a number of other GPCRs exist as dimers in biochemical, functional and pharmacological assays. It is with these new tools that we seek to understand the mechanisms and implications of dimerization. Initial results of these studies have demonstrated that the dimerization of opioid receptors may help consolidate several pharmacological findings that have remained unanswered. In this review we present biochemical, pharmacological and functional evidence for opioid receptor complexes and add evidence from our recent studies on opioid receptor dimerization. We believe a thorough understanding of receptor dimerization is crucial in clarifying the mechanism of action of opioids and other drugs and may serve a more practical purpose in aiding the development of novel therapeutic drugs. [Neruopsychopharmacology 23:S5-S18, 2000]
— id: 11484, year: 2000, vol: 23, page: S5, stat: Journal Article,

The C-terminal region of proSAAS is a potent inhibitor of prohormone convertase 1
Qian Y; Devi LA; Mzhavia N; Munzer S; Seidah NG; Fricker LD
2000 Aug 4;275(31):23596-23601, Journal of biological chemistry
ProSAAS is a recently discovered 26-kDa neuroendocrine protein that was previously found to inhibit prohormone convertase (PC) 1 and not PC2. In the present study, the specificity of proSAAS toward other members of the prohormone convertase family was determined. Two &mgr;m proSAAS selectively inhibits PC1 but not furin, PACE4, PC5A, or PC7. The PC1 inhibitory region of proSAAS was mapped to an 8-12-residue region near the C terminus that includes a critical Lys-Arg sequence. Synthetic peptides corresponding to this region are competitive inhibitors of PC1 with apparent K(i) values of 14-40 nm. The inhibition becomes more effective with incubation time, indicating that the inhibitor is slow binding. A fusion protein containing the inhibitory region of proSAAS linked to the C terminus of glutathione S-transferase binds the 71-kDa form but not the 85-kDa form of PC1. This binding, which occurs at pH 5.5 and not at pH 7.4, is stable to incubation at room temperature for 1 h in the presence or absence of 0.5% Triton X-100 and/or 0.5 m NaCl. The removal of Ca(2+) with chelating agents partially releases the bound PC1. High concentrations of the inhibitory peptide quantitatively release the bound PC1. Taken together, these data support the proposal that proSAAS functions as an endogenous inhibitor of PC1
— id: 18586, year: 2000, vol: 275, page: 23596, stat: Journal Article,

Role for C-tail residues in delta opioid receptor downregulation
Trapaidze N; Cvejic S; Nivarthi RN; Abood M; Devi LA
2000 Feb;19(2):93-101, DNA & cell biology
The delta opioid receptor, a member of the G-protein-coupled receptor superfamily, was used as a model system to characterize opioid receptor downregulation. Metabolic labeling followed by immunoprecipitation resulted in the isolation of the epitope-tagged mouse delta opioid receptor as a approximately 60-kDa protein. Prolonged agonist treatment with 100 nM d-Ala2, d-Leu5-enkephalin (DADLE) caused significant (approximately 60%) reduction in the level of receptor. The delta opioid receptor contains a number of phosphorylatable residues in the C tail. Point mutations of the majority of Ser/Thr sequences did not affect the level of downregulation, whereas mutation of Thr353 to Ala did. In order to test if phosphorylation at this site is involved in receptor downregulation, we generated a Thr353Glu mutant that would mimic the phosphorylated Thr at this site. This mutant exhibited a significantly higher extent of downregulation than the Thr353Ala mutant. In order to critically evaluate the requirement of Thr353 in receptor downregulation, we examined the downregulation of wildtype rat delta receptor (which does not contain Ala353) and an Ala353Thr point-mutant rat delta receptor. The wild-type receptor exhibited poor agonist-mediated downregulation, whereas Ala353Thr mutant exhibited increased downregulation. These results and results from additional studies with rat/mouse chimeric receptors support a role for phosphorylation of sites within the C tail in efficient downregulation of delta opioid receptors
— id: 8536, year: 2000, vol: 19, page: 93, stat: Journal Article,

Recycling and resensitization of delta opioid receptors
Trapaidze N; Gomes I; Bansinath M; Devi LA
2000 Apr;19(4):195-204, DNA & cell biology
Exposure to opioids results in the activation of opioid receptors; this is followed by receptor endocytosis. Previously, we showed that delta opioid receptors undergo rapid agonist-mediated internalization and that mutations in the C-tail result in a substantial loss of agonist-mediated internalization. In this study, we investigated the fate of receptors following rapid internalization. We found that the majority of the wild type receptors recycled back to the surface after acute agonist treatment. The kinetics of internalization and recycling of the receptor were virtually identical to the kinetics of internalization and recycling of the radiolabeled agonist. In contrast, the kinetics of internalization and recycling of a C-tail mutant receptor were substantially altered, suggesting an involvement of the C-tail in the recycling process. It is possible that in addition to agonist-mediated internalization, opioid receptors undergo constitutive, agonist-independent internalization. We directly examined this possibility using an antibody-prebinding assay. The wild type delta opioid receptors exhibited agonist-independent internalization via the clathrin-coated pit pathway. We also examined the role of receptor internalization and recycling in the modulation of its function by quantitating the level of opioid-stimulated phosphorylation of MAP kinase (MAPK) under conditions of receptor internalization and recycling. We found that agonist treatment caused a rapid increase in the level of phosphorylated MAPK that was rapidly desensitized. The removal of the agonist, which results in receptor recycling, led to the resensitization of the receptor, as evidenced by the agonist's ability to reinduce MAPK phosphorylation. Mutant receptors that underwent rapid recycling exhibited enhanced resensitization, suggesting a role for receptor recycling in the resensitization process. Taken together, these results indicate that agonist-mediated internalization and recycling modulate opioid receptor function and that the receptor C-tail plays an important role in both processes
— id: 11719, year: 2000, vol: 19, page: 195, stat: Journal Article,

Opioid receptor endocytosis and activation of MAP kinase pathway
Trapaidze N; Gomes I; Cvejic S; Bansinath M; Devi LA
2000 Mar 29;76(2):220-228, Brain research. Molecular brain research
Opioid receptors, members of the G-protein coupled receptor (GPCR) super family, bind to endogenous opioid peptides or opiate drugs and induce a wide variety of signal transduction processes by inhibiting adenylyl cyclase, modulating cation channels, and activating the mitogen-activated protein (MAP) kinases. Similar to other GPCRs, agonist binding causes rapid internalization and down-regulation of opioid receptors. The interdependence between receptor endocytosis and activation of MAP kinase pathway are increasingly being examined. We have examined these using ligands that exhibit differential extent of endocytosis as well as mutants of mu and delta opioid receptors that are unable to internalize. We find that ligands, including morphine, that do not induce receptor internalization are able to stimulate MAP kinase phosphorylation not only in heterologous cells but also in neuronal cell lines that express endogenous mu and delta receptors. Moreover, mutant receptors that fail to undergo agonist-mediated internalization are able to efficiently phosphorylate MAP kinases. Taken together, these data are consistent with the notion that the activation of MAP kinase pathway is an internalization independent phenomenon in the case of opioid receptors and that GPCR internalization and activation of MAP kinase are governed by complex regulatory mechanisms
— id: 11756, year: 2000, vol: 76, page: 220, stat: Journal Article,

