Moosa Mohammadi, Ph.D.

The Alternatively Spliced Acid Box Region Plays a Key Role in FGF Receptor Autoinhibition
Kalinina, Juliya; Dutta, Kaushik; Ilghari, Dariush; Beenken, Andrew; Goetz, Regina; Eliseenkova, Anna V; Cowburn, David; Mohammadi, Moosa
2012 Jan 11;20(1):77-88, Structure
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human malignancies necessitating multiple layers of self-regulatory control mechanisms. Fibroblast growth factor receptor (FGFR) autoinhibition mediated by the alternatively spliced immunoglobulin (Ig) domain 1 (D1) and the acid box (AB)-containing linker between D1 and Ig domain 2 (D2) serves as the first line of defense to minimize inadvertent FGF signaling. In this report, nuclear magnetic resonance and surface plasmon resonance spectroscopy are used to demonstrate that the AB subregion of FGFR electrostatically engages the heparan sulfate (HS)-binding site on the D2 domain in cis to directly suppress HS-binding affinity of FGFR. Furthermore, the cis electrostatic interaction sterically autoinhibits ligand-binding affinity of FGFR because of the close proximity of HS-binding and primary ligand-binding sites on the D2 domain. These data, together with the strong amino acid sequence conservation of the AB subregion among FGFR orthologs, highlight the universal role of the AB subregion in FGFR autoinhibition
— id: 149963, year: 2012, vol: 20, page: 77, stat: Journal Article,

Regulation of serum 1,25(OH)2 vitamin D3 levels by fibroblast growth factor 23 is mediated by FGF receptors 3 and 4
Gattineni, Jyothsna; Twombley, Katherine; Goetz, Regina; Mohammadi, Moosa; Baum, Michel
2011 Aug;301(2):F371-F377, American journal of physiology. Renal physiology
Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone implicated in the pathogenesis of several hypophosphatemic disorders. FGF23 causes hypophosphatemia by decreasing the expression of sodium phosphate cotransporters (NaPi-2a and NaPi-2c) and decreasing serum 1,25(OH)(2)Vitamin D(3) levels. We previously showed that FGFR1 is the predominant receptor for the hypophosphatemic actions of FGF23 by decreasing renal NaPi-2a and 2c expression while the receptors regulating 1,25(OH)(2)Vitamin D(3) levels remained elusive. To determine the FGFRs regulating 1,25(OH)(2)Vitamin D(3) levels, we studied FGFR3(-/-)FGFR4(-/-) mice as these mice have shortened life span and are growth retarded similar to FGF23(-/-) and Klotho(-/-) mice. Baseline serum 1,25(OH)(2)Vitamin D(3) levels were elevated in the FGFR3(-/-)FGFR4(-/-) mice compared with wild-type mice (102.2 +/- 14.8 vs. 266.0 +/- 34.0 pmol/l; P = 0.001) as were the serum levels of FGF23. Administration of recombinant FGF23 had no effect on serum 1,25(OH)(2)Vitamin D(3) in the FGFR3(-/-)FGFR4(-/-) mice (173.4 +/- 32.7 vs. 219.7 +/- 56.5 pmol/l; vehicle vs. FGF23) while it reduced serum 1,25(OH)(2)Vitamin D(3) levels in wild-type mice. Administration of FGF23 to FGFR3(-/-)FGFR4(-/-) mice resulted in a decrease in serum parathyroid hormone (PTH) levels and an increase in serum phosphorus levels mediated by increased renal phosphate reabsorption. These data indicate that FGFR3 and 4 are the receptors that regulate serum 1,25(OH)(2)Vitamin D(3) levels in response to FGF23. In addition, when 1,25(OH)(2)Vitamin D(3) levels are not affected by FGF23, as in FGFR3(-/-)FGFR4(-/-) mice, a reduction in PTH can override the effects of FGF23 on renal phosphate transport
— id: 137867, year: 2011, vol: 301, page: F371, stat: Journal Article,

Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice
Wang, Hongwei; Venkatesh, Madhukumar; Li, Hao; Goetz, Regina; Mukherjee, Subhajit; Biswas, Arunima; Zhu, Liang; Kaubisch, Andreas; Wang, Lei; Pullman, James; Whitney, Kathleen; Kuro-o, Makoto; Roig, Andres I; Shay, Jerry W; Mohammadi, Moosa; Mani, Sridhar
2011 Aug 1;121(8):3220-3232, Journal of clinical investigation
The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and 'normal' intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas
— id: 137955, year: 2011, vol: 121, page: 3220, stat: Journal Article,

Research resource: Comprehensive expression atlas of the fibroblast growth factor system in adult mouse
Fon Tacer, Klementina; Bookout, Angie L; Ding, Xunshan; Kurosu, Hiroshi; John, George B; Wang, Lei; Goetz, Regina; Mohammadi, Moosa; Kuro-o, Makoto; Mangelsdorf, David J; Kliewer, Steven A
2010 Oct;24(10):2050-2064, Molecular endocrinology
Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals
— id: 138019, year: 2010, vol: 24, page: 2050, stat: Journal Article,

Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation
Goetz, Regina; Nakada, Yuji; Hu, Ming Chang; Kurosu, Hiroshi; Wang, Lei; Nakatani, Teruyo; Shi, Mingjun; Eliseenkova, Anna V; Razzaque, Mohammed S; Moe, Orson W; Kuro-o, Makoto; Mohammadi, Moosa
2010 Jan 5;107(1):407-412, Proceedings of the National Academy of Sciences of the United States of America
Fibroblast growth factor (FGF) 23 inhibits renal phosphate reabsorption by activating FGF receptor (FGFR) 1c in a Klotho-dependent fashion. The phosphaturic activity of FGF23 is abrogated by proteolytic cleavage at the RXXR motif that lies at the boundary between the FGF core homology domain and the 72-residue-long C-terminal tail of FGF23. Here, we show that the soluble ectodomains of FGFR1c and Klotho are sufficient to form a ternary complex with FGF23 in vitro. The C-terminal tail of FGF23 mediates binding of FGF23 to a de novo site generated at the composite FGFR1c-Klotho interface. Consistent with this finding, the isolated 72-residue-long C-terminal tail of FGF23 impairs FGF23 signaling by competing with full-length ligand for binding to the binary FGFR-Klotho complex. Injection of the FGF23 C-terminal tail peptide into healthy rats inhibits renal phosphate excretion and induces hyperphosphatemia. In a mouse model of renal phosphate wasting attributable to high FGF23, the FGF23 C-terminal peptide reduces phosphate excretion, leading to an increase in serum phosphate concentration. Our data indicate that proteolytic cleavage at the RXXR motif abrogates FGF23 activity by a dual mechanism: by removing the binding site for the binary FGFR-Klotho complex that resides in the C-terminal region of FGF23, and by generating an endogenous inhibitor of FGF23. We propose that peptides derived from the C-terminal tail of FGF23 or peptidomimetics and small-molecule organomimetics of the C-terminal tail can be used as therapeutics to treat renal phosphate wasting
— id: 105946, year: 2010, vol: 107, page: 407, stat: Journal Article,