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases
Berman Y; Juliano L; Devi LA
1999 May;72(5):2120-2126, Journal of neurochemistry
The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites
— id: 8512, year: 1999, vol: 72, page: 2120, stat: Journal Article,

Dynorphin A processing enzyme: tissue distribution, isolation, and characterization
Berman, Y; Ageyeva, L; Veksler, B; Wood, D; Devi, L A
1999 Mar;125(3):641-647, Journal of biochemistry (Tokyo)
Limited proteolysis of the dynorphin precursor (prodynorphin) at dibasic and monobasic processing sites results in the generation of bioactive dynorphins. In the brain and neurointermediate lobe of the pituitary, prodynorphin is processed to produce alpha and beta neo endorphins, dynorphins (Dyn) A-17 and Dyn A-8, Dyn B-13, and leucine-enkephalin. The formation of Dyn A-8 from Dyn A-17 requires a monobasic cleavage between Ile and Arg. We have identified an enzymatic activity capable of processing at this monobasic site in the rat brain and neurointermediate lobe of the bovine pituitary; this enzyme is designated 'dynorphin A-17 processing enzyme.' In the rat brain and neurointermediate lobe, a majority of the Dyn A processing enzyme activity is membrane-associated and can be released by treatment with 1% Triton X-100. This enzyme has been purified to apparent homogeneity from the membrane extract of the neurointermediate lobe using preparative iso-electrofocussing in a granulated gel pH 3.5 to 10, FPLC using anion exchange chromatography, and non-denaturing electrophoresis. The Dyn A processing enzyme exhibits a pI of about 5.8 and a molecular mass of about 65 kDa under reducing conditions. The Dyn A processing enzyme is a metalloprotease and has a neutral pH optimum. It exhibits substantial sensitivity to metal chelating agents and thiol agents suggesting that this enzyme is a thiol-sensitive metalloprotease. Specific inhibitors of other metallopeptidases such as enkephalinase [EC 3.4.24.11], the enkephalin generating neutral endopeptidase [EC 3.4.24.15], or NRD convertase do not inhibit the Dyn A processing enzyme activity. In contrast, specific inhibitors of angiotensin converting enzyme inhibit the activity. The purified enzyme is able to process a number of neuropeptides at both monobasic and dibasic sites. These characteristics are consistent with a role for the Dyn A processing enzyme in the processing of Dyn A-17 and other neuropeptides in the brain
— id: 133223, year: 1999, vol: 125, page: 641, stat: Journal Article,

Stereoselective interaction of ketamine with recombinant mu, kappa, and delta opioid receptors expressed in Chinese hamster ovary cells
Hirota K; Okawa H; Appadu BL; Grandy DK; Devi LA; Lambert DG
1999 Jan;90(1):174-182, Anesthesiology
BACKGROUND: The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). METHODS: CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. RESULTS: Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. CONCLUSIONS: Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors
— id: 7347, year: 1999, vol: 90, page: 174, stat: Journal Article,

G-protein-coupled receptor heterodimerization modulates receptor function
Jordan BA; Devi LA
1999 Jun 17;399(6737):697-700, Nature
The opioid system modulates several physiological processes, including analgesia, the stress response, the immune response and neuroendocrine function. Pharmacological and molecular cloning studies have identified three opioid-receptor types, delta, kappa and mu, that mediate these diverse effects. Little is known about the ability of the receptors to interact to form new functional structures, the simplest of which would be a dimer. Structural and biochemical studies show that other G-protein-coupled receptors (GPCRs) interact to form homodimers. Moreover, two non-functional receptors heterodimerize to form a functional receptor, suggesting that dimerization is crucial for receptor function. However, heterodimerization between two fully functional receptors has not been documented. Here we provide biochemical and pharmacological evidence for the heterodimerization of two fully functional opioid receptors, kappa and delta. This results in a new receptor that exhibits ligand binding and functional properties that are distinct from those of either receptor. Furthermore, the kappa-delta heterodimer synergistically binds highly selective agonists and potentiates signal transduction. Thus, heterodimerization of these GPCRs represents a novel mechanism that modulates their function
— id: 8506, year: 1999, vol: 399, page: 697, stat: Journal Article,

Inflammatory mediators regulate cathepsin S in macrophages and microglia: A role in attenuating heparan sulfate interactions
Liuzzo JP; Petanceska SS; Moscatelli D; Devi LA
1999 May;5(5):320-333, Molecular medicine
BACKGROUND: Cathepsin S is a member of the family of cysteine lysosomal proteases. The distribution of cathepsin S is restricted to cells from the mononuclear lineage both in the brain and in the periphery. Also, its protease activity is uniquely stable at neutral pH. MATERIALS AND METHODS: We compared the expression of cathepsin S, B, and L mRNAs in various undifferentiated and differentiated cells of mononuclear origin, and examined the modulation of these mRNAs by inflammatory mediators (lipopolysaccharide and various cytokines). In addition, the effect of these agents on cathepsin S protein levels and protease activity was also determined. Lastly, the ability of cathepsin S to process basement membrane components such as heparan sulfate proteoglycans in vitro and in vivo was assessed. RESULTS: Cathepsin S, B, and L mRNAs are expressed in mature macrophages and microglial cells and not in undifferentiated monocytes. Activators of macrophages negatively regulate all three transcripts. Consistent with this, treatment with these agents leads to a decrease in intracellular cathepsin S protein levels and activity. However, the same treatments result in stimulation of secreted cathepsin S activity. Cathepsin S is capable of degrading heparan sulfate proteoglycans in vitro. Also, when expressed in endothelial cells, cathepsin S autocrinely attenuates the basic fibroblast growth factor (bFGF)-mediated binding of FGF receptor containing cells to endothelial cells, by acting on basement membrane proteoglycans. CONCLUSIONS: Taken together, these data imply that cathepsin S is a regulatable cysteine protease that plays a role in the degradation of extracellular proteins, whose secretion from macrophages and microglia is increased by signals that lead to activation of these cells, and may be important in regulating extracellular matrix interactions. http://link.springer-ny. com/link/service/journals/00020/bibs/5n5p320.html
— id: 6154, year: 1999, vol: 5, page: 320, stat: Journal Article,