Influence of Heparin Mimetics on Assembly of the FGF center dot FGFR4 Signaling Complex
Saxena, K; Schieborr, U; Anderka, O; Duchardt-Ferner, E; Elshorst, B; Gande, SL; Janzon, J; Kudlinzki, D; Sreeramulu, S; Dreyer, MK; Wendt, KU; Herbert, C; Duchaussoy, P; Bianciotto, M; Driguez, PA; Lassalle, G; Savi, P; Mohammadi, M; Bono, F; Schwalbe, H
2010 AUG 20 ;285(34):26628-26640, Journal of biological chemistry
Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF center dot FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1 center dot FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2 center dot FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1 center dot FGFR4 interaction site and a direct FGFR4 center dot FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF center dot FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2 center dot FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2 center dot HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF center dot FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs
— id: 111927, year: 2010, vol: 285, page: 26628, stat: Journal Article,

Nonsense Mutations in FGF8 Gene Causing Different Degrees of Human Gonadotropin-Releasing Deficiency
Trarbach, EB; Abreu, AP; Silveira, LFG; Garmes, HM; Baptista, MTM; Teles, MG; Costa, EMF; Mohammadi, M; Pitteloud, N; Mendonca, BB; Latronico, AC
2010 JUL ;95(7):3491-3496, Journal of clinical endocrinology & metabolism
Context: FGFR1 mutations cause isolated hypogonadotropic hypogonadism (IHH) with or without olfactory abnormalities, Kallmann syndrome, and normosmic IHH respectively. Recently, missense mutations in FGF8, a key ligand for fibroblast growth factor receptor (FGFR) 1 in the ontogenesis of GnRH, were identified in IHH patients, thus establishing FGF8 as a novel locus for human GnRH deficiency. Objective: Our objective was to analyze the clinical, hormonal, and molecular findings of two familial IHH patients due to FGF8 gene mutations. Methods and Patients: The entire coding region of the FGF8 gene was amplified and sequenced in two well-phenotyped IHH probands and their relatives. Results: Two unique heterozygous nonsense mutations in FGF8(p.R127X and p.R129X) were identified in two unrelated IHH probands, which were absent in 150 control individuals. These two mutations, mapped to the core domain of FGF8, impact all four human FGF8 isoforms, and lead to the deletion of a large portion of the protein, generating nonfunctional FGF8 ligands. The p.R127X mutation was identified in an 18-yr-old Kallmann syndrome female. Her four affected siblings with normosmic IHH or delayed puberty also carried the p.R127X mutation. Additional developmental anomalies, including cleft lip and palate and neurosensorial deafness, were also present in this family. The p.R129X mutation was identified in a 30-yr-old man with familial normosmic IHH and severe GnRH deficiency. Conclusions: We identified the first nonsense mutations in the FGF8 gene in familial IHH with variable degrees of GnRH deficiency and olfactory phenotypes, confirming that loss-of-function mutations in FGF8 cause human GnRH deficiency. (J Clin Endocrinol Metab 95: 3491-3496, 2010)
— id: 111334, year: 2010, vol: 95, page: 3491, stat: Journal Article,

New loci for congenital hypopituitarism: overlap with Kallmann syndrome
Avbelj, M; Romero, C; Tziaferi, V; McCabe, M; Zhang, CK; Sidis, Y; Plummer, L; Elting, M; Martin, C; Zou, QY; Mohammadi, M; Dattani, M; Radovick, S; Pitteloud, N
2009 NOV ;72(3):30-31, Hormone research
— id: 106178, year: 2009, vol: 72, page: 30, stat: Journal Article,

The FGF family: biology, pathophysiology and therapy
Beenken, Andrew; Mohammadi, Moosa
2009 Mar;8(3):235-253, Nature reviews. Drug discovery
The family of fibroblast growth factors (FGFs) regulates a plethora of developmental processes, including brain patterning, branching morphogenesis and limb development. Several mitogenic, cytoprotective and angiogenic therapeutic applications of FGFs are already being explored, and the recent discovery of the crucial roles of the endocrine-acting FGF19 subfamily in bile acid, glucose and phosphate homeostasis has sparked renewed interest in the pharmacological potential of this family. This Review discusses traditional applications of recombinant FGFs and small-molecule FGF receptor kinase inhibitors in the treatment of cancer and cardiovascular disease and their emerging potential in the treatment of metabolic syndrome and hypophosphataemic diseases
— id: 95478, year: 2009, vol: 8, page: 235, stat: Journal Article,

Graded levels of FGF protein span the midbrain and can instruct graded induction and repression of neural mapping labels
Chen, Yao; Mohammadi, Moosa; Flanagan, John G
2009 Jun 25;62(6):773-780, Neuron
Graded guidance labels are widely used in neural map formation, but it is not well understood which potential strategy leads to their graded expression. In midbrain tectal map development, FGFs can induce an entire midbrain, but their protein distribution is unclear, nor is it known whether they may act instructively to produce graded gene expression. Using a receptor-alkaline phosphatase fusion probe, we find a long-range posterior > anterior FGF protein gradient spanning the midbrain. Heparan sulfate proteoglycan (HSPG) is required for this gradient. To test whether graded FGF concentrations can instruct graded gene expression, a quantitative tectal explant assay was developed. Engrailed-2 and ephrin-As, normally in posterior > anterior tectal gradients, showed graded upregulation. Moreover, EphAs, normally in anterior > posterior countergradients, showed coordinately graded downregulation. These results provide a mechanism to establish graded mapping labels and more generally provide a developmental strategy to coordinately induce a structure and pattern its cell properties in gradients
— id: 100779, year: 2009, vol: 62, page: 773, stat: Journal Article,

Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma
Gartside, Michael G; Chen, Huaibin; Ibrahimi, Omar A; Byron, Sara A; Curtis, Amy V; Wellens, Candice L; Bengston, Ana; Yudt, Laura M; Eliseenkova, Anna V; Ma, Jinghong; Curtin, John A; Hyder, Pilar; Harper, Ursula L; Riedesel, Erica; Mann, Graham J; Trent, Jeffrey M; Bastian, Boris C; Meltzer, Paul S; Mohammadi, Moosa; Pollock, Pamela M
2009 Jan;7(1):41-54, Molecular cancer research
We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer
— id: 95479, year: 2009, vol: 7, page: 41, stat: Journal Article,

FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1
Gattineni, Jyothsna; Bates, Carlton; Twombley, Katherine; Dwarakanath, Vangipuram; Robinson, Michael L; Goetz, Regina; Mohammadi, Moosa; Baum, Michel
2009 Aug;297(2):F282-F291, American journal of physiology. Renal physiology
Fibroblast growth factor-23 (FGF23) is a phosphaturic hormone that contributes to several hypophosphatemic disorders by reducing the expression of the type II sodium-phosphate cotransporters (NaPi-2a and NaPi-2c) in the kidney proximal tubule and by reducing serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] levels. The FGF receptor(s) mediating the hypophosphatemic action of FGF23 in vivo have remained elusive. In this study, we show that proximal tubules express FGFR1, -3, and -4 but not FGFR2 mRNA. To determine which of these three FGFRs mediates FGF23's hypophosphatemic actions, we characterized phosphate homeostasis in FGFR3(-/-) and FGFR4(-/-) null mice, and in conditional FGFR1(-/-) mice, with targeted deletion of FGFR1 expression in the metanephric mesenchyme. Basal serum phosphorus levels and renal cortical brush-border membrane (BBM) NaPi-2a and NaPi-2c expression were comparable between FGFR1(-/-), FGFR3(-/-), and FGFR4(-/-) mice and their wild-type counterparts. Administration of FGF23 to FGFR3(-/-) mice induced hypophosphatemia in these mice (8.0 +/- 0.4 vs. 5.4 +/- 0.3 mg/dl; p < or = 0.001) and a decrease in renal BBM NaPi-2a and NaPi-2c protein expression. Similarly, in FGFR4(-/-) mice, administration of FGF23 caused a small but significant decrease in serum phosphorus levels (8.7 +/- 0.3 vs. 7.6 +/- 0.4 mg/dl; p < or = 0.001) and in renal BBM NaPi-2a and NaPi-2c protein abundance. In contrast, injection of FGF23 into FGFR1(-/-) mice had no effects on serum phosphorus levels (5.6 +/- 0.3 vs. 5.2 +/- 0.5 mg/dl) or BBM NaPi-2a and NaPi-2c expression. These data show that FGFR1 is the predominant receptor for the hypophosphatemic action of FGF23 in vivo, with FGFR4 likely playing a minor role
— id: 135000, year: 2009, vol: 297, page: F282, stat: Journal Article,

Crystal structure of a fibroblast growth factor homologous factor (FHF) defines a conserved surface on FHFs for binding and modulation of voltage-gated sodium channels
Goetz, Regina; Dover, Katarzyna; Laezza, Fernanda; Shtraizent, Nataly; Huang, Xiao; Tchetchik, Dafna; Eliseenkova, Anna V; Xu, Chong-Feng; Neubert, Thomas A; Ornitz, David M; Goldfarb, Mitchell; Mohammadi, Moosa
2009 Jun 26;284(26):17883-17896, Journal of biological chemistry
Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs
— id: 100603, year: 2009, vol: 284, page: 17883, stat: Journal Article,

Homodimerization controls the fibroblast growth factor 9 subfamily's receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix
Kalinina, Juliya; Byron, Sara A; Makarenkova, Helen P; Olsen, Shaun K; Eliseenkova, Anna V; Larochelle, William J; Dhanabal, Mohanraj; Blais, Steven; Ornitz, David M; Day, Loren A; Neubert, Thomas A; Pollock, Pamela M; Mohammadi, Moosa
2009 Sep;29(17):4663-4678, Molecular & cellular biology
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research
— id: 101445, year: 2009, vol: 29, page: 4663, stat: Journal Article,

Differential interactions of FGFs with heparan sulfate control gradient formation and branching morphogenesis
Makarenkova, Helen P; Hoffman, Matthew P; Beenken, Andrew; Eliseenkova, Anna V; Meech, Robyn; Tsau, Cindy; Patel, Vaishali N; Lang, Richard A; Mohammadi, Moosa
2009 ;2(88):ra55-ra55, Science signaling
The developmental activities of morphogens depend on the gradients that they form in the extracellular matrix. Here, we show that differences in the binding of fibroblast growth factor 7 (FGF7) and FGF10 to heparan sulfate (HS) underlie the formation of different gradients that dictate distinct activities during branching morphogenesis. Reducing the binding affinity of FGF10 for HS by mutating a single residue in its HS-binding pocket converted FGF10 into a functional mimic of FGF7 with respect to gradient formation and regulation of branching morphogenesis. In particular, the mutant form of FGF10 caused lacrimal and salivary gland epithelium buds to branch rather than to elongate. In contrast, mutations that reduced the affinity of the FGF10 for its receptor affected the extent, but not the nature, of the response. Our data may provide a general model for understanding how binding to HS regulates other morphogenetic gradients
— id: 111955, year: 2009, vol: 2, page: ra55, stat: Journal Article,