Cloning, expression, and characterization of human metalloprotease 1: a novel member of the pitrilysin family of metalloendoproteases
Mzhavia N; Berman YL; Qian Y; Yan L; Devi LA
1999 May;18(5):369-380, DNA & cell biology
A novel cDNA, designated human metalloendoprotease 1 (hMP1), was identified on the basis of homology to known metalloendoproteases of the pitrilysin family. The full-length MP1 codes for a protein with an open reading frame of 1038 amino acids. The N-terminal region contains the HXXEH(X)76E catalytic domain that is conserved in the members of pitrilysin family, namely insulin-degrading enzyme and NRD convertase. The hMP1 mRNA is expressed in a number of cell lines and tissues as a single species of about 3.4 kb. The expression of hMP1 mRNA is higher in muscle and heart than in brain, pancreas, liver, lung, and placenta. The full-length hMP1 was expressed in the baculovirus system and purified to homogeneity using isoelectrofocusing and ion-exchange chromatography. The enzyme exhibited a neutral pH optimum and high sensitivity to thiol reagents. HMP1 was inactivated by 1,10-phenanthroline, a specific inhibitor of Zn(+2)-dependent metalloproteases. The enzyme was not inhibited by agents that inhibit neutral metalloendoproteases of the thermolysin family such as thimet endo-oligopeptidase, enkephalinase, or angiotensin-converting enzyme. HMP1 cleaved a prodynorphin-derived peptide, leumorphin, N-terminal to Arg in the monobasic processing site, as evidenced by MALDI-TOF mass spectrometry. However, the enzyme did not exhibit strict monobasic cleavage specificity, as peptide substrates with amino acid substitutions around the monobasic site was cleaved efficiently by hMP1. Taken together, these results suggest that hMP1 is a novel member of the metalloendoprotease superfamily with ubiquitous distribution that could play a broad role in general cellular regulation
— id: 12006, year: 1999, vol: 18, page: 369, stat: Journal Article,

Effect of lidocaine on cAMP formation with recombinant opioid receptors expressed in Chinese hamster ovary cells
Hirota, K; Appadu, BL; Grandy, DK; Devi, LA; Lambert, DG
1998 SEP ;89(3A):U756-U756, Anesthesiology
— id: 53757, year: 1998, vol: 89, page: U756, stat: Journal Article,

Interaction of local anesthetics with recombinant opioid receptors expressed in Chinese hamster ovary cells
Hirota, K; Appadu, BL; Grandy, DK; Devi, LA; Lambert, DG
1998 SEP ;89(3A):U722-U722, Anesthesiology
— id: 53756, year: 1998, vol: 89, page: U722, stat: Journal Article,

Effects of C-terminal truncation of the recombinant delta-opioid receptor on phospholipase C and adenylyl cyclase coupling
Hirst RA; Smart D; Devi LA; Lambert DG
1998 Jun;70(6):2273-2278, Journal of neurochemistry
Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. We have generated two C-terminal truncation mutants of the delta-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. D-[Pen(2,5)]-enkephalin (DPDPE) mediated Ins(1,4,5)P3 formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P3 mass formation in Chinese hamster ovary (CHO) cells expressing the delta(wt), delta15, and delta37 receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHO delta(wt), 60 s in CHO delta15, and 120 s in CHO delta37) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P3 levels. Whereas the dose-response curves to Ins(1,4,5)P3 formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC50 values, with cyclic AMP inhibition being the more potent, perhaps indicating G(i alpha) coupling to adenylyl cyclase and G(i beta/gamma) coupling to PLC. Collectively, these data indicate that the C terminus of the delta-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal 'strong' coupling of receptor to Gi and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples
— id: 7603, year: 1998, vol: 70, page: 2273, stat: Journal Article,

Molecular mechanisms of opioid receptor signal transduction
Jordan B; Devi LA
1998 Jul;81(1):12-19, British journal of anaesthesia
— id: 7625, year: 1998, vol: 81, page: 12, stat: Journal Article,

Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type
Wei L; Berman Y; Castano EM; Cadene M; Beavis RC; Devi L; Levy E
1998 May 8;273(19):11806-11814, Journal of biological chemistry
A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation
— id: 7979, year: 1998, vol: 273, page: 11806, stat: Journal Article,

Bax cleavage is mediated by calpain during drug-induced apoptosis
Wood DE; Thomas A; Devi LA; Berman Y; Beavis RC; Reed JC; Newcomb EW
1998 Sep 3;17(9):1069-1078, Oncogene
The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates
— id: 7860, year: 1998, vol: 17, page: 1069, stat: Journal Article,

Chronic food restriction and streptozotocin-induced diabetes differentially alter prodynorphin mRNA levels in rat brain regions
Berman Y; Devi L; Spangler R; Kreek MJ; Carr KD
1997 Jun;46(1-2):25-30, Brain research. Molecular brain research
It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative solution hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/microg total RNA), bed nucleus of the stria terminalis (4.18 pg/microg), and in caudate nucleus (3.51 pg/microg). Lower levels were observed in the lateral hypothalamus (1.87 pg/microg) and central nucleus of the amygdala (1.22 pg/microg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need
— id: 7114, year: 1997, vol: 46, page: 25, stat: Journal Article,

Dimerization of the delta opioid receptor: implication for a role in receptor internalization
Cvejic S; Devi LA
1997 Oct 24;272(43):26959-26964, Journal of biological chemistry
Dimerization of G-protein-coupled receptors has been increasingly noted in the regulation of their biological activity. However, its involvement in agonist-induced receptor internalization is not well understood. In this study, we examined the ability of mouse delta-opioid receptors to dimerize and the role of receptor dimerization in agonist-induced internalization. Using differentially (Flag and c-Myc) epitope-tagged receptors we show that delta-opioid receptors exist as dimers. The level of dimerization is agonist dependent. Increasing concentrations of agonists reduce the levels of dimer with a corresponding increase in the levels of monomer. Interestingly, morphine does not affect the levels of either form. It has been shown that morphine, unlike other opioid agonists, does not induce receptor internalization. This suggests a relationship between the ability of agonists to reduce the levels of dimer and to induce receptor internalization. The time course of the agonist-induced decrease of delta-opioid receptor dimers is shorter than the time course of internalization, suggesting that monomerization precedes the agonist-induced internalization of the receptor. Furthermore, we found that a mutant delta-opioid receptor, with a 15-residue C-terminal deletion, does not exhibit dimerization. This mutant receptor has been shown to lack the ability to undergo agonist-induced internalization. These results suggest that the interconversion between the dimeric and monomeric forms plays a role in opioid receptor internalization
— id: 7135, year: 1997, vol: 272, page: 26959, stat: Journal Article,

Thr353, located within the COOH-terminal tail of the delta opiate receptor, is involved in receptor down-regulation
Cvejic S; Trapaidze N; Cyr C; Devi LA
1996 Feb 23;271(8):4073-4076, Journal of biological chemistry
Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of receptors, or down-regulation. The molecular mechanism of the opiate receptor down-regulation is not known. In order to address this, we generated a number of mutants of the delta opiate receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated receptor does not show down-regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type receptor and the receptor with the deletion of only 15 COOH-terminal residues show substantial down-regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the receptor down-regulation. In order to identify residues that play a key role in down-regulation, point mutations of residues within this region were examined for their ability to modulate receptor down-regulation. The receptor with a mutation of Thr353 to Ala does not down-regulate, whereas the receptor with a mutation of Ser344 to Gly down-regulates with a time course similar to that of the wild type receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-regulation and that Thr353 is critical for the agonist-mediated down-regulation of the delta opiate receptor
— id: 6898, year: 1996, vol: 271, page: 4073, stat: Journal Article,

Heterologous desensitization of the human endothelin A and neurokinin A receptors in Xenopus laevis oocytes
Cyr CR; Devi LA; Rudy B; Kris RM
1996 ;6(2):99-109, Receptors & signal transduction
Endothelin 1 (ET1) desensitizes endothelin A receptor for 90-110 min while neurokinin A (NKA) desensitizes neurokinin A receptor for 25-35 min in Xenopus laevis oocytes. In the present study, endothelin A receptor and neurokinin A receptor were coexpressed in Xenopus laevis oocytes in an effort to characterize heterologous desensitization of the receptors that activate phospholipase C-beta. ET1 desensitizes both the endothelin A receptor and the neurokinin A receptor for 90-110 min, whereas stimulation with NKA desensitizes the same two receptors for only 25-35 min. Homologous and heterologous desensitization experiments were also carried out with endothelin 3 (ET3), a ligand that exhibits lower affinity to the endothelin A receptor and a quicker dissociation rate than ET1. ET3 was unable to desensitize endothelin A receptor and the neurokinin A receptor; this is in contrast to ET1 that desensitizes both receptors. These results suggests that the receptors that undergo homologous desensitization are able to heterologously desensitize other receptors that activate PLC-beta. Furthermore, the agonist-specific dissociation constant dictates the extent of desensitization and time of recovery of the receptor-mediated response
— id: 12660, year: 1996, vol: 6, page: 99, stat: Journal Article,

Carboxypeptidase E activity is deficient in mice with the fat mutation. Effect on peptide processing
Fricker LD; Berman YL; Leiter EH; Devi LA
1996 Nov 29;271(48):30619-30624, Journal of biological chemistry
Carboxypeptidase E (CPE) is involved in the biosynthesis of many peptide hormones and neurotransmitters. Mice with the fat mutation have previously been found to have a point mutation in the cpe gene, and to have greatly reduced levels of CPE-like enzyme activity in the pituitary and pancreatic islets (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). In the present report, we examined CPE-like activity and peptide processing in several tissues of C57BLKS/LtJ-Cpefat/Cpefat mutant (Cpefat/Cpefat) mice. Whereas CPE-like activity is detected in homogenates of Cpefat/Cpefat mouse tissues, the majority of this activity is not due to CPE based on the sensitivity to p-chloromercuriphenyl sulfonate. In addition, the Cpefat/Cpefat activity does not bind to a substrate affinity column under conditions that bind CPE. Furthermore, the enzyme activity and immunoreactive properties of the activity purified from Cpefat/Cpefat brain are distinct from those of CPE. Taken together, these data suggest that CPE is completely inactive in the Cpefat/Cpefat mice, and that all of the CPE-like activity is due to other carboxypeptidases such as carboxypeptidase D. Levels of Leu-enkephalin in Cpefat/Cpefat mouse brain are approximately 5-fold lower than those in control brain. Treatment of the Cpefat/Cpefat brain extract with carboxypeptidase B restores the level of Leu-enkephalin to the level in control brain. Interestingly, the large molecular weight enkephalin-containing peptides are elevated 2-3-fold in Cpefat/Cpefat mouse brain. These data indicate that CPE plays an important role in the processing of peptide hormones in various tissues, but that other carboxypeptidases also contribute to peptide processing. Furthermore, the increase in levels of high molecular weight enkephalin peptides in the Cpefat/Cpefat mouse suggests that CPE is required for efficient peptide processing by the endopeptidases
— id: 18588, year: 1996, vol: 271, page: 30619, stat: Journal Article,

Expression of rat cathepsin S in phagocytic cells
Petanceska S; Canoll P; Devi LA
1996 Feb 23;271(8):4403-4409, Journal of biological chemistry
Cysteine lysosomal proteases are essential for turnover of intracellular and extracellular proteins. These enzymes are strongly implicated in normal and pathological processes involving tissue remodeling. Among the cysteine proteases, cathepsin S seems to be best suited for such a process since it retains most of its enzymatic activity at neutral pH. In situ hybridization analyses of the adult rat brain, spleen, and lung reveal that cathepsin S mRNA is preferentially expressed in cells of mononuclear-phagocytic origin. After entorhinal cortex lesion of adult rat brain (a paradigm for neuronal degeneration and reactive synaptogenesis), cathepsin S mRNA is dramatically increased in activated microglia in the deafferented dentate gyrus and in macrophages at the wound site, suggesting a role in lesion-induced tissue remodeling. This possibility is further supported by the finding that cathepsin S degrades a number of extracellular matrix molecules at neutral pH and by the finding that inflammatory mediators stimulate its secretion from the microglia and macrophages. These data suggest that cathepsin S is an important player in degenerative disorders associated with the cells of the mononuclear phagocytic system
— id: 6945, year: 1996, vol: 271, page: 4403, stat: Journal Article,

Sequestration of the delta opioid receptor. Role of the C terminus in agonist-mediated internalization
Trapaidze N; Keith DE; Cvejic S; Evans CJ; Devi LA
1996 Nov 15;271(46):29279-29285, Journal of biological chemistry
The primary structure of the opioid receptors have revealed that many of the structural features that are conserved in other G protein-coupled receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, whereas other structurally homologous G protein-coupled receptors do not. It is not known whether opioid receptors are regulated by rapid endocytosis. In transfected Chinese hamster ovary cells expressing the epitope-tagged wild type delta opioid receptor, exposure to 100 nM [D-Ala2,D-Leu5]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy. The rate of internalization of the wild type receptor is rapid with a half-maximal reduction by about 10 min, as determined by the reduction in mean surface receptor fluorescence intensity measured using flow cytometry. In contrast, the cells expressing receptors lacking the C-terminal 15 or 37 amino acids exhibit a substantially slower rate of internalization. Furthermore, the cells expressing receptors with point mutations of any of the Ser/Thr between Ser344 and Ser363 in the C-terminal tail exhibit a significant reduction in the rate of receptor internalization. These results suggest that a portion of the C-terminal tail is involved in receptor internalization. Agents that block the formation of clathrin-coated pits considerably reduce the extent of agonist-mediated internalization of the wild type receptor. Taken together, these results suggest that the wild type opioid receptor undergoes rapid agonist-mediated internalization via a classic endocytic pathway and that a portion of the C-terminal tail plays an important role in this internalization process
— id: 12474, year: 1996, vol: 271, page: 29279, stat: Journal Article,