In vivo genetic evidence for klotho-dependent, fibroblast growth factor 23 (Fgf23) -mediated regulation of systemic phosphate homeostasis
Nakatani, Teruyo; Sarraj, Bara; Ohnishi, Mutsuko; Densmore, Michael J; Taguchi, Takashi; Goetz, Regina; Mohammadi, Moosa; Lanske, Beate; Razzaque, M Shawkat
2009 Feb;23(2):433-441, FASEB journal
A major breakthrough in systemic phosphate homeostasis regulation was achieved by the demonstration of strikingly similar physical, morphological, and biochemical phenotypes of fibroblast growth factor 23 (Fgf23) and klotho ablated mice, which led to identification of klotho as an Fgf23 signaling cofactor. Here, we generated Fgf23 and klotho double-knockout (Fgf23(-/-)/klotho(-/-)) mice to test the hypothesis whether Fgf23 has a klotho-independent function. Fgf23(-/-)/klotho(-/-) mice are viable and have high serum phosphate levels, similar to Fgf23(-/-) and klotho(-/-) single-knockout mice. In addition, the Fgf23(-/-)/klotho(-/-) mice have increased renal expression of the sodium/phosphate cotransporter NaP(i)2a and of 1- alpha-hydroxylase concomitant with increased serum levels of 1,25-dihydroxyvitamin-D, as also observed in the Fgf23(-/-) and klotho(-/-) mice. Moreover, Fgf23(-/-)/klotho(-/-) mice show soft tissue and vascular calcification, severe muscle wasting, hypogonadism, pulmonary emphysema, distention of intestinal wall, and skin atrophy, all of which are also seen in Fgf23(-/-) and klotho(-/-) mice. Notably, injection of bioactive FGF23 protein into Fgf23(-/-)/klotho(-/-) and klotho(-/-) mice does not lower serum phosphate, whereas in wild-type and Fgf23(-/-) mice, it reduces serum phosphate. Together, these results provide compelling evidence that Fgf23 does not have a klotho-independent role in the regulation of systemic phosphate and vitamin D homeostasis
— id: 95480, year: 2009, vol: 23, page: 433, stat: Journal Article,

FGF21 induces PGC-1alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response
Potthoff, Matthew J; Inagaki, Takeshi; Satapati, Santhosh; Ding, Xunshan; He, Tianteng; Goetz, Regina; Mohammadi, Moosa; Finck, Brian N; Mangelsdorf, David J; Kliewer, Steven A; Burgess, Shawn C
2009 Jun 30;106(26):10853-10858, Proceedings of the National Academy of Sciences of the United States of America
The liver plays a crucial role in mobilizing energy during nutritional deprivation. During the early stages of fasting, hepatic glycogenolysis is a primary energy source. As fasting progresses and glycogen stores are depleted, hepatic gluconeogenesis and ketogenesis become major energy sources. Here, we show that fibroblast growth factor 21 (FGF21), a hormone that is induced in liver by fasting, induces hepatic expression of peroxisome proliferator-activated receptor gamma coactivator protein-1alpha (PGC-1alpha), a key transcriptional regulator of energy homeostasis, and causes corresponding increases in fatty acid oxidation, tricarboxylic acid cycle flux, and gluconeogenesis without increasing glycogenolysis. Mice lacking FGF21 fail to fully induce PGC-1alpha expression in response to a prolonged fast and have impaired gluconeogenesis and ketogenesis. These results reveal an unexpected relationship between FGF21 and PGC-1alpha and demonstrate an important role for FGF21 in coordinately regulating carbohydrate and fatty acid metabolism during the progression from fasting to starvation
— id: 133692, year: 2009, vol: 106, page: 10853, stat: Journal Article,

Impaired fibroblast growth factor receptor 1 signaling as a cause of normosmic idiopathic hypogonadotropic hypogonadism
Raivio, Taneli; Sidis, Yisrael; Plummer, Lacey; Chen, Huaibin; Ma, Jinghong; Mukherjee, Abir; Jacobson-Dickman, Elka; Quinton, Richard; Van Vliet, Guy; Lavoie, Helene; Hughes, Virginia A; Dwyer, Andrew; Hayes, Frances J; Xu, Shuyun; Sparks, Susan; Kaiser, Ursula B; Mohammadi, Moosa; Pitteloud, Nelly
2009 Nov;94(11):4380-4390, Journal of clinical endocrinology & metabolism
CONTEXT: FGFR1 mutations have been identified in about 10% of patients with Kallmann syndrome. Recently cases of idiopathic hypogonadotropic hypogonadism (IHH) with a normal sense of smell (nIHH) have been reported. AIMS: The objective of the study was to define the frequency of FGFR1 mutations in a large cohort of nIHH, delineate the spectrum of reproductive phenotypes, assess functionality of the FGFR1 mutant alleles in vitro, and investigate genotype-phenotype relationships. DESIGN: FGFR1 sequencing of 134 well-characterized nIHH patients (112 men and 22 women) and 270 healthy controls was performed. The impact of the identified mutations on FGFR1 function was assessed using structural prediction and in vitro studies. RESULTS: Nine nIHH subjects (five males and four females; 7%) harbor a heterozygous mutation in FGFR1 and exhibit a wide spectrum of pubertal development, ranging from absent puberty to reversal of IHH in both sexes. All mutations impair receptor function. The Y99C, Y228D, and I239T mutants impair the tertiary folding, resulting in incomplete glycosylation and reduced cell surface expression. The R250Q mutant reduces receptor affinity for FGF. The K618N, A671P, and Q680X mutants impair tyrosine kinase activity. However, the degree of functional impairment of the mutant receptors did not always correlate with the reproductive phenotype, and variable expressivity of the disease was noted within family members carrying the same FGFR1 mutation. These discrepancies were partially explained by additional mutations in known IHH loci. CONCLUSIONS: Loss-of-function mutations in FGFR1 underlie 7% of nIHH with different degrees of impairment in vitro. These mutations act in concert with other gene defects in several cases, consistent with oligogenicity
— id: 133728, year: 2009, vol: 94, page: 4380, stat: Journal Article,

Compositional Analysis of Heparin/Heparan Sulfate Interacting with Fibroblast Growth Factor.Fibroblast Growth Factor Receptor Complexes
Zhang, FM; Zhang, ZQ; Lin, XF; Beenken, A; Eliseenkova, AV; Mohammadi, M; Linhardt, RJ
2009 SEP 8 ;48(35):8379-8386, Biochemistry
Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. One major function of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and form FGF.HS.FGFR signaling complexes. Past studies primarily examined the selectivity of HS for FGF or FGFR. In this report, we used a new strategy to study the structural specificity of HS binding to 10 different FGF.FGFR complexes. Oligosaccharide libraries prepared from heparin, 6-desufated heparin, and HS were used for the interaction studies by solution competition surface plasmon resonance (SPR) and filter trapping assays. specific oligosaccharides binding to FGF.FGFR complexes were subjected to polyacrylamide gel electrophoresis (PAGE) analysis and disaccharide compositional analysis using liquid chromatography and mass spectrometry. The competition SPR studies using sized oligosaccharide mixtures showed that binding of each of the tested FGFs or FGF.FGFR complexes to heparin immobilized to an SPR chip was size-dependent. The 6-desulfated heparin oligosaccharides exhibited a reduced level of inhibition of FGF and FGF.FGFR complex binding to heparin in the competition experiments. Heparin and the 6-desulfated heparin exhibited higher levels of inhibition of the FGF.FGFR complex binding to heparin than of FGF binding to heparin. In the filter trapping experiments, PAGE analysis showed different affinities between the FGF.FGFR complexes kind oligosaccharides. Disaccharide analysis showed that HS disaccharides with a degree of polymerization of 10(dp10) had high binding selectivity, while dp10 heparin and dp10 6-desulfated heparin showed reduced or no selectivity for the different FGF.FGFR complexes tested
— id: 102304, year: 2009, vol: 48, page: 8379, stat: Journal Article,

Somatic FGF9 mutations in colorectal and endometrial carcinomas associated with membranous beta-catenin
Abdel-Rahman, Wael M; Kalinina, Juliya; Shoman, Soheir; Eissa, Saad; Ollikainen, Miina; Elomaa, Outi; Eliseenkova, Anna V; Butzow, Ralf; Mohammadi, Moosa; Peltomaki, Paivi
2008 Mar;29(3):390-397, Human mutation
We previously described striking molecular features including high frequency of membranous beta-catenin in subsets of familial colon cancers with as yet unknown predisposition. We hypothesized that such tumors might carry mutations in Wnt/beta-catenin target genes. Fibroblast growth factor 9 (FGF9) was an attractive target, as it maps to a common area of loss of heterozygosity (LOH) in colorectal carcinomas on 13q12.11. Here, we report, for the first time, the occurrence of FGF9 mutations in human cancers. We found a total of six distinct FGF9 mutations including one frameshift, four missense, and one nonsense, in 10 (six colorectal and four endometrial) out of 203 tumors and cell lines. The frameshift mutation was detected in five different tumors. Mapping of these mutations onto the crystal structure of FGF9 predicted that they should all lead to loss of function albeit through variable mechanisms. The p.R173K mutation should diminish ligand affinity for heparin/heparan sulfate, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations should negatively impact ligand's interaction with receptor, while p.G84E and p.E142X (FGF9(Delta142-208)) mutations should interfere with ligand folding. Consistent with these structural predictions, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations impaired the ability of ligand to activate mitogen-activated protein kinase (MAPK) cascade in cultured cells expressing FGF receptors. LOH was observed in seven out of nine FGF9 mutant tumors, supporting the predicted loss of function. Interestingly, eight out of 10 (80%) of the FGF9 mutant tumors showed normal membranous beta-catenin expression and the absence of mutation in the beta-catenin gene (CTNNB1). These data suggest that FGF9 plays a role in colorectal and endometrial carcinogenesis
— id: 95484, year: 2008, vol: 29, page: 390, stat: Journal Article,

FGF9 mutations in colorectal and endometrial cancers
Abdel-Rahman, WM; Kalinina, J; Eliseenkova, A; Eissa, S; Ollikainen, M; Elomaa, O; Butzow, R; Shoman, S; Mohammadi, M; Peltomaki, P
2008 JUL ;6(9):48-48, EJC Supplements
— id: 86831, year: 2008, vol: 6, page: 48, stat: Journal Article,

A crystallographic snapshot of tyrosine trans-phosphorylation in action
Chen, Huaibin; Xu, Chong-Feng; Ma, Jinghong; Eliseenkova, Anna V; Li, Wanqing; Pollock, Pamela M; Pitteloud, Nelly; Miller, W Todd; Neubert, Thomas A; Mohammadi, Moosa
2008 Dec 16;105(50):19660-19665, Proceedings of the National Academy of Sciences of the United States of America
Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 A away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs
— id: 90927, year: 2008, vol: 105, page: 19660, stat: Journal Article,

Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice
Falardeau, John; Chung, Wilson C J; Beenken, Andrew; Raivio, Taneli; Plummer, Lacey; Sidis, Yisrael; Jacobson-Dickman, Elka E; Eliseenkova, Anna V; Ma, Jinghong; Dwyer, Andrew; Quinton, Richard; Na, Sandra; Hall, Janet E; Huot, Celine; Alois, Natalie; Pearce, Simon H S; Cole, Lindsay W; Hughes, Virginia; Mohammadi, Moosa; Tsai, Pei; Pitteloud, Nelly
2008 Aug;118(8):2822-2831, Journal of clinical investigation
Idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome; KS) or with a normal sense of smell (normosmic IHH; nIHH) are heterogeneous genetic disorders associated with deficiency of gonadotropin-releasing hormone (GnRH). While loss-of-function mutations in FGF receptor 1 (FGFR1) cause human GnRH deficiency, to date no specific ligand for FGFR1 has been identified in GnRH neuron ontogeny. Using a candidate gene approach, we identified 6 missense mutations in FGF8 in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism. Four mutations affected all 4 FGF8 splice isoforms (FGF8a, FGF8b, FGF8e, and FGF8f), while 2 mutations affected FGF8e and FGF8f isoforms only. The mutant FGF8b and FGF8f ligands exhibited decreased biological activity in vitro. Furthermore, mice homozygous for a hypomorphic Fgf8 allele lacked GnRH neurons in the hypothalamus, while heterozygous mice showed substantial decreases in the number of GnRH neurons and hypothalamic GnRH peptide concentration. In conclusion, we identified FGF8 as a gene implicated in GnRH deficiency in both humans and mice and demonstrated an exquisite sensitivity of GnRH neuron development to reductions in FGF8 signaling
— id: 95482, year: 2008, vol: 118, page: 2822, stat: Journal Article,