Effects of streptozotocin-induced diabetes on prodynorphin-derived peptides in rat brain regions
Berman Y; Devi L; Carr KD
1995 Jul 10;685(1-2):129-134, Brain research
Pharmacological studies suggest that diabetes produces changes in the brain opioid system, affecting several behavioral functions including analgesia, feeding and self-stimulation. Previous investigations of opioid receptor binding have failed to explain the unusual opioid pharmacology of the diabetic animal. In the present study, the effects of streptozotocin-induced diabetes on levels of three immunoreactive (ir)-prodynorphin-derived peptides, ir-dynorphin A1-17 (A1-17), ir-dynorphin A1-8 (A1-8) and ir-dynorphin B1-13 (B1-13), were determined in eleven brain regions known to be involved in appetite, taste and reward. Diabetes was found to increase levels of A1-17 in the ventromedial and dorsomedial hypothalamic nuclei (+60% and +25%, respectively) and levels of A1-8 in the dorsomedial and lateral hypothalamus (+45% and +35%, respectively). The possible significance of these results is discussed in relation to (i) diabetic hyperphagia, (ii) medial hypothalamic transduction of circulating insulin levels, and (iii) the potentiation of reward by metabolic need states
— id: 6887, year: 1995, vol: 685, page: 129, stat: Journal Article,

Purification and characterization of a dynorphin-processing endopeptidase
Berman YL; Juliano L; Devi LA
1995 Oct 6;270(40):23845-23850, Journal of biological chemistry
Dynorphin B (Dyn B-13, also known as rimorphin) is generated from Dyn B-29 (leumorphin) by the cleavage at a single Arg residue. An enzymatic activity capable of processing at this monobasic site has been previously reported in neurosecretory vesicles of the bovine pituitary and pituitary-derived cell lines. This enzyme termed 'the dynorphin-converting enzyme' (DCE) has been purified to apparent homogeneity from the neurointermediate lobe of the bovine pituitary using hydrophobic chromatography on phenyl-Sepharose, preparative isoelectrofocusing in a granulated gel between pH 4 to 6.5, and non-denaturing electrophoresis on 5% polyacrylamide gel. DCE exhibits a pI of about 5.1 and a molecular mass of about 54 kDa under reducing conditions. DCE is a metallopeptidase and exhibits a neutral pH optimum. Specific Inhibitors of soluble metallopeptidases such as enkephalinase (EC 3.4.24.11) or enkephalin generating neutral endopeptidase (EC 3.4.24.15) do not inhibit DCE activity indicating that DCE is distinct from these two enzymes. Cleavage site determination with matrix-assisted laser desorption ionization time of flight (MALDITOF) mass spectrometry shows that DCE cleaves the Dyn B-29 N terminus to the Arg14 generating Dyn B-13 and Dyn B-(14-29). Among other peptides derived from Dyn B-29, DCE cleaves only those peptides that fit the predicted 'consensus motif' for monobasic processing. These data are consistent with a broader role for the dynorphin converting enzyme in the biosynthesis of many peptide hormones and neuropeptides by processing at monobasic sites
— id: 6888, year: 1995, vol: 270, page: 23845, stat: Journal Article,

Conserved cysteine residues in the cytoplasmic trail of the human neurokinin A receptor are involved in receptor desensitization
Cyr, C. R.; Josiah, S.; Rudy, B.; Devi, L.; Kris, R. M.
1995 ;21(1-3):1595-1595, Abstracts (Society for Neuroscience)
— id: 92551, year: 1995, vol: 21, page: 1595, stat: Journal Article,

Effects of chronic food restriction on prodynorphin-derived peptides in rat brain regions
Berman Y; Devi L; Carr KD
1994 Nov 21;664(1-2):49-53, Brain research
Chronic food restriction produces a variety of physiological and behavioral adaptations including a potentiation of the reinforcing effect of food, drugs and lateral hypothalamic electrical stimulation. Previous work in this laboratory has revealed that the lowering of self-stimulation threshold by food restriction is reduced by mu- and kappa-selective opioid antagonists. In the present study, the effect of chronic food restriction on levels of three prodynorphin-derived peptides, namely dynorphin A1-17 (A1-17), dynorphin A1-8 (A1-8) and dynorphin B1-13 (B1-13) were measured in eleven brain regions known to be involved in appetite, taste and reward. Food restriction increased levels of A1-17 in dorsal medial (+19.6%), ventral medial (+24.2%) and medial preoptic (+82.9%) hypothalamic areas. Levels of A1-17 decreased in the central nucleus of the amygdala (-35.1%). Food restriction increased levels of A1-8 in nucleus accumbens (+34.4%), bed nucleus of the stria terminalis (+24.5%) and lateral hypothalamus (+41.9%). Food restriction had no effect on levels of B1-13. A1-17 is highly kappa-preferring and the brain regions in which levels increased all have a high ratio of kappa: mu and delta receptors. A1-8 is less discriminating among opioid receptor types and the brain regions in which levels increased have a low ratio of kappa: mu and delta receptors. The present results suggest that food restriction alters posttranslational processing within the dynorphin A domain of the prodynorphin precursor, possibly leading to a change in the balance between kappa and non-kappa opioid receptor stimulation in specific brain regions
— id: 6573, year: 1994, vol: 664, page: 49, stat: Journal Article,

Regional distribution of neuropeptide processing endopeptidases in adult rat brain
Berman YL; Rattan AK; Carr K; Devi L
1994 ;76(3-4):245-250, Biochimie
Many peptide hormone and neuropeptide precursors undergo post-translational processing at mono- and/or dibasic residues. An enzymatic activity capable of processing prodynorphin at a monobasic processing site designated 'dynorphin converting enzyme' has been previously reported in rat rain and bovine pituitary. In this study the distribution of dynorphin converting enzyme activity in ten regions of rat brain has been compared with the distribution of subtilisin-like processing enzymes and with the immuno-reactive dynorphin peptides. The distribution of dynorphin converting enzyme activity generally matches the distribution of immuno-reactive dynorphin B-13 in most but not all brain regions. The regions that are known to have a relatively large number of immuno-reactive dynorphin-neurons also contain high levels of dynorphin converting enzyme activity. The distribution of dynorphin converting enzyme activity does not match the distribution of subtilisin-like processing enzyme or carboxypeptidase E activities. Taken together the data support the possibility that the dynorphin converting enzyme is involved in the maturation of dynorphin, as well as other neuropeptides, and peptide hormones
— id: 6574, year: 1994, vol: 76, page: 245, stat: Journal Article,

Effects of chronic food restriction on prodynorphin-derived peptides in rat brain regions
Berman, Y.; Devi, L.; Carr, K. D.
1994 ;20(1-2):752-752, Abstracts (Society for Neuroscience)
— id: 92222, year: 1994, vol: 20, page: 752, stat: Journal Article,

Regulation of neuropeptide-processing enzymes by nitric oxide in cultured astrocytes
Devi L; Petanceska S; Liu R; Arbabha B; Bansinath M; Garg U
1994 Jun;62(6):2387-2393, Journal of neurochemistry
Nitric oxide (NO), a recently discovered neurotransmitter, has been shown to have a cytostatic effect on cultured glia. A NO-generating agent, S-nitroso-N-acetyl-penicillamine (SNAP), was used to treat C6 glioma and primary cortical astrocytes. The levels of a monobasic peptide-processing enzyme activity and carboxypeptidase E activity were examined. The cellular levels of these two enzymes are specifically reduced in response to treatment with SNAP. A decrease of approximately 30-50% in these two enzyme activities was seen in both primary astrocytes and C6 glioma cells. This decrease in cellular enzyme activities is not due to increased secretion because the secreted activity is also reduced in response to SNAP treatment in both the glioma cells and the primary astrocytes. Removal of SNAP treatment causes the carboxypeptidase enzyme activity to return to control levels within 3 days. Northern and western blot analyses indicate that the reduced cellular level of carboxypeptidase E is not due to reduced expression of the messenger RNA or protein, suggesting that the SNAP treatment is affecting factors that influence carboxypeptidase E activity. Taken together, these results imply that NO is involved in the regulation of peptide biosynthetic enzymes and this could lead to the antimitogenic action of SNAP on glia
— id: 6356, year: 1994, vol: 62, page: 2387, stat: Journal Article,

Differential distribution of messenger RNAs for cathepsins B, L and S in adult rat brain: an in situ hybridization study
Petanceska S; Burke S; Watson SJ; Devi L
1994 Apr;59(3):729-738, Neuroscience
The cysteine lysosomal proteases comprise a large family of highly conserved enzymes which are essential for intracellular protein turnover. These proteases are very efficient in their ability to degrade components of the extracellular matrix, and have been implicated in processes of cell growth, malignant transformation and inflammation. There is also a growing body of evidence for their involvement in the metabolism of the amyloid precursor protein. The production of insoluble beta A4 amyloid peptide is thought to be one of the key events that lead to the development of Alzheimer's pathology. To see the physiological role these enzymes play in the brain, we studied the relative abundance and distribution of the messenger RNAs for three lysosomal cysteine proteases, cathepsins B and L and cathepsin S, by in situ hybridization histochemistry in rat brain. All three enzymes are capable of degrading components of the extracellular matrix but they have different substrate preferences and resistances to neutral pH. We found that the mRNAs for cathepsins B, L, and S have different expression patterns in brain. Cathepsin B mRNA shows the highest level of expression. It has a wide distribution, and is preferentially expressed in neurons. The expression patterns of cathepsin B and cathepsin L mRNA overlap in many brain regions; in some areas they complement each other. Cathepsin B and L mRNAs are highly expressed in the choroid plexus, a structure that is instrumental in brain development. Both transcripts are also abundant in the neuropeptide synthesizing hypothalamic nuclei. Cathepsin S mRNA has wide expression pattern throughout brain, in grey and white matter. A great number of cells that express cathepsin S have microglial morphology. Regions that are known to contain the highest amounts of the amyloid precursor protein express highest levels of cathepsin B and cathepsin L mRNA. Also, all three transcripts are highly represented in regions that are most prone to degeneration in Alzheimer's disease. These results suggest a role for these lysosomal hydrolases released from degenerating cells in the development of Alzheimer's pathology
— id: 6484, year: 1994, vol: 59, page: 729, stat: Journal Article,

Distribution and characterization of a neuropeptide processing enzyme in adult rat brain
Berman, E.; Carr, K.; Fricker, L. D.; Devi, L.
1993 ;19(1-3):1362-1362, Abstracts (Society for Neuroscience)
— id: 92223, year: 1993, vol: 19, page: 1362, stat: Journal Article,

Tissue distribution of a dynorphin-processing endopeptidase
Devi L
1993 Mar;132(3):1139-1144, Endocrinology
A number of peptide hormones and neurotransmitters require post-translational processing at monobasic cleavage sites. An enzymatic activity capable of processing prodynorphin at a monobasic processing site has been previously reported in rat brain and bovine pituitary. This dynorphin (Dyn)-converting enzyme (DCE) activity is capable of converting Dyn-B-29 (leumorphin) to Dyn-B-13 (rimorphin). The tissue distribution of the DCE activity in the adult rat shows that the activity is present at high levels in the brain, ileum, neurointermediate pituitary, and adrenal. Lower levels of activity are found in the anterior pituitary, liver, heart, ovary, kidney, lung, and serum. In the rat pituitary, the anterior lobe has 10-fold lower specific activity than the neurointermediate lobe. The protease inhibitor profile shows that the activity in various tissues is considerably inhibited by the thiol protease inhibitor p-chloromercuriphenyl sulfonic acid (PCMPS), suggesting that the Dyn-converting activity is due to a putative thiol protease. The Dyn-converting activity in the rat ileum and brain was subjected to ion exchange chromatography on diethylaminoethyl-cellulose; the majority of activity eluted around 0.3 M NaCl, as did bovine pituitary DCE. This chromatography behavior, peptide inhibitor profile, and pH optima are consistent with those of the previously reported enzyme activity from bovine pituitary and pituitary-derived cell lines. In the bovine brain, the distribution of activity generally matches that of Dyn-B-13. In the bovine adrenal medulla, the activity is localized to secretory vesicles that also contain carboxypeptidase-E activity, an enzyme thought to be involved with peptide processing. Taken together, the tissue distribution and enzyme properties support the possibility that the DCE is involved in the maturation of Dyn as well as many peptide hormones and neuropeptides
— id: 13243, year: 1993, vol: 132, page: 1139, stat: Journal Article,

Plasma beta-endorphin levels, naltrexone, and haloperidol in autistic children
Ernst M; Devi L; Silva RR; Gonzalez NM; Small AM; Malone RP; Campbell M
1993 ;29(2):221-227, Psychopharmacology bulletin
Plasma beta-endorphin levels were measured in 13 autistic children, aged 3.67 to 11.67 years at the end of treatment (naltrexone, haloperidol, pimozide, or placebo) and in 5 of the 13 children also at baseline. Baseline plasma beta-endorphin levels were lower than those reported in the literature. There was a strong correlation between plasma beta-endorphin levels and severity of sterotypies in all children. Naltrexone did not seem to have a specific effect on plasma beta-endorphin levels; short-term haloperidol treatment was associated with an increase, whereas long-term haloperidol treatment seemed to have a depressive effect on plasma beta-endorphin levels, which rose after withdrawal of haloperidol
— id: 6366, year: 1993, vol: 29, page: 221, stat: Journal Article,

Posttranslational processing of carboxypeptidase E, a neuropeptide-processing enzyme, in AtT-20 cells and bovine pituitary secretory granules
Fricker LD; Devi L
1993 Oct;61(4):1404-1415, Journal of neurochemistry
Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, 'proCPE.' To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [35S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37 degrees C but was unaffected when cells were chased at either 20 or 15 degrees C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCl2. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme
— id: 18589, year: 1993, vol: 61, page: 1404, stat: Journal Article,