Inhibition of growth hormone signaling by the fasting-induced hormone FGF21
Inagaki, Takeshi; Lin, Vicky Y; Goetz, Regina; Mohammadi, Moosa; Mangelsdorf, David J; Kliewer, Steven A
2008 Jul;8(1):77-83, Cell metabolism
Starvation blocks the actions of growth hormone (GH) and inhibits growth through mechanisms that are not well understood. In this report, we demonstrate that fibroblast growth factor 21 (FGF21), a hormone induced by fasting, causes GH resistance. In liver, FGF21 reduces concentrations of the active form of signal transducer and activator of transcription 5 (STAT5), a major mediator of GH actions, and causes corresponding decreases in the expression of its target genes, including insulin-like growth factor 1 (IGF-1). FGF21 also induces hepatic expression of IGF-1 binding protein 1 and suppressor of cytokine signaling 2, which blunt GH signaling. Chronic exposure to FGF21 markedly inhibits growth in mice. These data suggest a central role for FGF21 in inhibiting growth as part of its broader role in inducing the adaptive response to starvation
— id: 95483, year: 2008, vol: 8, page: 77, stat: Journal Article,

FGF-23: Beyond PI regulation
Medici, D; Sitara, D; DeLuca, S; Mohammadi, M; Kuro-o, M; Razzaque, MS; Olsen, BR; Erben, RG; Lanske, B
2008 JUN ;82(6):S23-S24, Calcified tissue international
— id: 86859, year: 2008, vol: 82, page: S23, stat: Journal Article,

FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis
Medici, Damian; Razzaque, Mohammed S; Deluca, Stephelynn; Rector, Trent L; Hou, Bo; Kang, Kihwa; Goetz, Regina; Mohammadi, Moosa; Kuro-O, Makoto; Olsen, Bjorn R; Lanske, Beate
2008 Aug 11;182(3):459-465, Journal of cell biology
Fibroblast growth factor 23 (FGF-23) and Klotho are secretory proteins that regulate mineral-ion metabolism. Fgf-23(-/-) or Klotho(-/-) knockout mice exhibit several pathophysiological processes consistent with premature aging including severe atrophy of tissues. We show that the signal transduction pathways initiated by FGF-23-Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1alpha-hydroxylase expression and phosphoinositide-3 kinase-dependent inhibition of caspase activity. These data provide new insights into the physiological roles of FGF-23 and Klotho
— id: 95481, year: 2008, vol: 182, page: 459, stat: Journal Article,

CARB 100-Structural analysis on heparin/heparan sulfate interacting with FGF center dot FGFR complexes
Zhang, FM; Zhang, ZQ; Beenken, A; Mohammadi, M; Linhardt, RJ
2008 AUG 17 ;236(2):94-94, Abstracts of papers (American Chemical Society)
— id: 106238, year: 2008, vol: 236, page: 94, stat: Journal Article,

The parathyroid is a target organ for FGF23 in rats
Ben-Dov, Iddo Z; Galitzer, Hillel; Lavi-Moshayoff, Vardit; Goetz, Regina; Kuro-o, Makoto; Mohammadi, Moosa; Sirkis, Roy; Naveh-Many, Tally; Silver, Justin
2007 Dec;117(12):4003-4008, Journal of clinical investigation
Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. FGF23 is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of vitamin D. FGF23 signals with highest efficacy through several FGF receptors (FGFRs) bound by the transmembrane protein Klotho as a coreceptor. Since most tissues express FGFR, expression of Klotho determines FGF23 target organs. Here we identify the parathyroid as a target organ for FGF23 in rats. We show that the parathyroid gland expressed Klotho and 2 FGFRs. The administration of recombinant FGF23 led to an increase in parathyroid Klotho levels. In addition, FGF23 activated the MAPK pathway in the parathyroid through ERK1/2 phosphorylation and increased early growth response 1 mRNA levels. Using both rats and in vitro rat parathyroid cultures, we show that FGF23 suppressed both parathyroid hormone (PTH) secretion and PTH gene expression. The FGF23-induced decrease in PTH secretion was prevented by a MAPK inhibitor. These data indicate that FGF23 acts directly on the parathyroid through the MAPK pathway to decrease serum PTH. This bone-parathyroid endocrine axis adds a new dimension to the understanding of mineral homeostasis
— id: 95485, year: 2007, vol: 117, page: 4003, stat: Journal Article,

A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases
Chen, Huaibin; Ma, Jinghong; Li, Wanqing; Eliseenkova, Anna V; Xu, Chongfeng; Neubert, Thomas A; Miller, W Todd; Mohammadi, Moosa
2007 Sep 7;27(5):717-730, Molecular cell
Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly
— id: 73939, year: 2007, vol: 27, page: 717, stat: Journal Article,

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members
Goetz, Regina; Beenken, Andrew; Ibrahimi, Omar A; Kalinina, Juliya; Olsen, Shaun K; Eliseenkova, Anna V; Xu, ChongFeng; Neubert, Thomas A; Zhang, Fuming; Linhardt, Robert J; Yu, Xijie; White, Kenneth E; Inagaki, Takeshi; Kliewer, Steven A; Yamamoto, Masaya; Kurosu, Hiroshi; Ogawa, Yasushi; Kuro-o, Makoto; Lanske, Beate; Razzaque, Mohammed S; Mohammadi, Moosa
2007 May;27(9):3417-3428, Molecular & cellular biology
Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors
— id: 71392, year: 2007, vol: 27, page: 3417, stat: Journal Article,