Dopamine antagonist haloperidol increases carboxypeptidase E mRNA in rat neurointermediate pituitary but not in various other rat tissues
Grigoriants O; Devi L; Fricker LD
1993 Jul;19(1-2):161-164, Brain research. Molecular brain research
Carboxypeptidase E (CPE) is involved with the biosynthesis of many neuropeptides, including several whose genes are regulated by haloperidol treatments. In this study, we examined whether haloperidol alters CPE mRNA levels in a variety of tissues. Rats were treated for either 1, 3, 7, 14, or 21 days with 2 mg/kg haloperidol, and then Northern blot analysis performed on RNA from neurointermediate pituitary, anterior pituitary, hypothalamus, striatum, cerebellum, and adrenal. The 14 and 21 day treatments produced a significant 90-110% elevation of CPE mRNA in neurointermediate pituitary. However, the levels of CPE mRNA in the other tissues were not significantly influenced by the haloperidol treatments. This finding indicates that CPE is not co-regulated with peptide hormone mRNAs in all tissues
— id: 18590, year: 1993, vol: 19, page: 161, stat: Journal Article,

Processing of prodynorphin in BRL-3A cells, a rat liver-derived cell line: implications for the specificity of neuropeptide-processing enzymes
Petanceska S; Zikherman J; Fricker LD; Devi L
1993 Jul;94(1):37-45, Molecular & cellular endocrinology
Prodynorphin is post-translationally processed into dynorphin B-13 and other peptides by the action of endopeptidases that cleave at pairs of basic amino acids and at single basic residues, followed by a carboxypeptidase that removes the C-terminal basic residues. To evaluate the specificity of neuropeptide processing enzymes, rat prodynorphin was transfected into BRL-3A cells, a rat liver-derived cell line which produces insulin-like growth factor II, but does not normally express prodynorphin. The transfected prodynorphin was post-translationally processed at both monobasic and dibasic cleavage sites, with the formation of dynorphin B-13 and other peptides. This finding indicates that BRL-3A cells express prodynorphin-processing enzymes. These cells were found to secrete two enzyme activities previously implicated in the processing of dynorphin, a monobasic cleaving 'dynorphin converting enzyme' and 'carboxypeptidase E', based on inhibitor sensitivities and pH optima. The dynorphin converting enzyme secreted from BRL-3A cells elutes from an anion exchange column under the same conditions as the enzyme secreted from pituitary-derived cell lines (AtT-20, GH4C1). Northern blot analysis indicates that BRL-3A cells express carboxypeptidase E mRNA in addition to mRNA encoding furin, a prohormone-processing endopeptidase. The mRNAs for two other related endopeptidases, prohormone convertase 1 and 2, were not detected on Northern blots, suggesting that these enzymes are not required for the processing of prodynorphin. The expression of carboxypeptidase E, furin, and dynorphin converting enzyme in BRL-3A cells suggests that these peptide processing enzymes are not specific for neuropeptides, but are also present in cells which process peptide growth factors
— id: 13114, year: 1993, vol: 94, page: 37, stat: Journal Article,

Secretion and regulation of a neuropeptide-processing enzyme by AtT-20 cells
Devi L
1992 Oct;131(4):1930-1935, Endocrinology
The mouse anterior pituitary-derived cell line AtT-20 has been widely used to study the biosynthesis and secretion of peptide hormones, such as ACTH, and peptide-processing enzymes, such as carboxypeptidase-E (CPE). Although AtT-20 cells do not express dynorphin (Dyn), previous studies using gene transfer have revealed that these cells are capable of processing pro-Dyn into peptides such as Dyn-B-13. A Dyn-converting enzyme (DCE) has been identified in AtT-20 cells; this enzyme processes Dyn-B-29 to Dyn-B-13. By several criteria, the enzyme activity secreted from AtT-20 cells is similar to the previously characterized enzyme activity in rat brain and bovine pituitary. In AtT-20 cells, the DCE activity and CPE activity are localized to a similar secretory compartment. Upon stimulation with a beta-adrenergic agonist, a phorbol ester, a calcium ionophore, or forskolin, the secretion of DCE activity was increased; this rise was parallel to the secretion of CPE activity and ACTH. These data suggest that DCE activity is in the regulated pathway of secretion. In AtT-20 cells treated with glucocorticoid for up to 7 days, cellular levels of beta-endorphin decreased to half the control levels. In contrast, the levels of DCE activity did not decline in response to this treatment, suggesting that the enzyme activity was not coregulated with the endogenous hormone. Taken together, the data presented here support a role for DCE in posttranslational processing of regulatory peptides
— id: 13433, year: 1992, vol: 131, page: 1930, stat: Journal Article,

Effect of nitric oxide on mitogenesis and proliferation of cerebellar glial cells
Garg UC; Devi L; Turndorf H; Goldfrank LR; Bansinath M
1992 Oct 2;592(1-2):208-212, Brain research
In the brain, nitric oxide (NO) has been identified as a messenger molecule and a mediator of excitatory amino acid-induced neurotoxicity. In this study, the effects of NO on serum-induced mitogenesis and cell proliferation of the cerebellar glial cells were assessed. NO-generating agent, S-nitroso-N-acetylpenicillamine (SNAP) increased intracellular cyclic guanosine monophosphate (cGMP) levels. Furthermore, 2 chemically dissimilar NO-generating agents, SNAP and sodium nitroprusside (SNP) inhibited serum-induced thymidine incorporation and cell proliferation. The antimitogenic effect of NO was mimicked by 8-bromo-cGMP and blocked by hemoglobin, a known inhibitor of NO. The effect of NO was not cytotoxic, since the cells were not stained with Trypan blue and did not show increased release of lactate dehydrogenase in the culture supernatants. However, NO-treated cells showed decreased conversion of tetrazolium to blue formazan suggesting that NO inhibited mitochondrial activity in the glial cells. These results demonstrate that NO inhibits serum-induced mitogenesis and cell proliferation of cultured rat cerebellar glial cells
— id: 13398, year: 1992, vol: 592, page: 208, stat: Journal Article,

Dynorphin-processing endopeptidase in the rat anterior pituitary lactotrophic cell line, GH4C1
Greco L; Daly L; Kim S; Devi L
1992 Mar;55(3):351-356, Neuroendocrinology
Several peptide hormones and neurotransmitters are produced by cleavage at the monobasic processing sites. An endoprotease capable of cleaving a dynorphin peptide at the monobasic processing site is secreted from the rat anterior pituitary lactotrophic cell line, GH4C1. When characterized by fast protein liquid chromatography using an ion exchange column, the majority of the endoprotease activity elutes as a single symmetrical peak around 0.3 M NaCl. The protease inhibitor profile suggests that the activity is due to putative thiol protease. These enzymatic properties are similar to a monobasic processing enzyme previously found in bovine pituitary and in the rat brain. The secretory pathway which contains the enzyme activity in GH4C1 cells was characterized by stimulation of secretion by thyrotropin releasing hormone, forskolin, phorbol ester, or potassium chloride. The secretion of the enzyme activity was substantially increased by these compounds suggesting that the GH4C1 cells secrete the activity via the regulated pathway. A hormonal treatment of the GH4C1 cells which has been previously shown to produce a substantial increase in the number of secretory granules and ir-prolactin has been found in this study to elevate this enzyme activity 2-fold. This increase is similar to that seen in the carboxypeptidase E activity, another putative peptide hormone processing enzyme activity. These data suggest that the peptide processing activity is regulated to a small but significant extent and is coordinately regulated with carboxypeptidase E activity
— id: 13685, year: 1992, vol: 55, page: 351, stat: Journal Article,

Sequence analysis, tissue distribution, and expression of rat cathepsin S
Petanceska S; Devi L
1992 Dec 25;267(36):26038-26043, Journal of biological chemistry
Cysteine proteases are involved in many diverse cellular processes ranging from processing of precursor proteins to intracellular degradation. In an effort to identify novel cysteine proteases, we used the polymerase chain reaction and primers directed against the catalytic sites of previously cloned cysteine proteases. From rat brain mRNA, a 600-base pair band was amplified; cloning and partial sequence analysis of this band resulted in the identification of cathepsins B and L and five novel sequences. The novel cDNAs contained a number of residues conserved in lysosomal cysteine proteases, including the active site residue His159 (papain numbering). In addition, the amino acid homology between the novel sequences and either cathepsins B, L, or H, ranged from 63 to 32%. The insert with highest homology was used to screen a rat brain cDNA library; a 1334-base pair cDNA was isolated and the nucleotide sequence determined. This sequence encodes an open reading frame of 330 amino acids which is 82% homologous to human cathepsin S, suggesting that this sequence represents rat cathepsin S. Northern blot analysis for rat cathepsin S revealed tissue-specific expression distinct from the distribution of cathepsin B and L. The regulation of expression of rat cathepsin S mRNA in response to thyroid-stimulating hormone was studied in a rat thyroid cell line FRTL-5. The level of cathepsin S mRNA was substantially increased in response to thyroid-stimulating hormone, whereas cathepsin B and cathepsin L mRNA levels were not altered by this treatment. A portion of cDNA encoding the predicted mature protein of rat cathepsin S was expressed as a glutathione S-transferase-fusion protein. The affinity-purified protein exhibited proteolytic activity with properties similar to bovine cathepsin S. Taken together, these results imply highly specific functions for cathepsin S
— id: 13340, year: 1992, vol: 267, page: 26038, stat: Journal Article,

Consensus sequence for processing of peptide precursors at monobasic sites
Devi L
1991 Mar 25;280(2):189-194, FEBS letters
Many regulatory peptide precursors undergo post-translational processing at mono- and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid is present at either 3, 5, or 7 amino acids N-terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C-terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. When compared with a large number of sequences containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many of these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites
— id: 14096, year: 1991, vol: 280, page: 189, stat: Journal Article,

Subcellular localization, partial purification, and characterization of a dynorphin processing endopeptidase from bovine pituitary
Devi L; Gupta P; Fricker LD
1991 Jan;56(1):320-329, Journal of neurochemistry
An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues
— id: 14232, year: 1991, vol: 56, page: 320, stat: Journal Article,

Comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase E (EC 3.4.17.10) and other carboxypeptidase B-like enzymes
Fricker LD; Devi L
1990 Jan;184(1):21-27, Analytical biochemistry
Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme involved in the biosynthesis of numerous peptide hormones and neurotransmitters. A sensitive assay for CPE and other carboxypeptidase B-like enzymes has been developed using 125I-acetyl-Tyr-Ala-Arg (125I-AcYAR) as the substrate. This peptide is poorly soluble in ethyl acetate whereas the product of carboxypeptidase B-like enzymatic activity (125I-AcYA) can be quantitatively extracted with this solvent, allowing the rapid separation of product from substrate. This radiometric assay can detect less than 1 pg of either CPE or carboxypeptidase B. For CPE, the assay with 125I-AcYAR is approximately 1000 times more sensitive than a fluorescent assay using dansyl-Phe-Ala-Arg (dans-FAR), and 6000 times more sensitive than a spectrophotometric assay using hippuryl-Arg (hipp-R). CPE hydrolyzes the three substrates with Kcat values of 16 s-1 for AcYAR, 13 s-1 for dans-FAR, and 8.5 s-1 for hipp-R. The Km values for CPE with AcYAR (28 microM) and dans-FAR (34 microM) are similar, and are much lower than the Km with hipp-R (400 microM). Thus, the primary reason for the increased sensitivity of the 125I-AcYAR assay over the fluorescent assay is not a result of kinetic differences but is due to the detection limit of iodinated product (10(-15) mol), compared to the fluorescent product (5 x 10(-11) mol). Applications of this rapid and sensitive radiometric assay to detect CPE in cultured cells and in subcellular fractions of the pituitary are described
— id: 18591, year: 1990, vol: 184, page: 21, stat: Journal Article,

Expression and posttranslational processing of preprodynorphin complementary DNA in the mouse anterior pituitary cell line AtT-20
Devi L; Gupta P; Douglass J
1989 Nov;3(11):1852-1860, Molecular endocrinology
A recombinant plasmid containing the rat prodynorphin cDNA was introduced into the mouse anterior pituitary corticotroph cell line AtT-20. These cells normally express and posttranslationally process proopiomelanocortin, but not prodynorphin. Stable transformants were isolated and analyzed for the expression and processing of prodynorphin. The stably transformed AtT-20 cells that expressed a 1.3-kilobase prodynorphin mRNA also expressed prodynorphin protein and processed it to dynorphin peptides. The peptides included leucine-enkephalin, beta-neoendorphin, dynorphin-A8, and dynorphin-B, as identified by gel filtration and reverse phase HPLC followed by RIA using peptide-specific antisera. These results demonstrate that AtT-20 cells efficiently and accurately process prodynorphin at both dibasic sites and monobasic cleavage sites, indicating that the AtT-20 cells contain enzymes capable of cleaving the precursor not only at dibasic residues but also at monobasic residues. The release of prodynorphin-derived peptides paralleled secretion of endogenous proopiomelanocortin-derived peptides when stimulated by CRF, a natural secretagogue for ACTH
— id: 10445, year: 1989, vol: 3, page: 1852, stat: Journal Article,

Expression and Post-Translational Processing of Preprodynorphin in the Rat Anterior Pituitary Cell Line, GH4C1
Devi, L; Gupta, P
1989 Oct 1;1(5):363-368, Journal of neuroendocrinology
Abstract A recombinant plasmid containing the rat preprodynorphin cDNA was introduced into the rat anterior pituitary cell line, GH4C1. These cells normally express growth hormone and prolactin but not prodynorphin. Stable transformants were isolated and analyzed for the expression and processing of prodynorphin. Chromatographic analyses demonstrated that the prodynorphin was incompletely processed in GH4C1 cells. Analyses of the peptides by specific radioimmunoassays to chemically synthesized peptides showed that the cells have the ability to process both at dibasic and monobasic cleavage sites. The release of prodynorphin-derived peptides paralleled that of prolactin upon stimulation with thyrotropin-releasing hormone, forskolin or carbachol suggesting that the prodynorphin-derived peptides and prolactin are sequestered in similar physiologically responsive compartments. These data suggest that the GH4C1 cells incompletely process prodynorphin. The processing in GH4C1 cells occurs both at monobasic and dibasic cleavage sites
— id: 105918, year: 1989, vol: 1, page: 363, stat: Journal Article,