A homozygous missense mutation in human Klotho causes severe tumoral calcinosis, but not premature aging
Ichikawa, S; Imel, EA; Kreiter, ML; Yu, X; Mackenzie, DS; Sorenson, AH; Goetz, R; Mohammadi, M; White, KE; Econs, MJ
2007 SEP ;22(1):S33-S33, Journal of bone & mineral research
— id: 75795, year: 2007, vol: 22, page: S33, stat: Journal Article,

A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis
Ichikawa, Shoji; Imel, Erik A; Kreiter, Mary L; Yu, Xijie; Mackenzie, Donald S; Sorenson, Andrea H; Goetz, Regina; Mohammadi, Moosa; White, Kenneth E; Econs, Michael J
2007 Sep;117(9):2684-2691, Journal of clinical investigation
Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans
— id: 95486, year: 2007, vol: 117, page: 2684, stat: Journal Article,

Endocrine regulation of the fasting response by PPARalpha-mediated induction of fibroblast growth factor 21
Inagaki, Takeshi; Dutchak, Paul; Zhao, Guixiang; Ding, Xunshan; Gautron, Laurent; Parameswara, Vinay; Li, Yong; Goetz, Regina; Mohammadi, Moosa; Esser, Victoria; Elmquist, Joel K; Gerard, Robert D; Burgess, Shawn C; Hammer, Robert E; Mangelsdorf, David J; Kliewer, Steven A
2007 Jun;5(6):415-425, Cell metabolism
Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the utilization of fat as an energy source during starvation and is the molecular target for the fibrate dyslipidemia drugs. Here, we identify the endocrine hormone fibroblast growth factor 21 (FGF21) as a mediator of the pleiotropic actions of PPARalpha. FGF21 is induced directly by PPARalpha in liver in response to fasting and PPARalpha agonists. FGF21 in turn stimulates lipolysis in white adipose tissue and ketogenesis in liver. FGF21 also reduces physical activity and promotes torpor, a short-term hibernation-like state of regulated hypothermia that conserves energy. These findings demonstrate an unexpected role for the PPARalpha-FGF21 endocrine signaling pathway in regulating diverse metabolic and behavioral aspects of the adaptive response to starvation
— id: 95488, year: 2007, vol: 5, page: 415, stat: Journal Article,

Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21
Kurosu, Hiroshi; Choi, Mihwa; Ogawa, Yasushi; Dickson, Addie S; Goetz, Regina; Eliseenkova, Anna V; Mohammadi, Moosa; Rosenblatt, Kevin P; Kliewer, Steven A; Kuro-o, Makoto
2007 Sep 14;282(37):26687-26695, Journal of biological chemistry
The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1-4). We demonstrated that Klotho and betaKlotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires betaKlotho. Both FGF19 and FGF21 can signal through FGFR1-3 bound by betaKlotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the betaKlotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of betaKlotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21
— id: 95487, year: 2007, vol: 282, page: 26687, stat: Journal Article,

BetaKlotho is required for metabolic activity of fibroblast growth factor 21
Ogawa, Yasushi; Kurosu, Hiroshi; Yamamoto, Masaya; Nandi, Animesh; Rosenblatt, Kevin P; Goetz, Regina; Eliseenkova, Anna V; Mohammadi, Moosa; Kuro-o, Makoto
2007 May 1;104(18):7432-7437, Proceedings of the National Academy of Sciences of the United States of America
Fibroblast growth factor 21 (FGF21) is a liver-derived endocrine factor that stimulates glucose uptake in adipocytes. Here, we show that FGF21 activity depends on betaKlotho, a single-pass transmembrane protein whose expression is induced during differentiation from preadipocytes to adipocytes. BetaKlotho physically interacts with FGF receptors 1c and 4, thereby increasing the ability of these FGF receptors to bind FGF21 and activate the MAP kinase cascade. Knockdown of betaKlotho expression by siRNA in adipocytes diminishes glucose uptake induced by FGF21. Importantly, administration of FGF21 into mice induces MAP kinase phosphorylation in white adipose tissue and not in tissues without betaKlotho expression. Thus, betaKlotho functions as a cofactor essential for FGF21 activity
— id: 95489, year: 2007, vol: 104, page: 7432, stat: Journal Article,

Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism
Pitteloud, Nelly; Quinton, Richard; Pearce, Simon; Raivio, Taneli; Acierno, James; Dwyer, Andrew; Plummer, Lacey; Hughes, Virginia; Seminara, Stephanie; Cheng, Yu-Zhu; Li, Wei-Ping; Maccoll, Gavin; Eliseenkova, Anna V; Olsen, Shaun K; Ibrahimi, Omar A; Hayes, Frances J; Boepple, Paul; Hall, Janet E; Bouloux, Pierre; Mohammadi, Moosa; Crowley, William
2007 Feb;117(2):457-463, Journal of clinical investigation
Idiopathic hypogonadotropic hypogonadism (IHH) due to defects of gonadotropin-releasing hormone (GnRH) secretion and/or action is a developmental disorder of sexual maturation. To date, several single-gene defects have been implicated in the pathogenesis of IHH. However, significant inter- and intrafamilial variability and apparent incomplete penetrance in familial cases of IHH are difficult to reconcile with the model of a single-gene defect. We therefore hypothesized that mutations at different IHH loci interact in some families to modify their phenotypes. To address this issue, we studied 2 families, one with Kallmann syndrome (IHH and anosmia) and another with normosmic IHH, in which a single-gene defect had been identified: a heterozygous FGF receptor 1 (FGFR1) mutation in pedigree 1 and a compound heterozygous gonadotropin-releasing hormone receptor (GNRHR) mutation in pedigree 2, both of which varied markedly in expressivity within and across families. Further candidate gene screening revealed a second heterozygous deletion in the nasal embryonic LHRH factor (NELF) gene in pedigree 1 and an additional heterozygous FGFR1 mutation in pedigree 2 that accounted for the considerable phenotypic variability. Therefore, 2 different gene defects can synergize to produce a more severe phenotype in IHH families than either alone. This genetic model could account for some phenotypic heterogeneity seen in GnRH deficiency
— id: 95491, year: 2007, vol: 117, page: 457, stat: Journal Article,

Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes
Pollock, PM; Gartside, MG; Dejeza, LC; Powell, MA; Mallon, MA; Davies, H; Mohammadi, M; Futreal, PA; Stratton, MR; Trent, JM; Goodfellow, PJ
2007 NOV 1 ;26(50):7158-7162, Oncogene
Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 ( 30%) of endometrial cell lines and 19/187 ( 10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype ( 18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare-Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W ( in eight tumors) and N550K ( in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma
— id: 75124, year: 2007, vol: 26, page: 7158, stat: Journal Article,

Impaired FGF signaling contributes to cleft lip and palate
Riley, Bridget M; Mansilla, M Adela; Ma, Jinghong; Daack-Hirsch, Sandra; Maher, Brion S; Raffensperger, Lisa M; Russo, Erilynn T; Vieira, Alexandre R; Dode, Catherine; Mohammadi, Moosa; Marazita, Mary L; Murray, Jeffrey C
2007 Mar 13;104(11):4512-4517, Proceedings of the National Academy of Sciences of the United States of America
Nonsyndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from a combination of genetic and environmental factors. Several members of the FGF and FGFR families are expressed during craniofacial development and can rarely harbor mutations that result in human clefting syndromes. We hypothesized that disruptions in this pathway might also contribute to NS CLP. We sequenced the coding regions and performed association testing on 12 genes (FGFR1, FGFR2, FGFR3, FGF2, FGF3, FGF4, FGF7, FGF8, FGF9, FGF10, FGF18, and NUDT6) and used protein structure analyses to predict the function of amino acid variants. Seven likely disease-causing mutations were identified, including: one nonsense mutation (R609X) in FGFR1, a de novo missense mutation (D73H) in FGF8, and other missense variants in FGFR1, FGFR2, and FGFR3. Structural analysis of FGFR1, FGFR2, and FGF8 variants suggests that these mutations would impair the function of the proteins, albeit through different mechanisms. Genotyping of SNPs in the genes found associations between NS CLP and SNPs in FGF3, FGF7, FGF10, FGF18, and FGFR1. The data suggest that the FGF signaling pathway may contribute to as much as 3-5% of NS CLP and will be a consideration in the clinical management of CLP
— id: 95490, year: 2007, vol: 104, page: 4512, stat: Journal Article,

Hedgehogs like it sweet, too
Beenken, Andrew; Mohammadi, Moosa
2006 Nov 14;103(46):17069-17070, Proceedings of the National Academy of Sciences of the United States of America
— id: 69697, year: 2006, vol: 103, page: 17069, stat: Journal Article,

The FGF signaling pathway and human disease
Ibrahimi, OA; Mohammadi, M
2006 MAR 6 ;20(4):A694-A694, FASEB journal
— id: 63859, year: 2006, vol: 20, page: A694, stat: Journal Article,

A single amino acid substitution in the activation loop defines the decoy characteristic of VEGFR-1/FLT-1
Meyer, Rosana D; Mohammadi, Moosa; Rahimi, Nader
2006 Jan 13;281(2):867-875, Journal of biological chemistry
VEGFR-1 is a kinase-defective receptor tyrosine kinase (RTK) and negatively modulates angiogenesis by acting as a decoy receptor. The decoy characteristic of VEGFR-1 is required for normal development and angiogenesis. To date, there is no molecular explanation for this unusual characteristic of VEGFR-1. Here we show that the molecular mechanisms underlying the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue in the activation loop. This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid, but in VEGFR-1 it is substituted to asparagine. Mutation of asparagine (Asn(1050)) within the activation loop to aspartic acid promoted enhanced ligand-dependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. The mutant VEGFR-1 (Asp(1050)) promoted endothelial cell proliferation but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. Furthermore, mutation of the invariable aspartic acid to asparagine in VEGFR-2 lowered the autophosphorylation of activation loop tyrosines 1052 and 1057. We propose that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines, and its absence renders RTK to a less potent enzyme by disfavoring transphosphorylation of activation loop tyrosines
— id: 68522, year: 2006, vol: 281, page: 867, stat: Journal Article,

Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain
Olsen, Shaun K; Li, James Y H; Bromleigh, Carrie; Eliseenkova, Anna V; Ibrahimi, Omar A; Lao, Zhimin; Zhang, Fuming; Linhardt, Robert J; Joyner, Alexandra L; Mohammadi, Moosa
2006 Jan 15;20(2):185-198, Genes & development
Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the 'c' splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity of FGF8a and FGF8b, the 'b' isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8b(F32A) mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution
— id: 62746, year: 2006, vol: 20, page: 185, stat: Journal Article,

Mutations in fibroblast growth factor receptor 1 cause both Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism
Pitteloud, Nelly; Acierno, James S Jr; Meysing, Astrid; Eliseenkova, Anna V; Ma, Jinghong; Ibrahimi, Omar A; Metzger, Daniel L; Hayes, Frances J; Dwyer, Andrew A; Hughes, Virginia A; Yialamas, Maria; Hall, Janet E; Grant, Ellen; Mohammadi, Moosa; Crowley, William F Jr
2006 Apr 18;103(16):6281-6286, Proceedings of the National Academy of Sciences of the United States of America
Mutations in KAL1 and FGFR1 cause Kallmann syndrome (KS), whereas mutations in the GNRHR and GPR54 genes cause idiopathic hypogonadotropic hypogonadism with normal olfaction (nIHH). Mixed pedigrees containing both KS and nIHH have also been described; however, the genetic cause of these rare cases is unknown. We examined the FGFR1 gene in seven nIHH subjects who either belonged to a mixed pedigree (n = 5) or who had associated midline defects (n = 2). Heterozygous FGFR1 mutations were found in three of seven unrelated nIHH probands with normal MRI of the olfactory system: (i) G237S in an nIHH female and a KS brother; (ii) (P722H and N724K) in an nIHH male missing two teeth and his mother with isolated hyposmia; and (iii) Q680X in a nIHH male with cleft lip/palate and missing teeth, his brother with nIHH, and his father with delayed puberty. We show that these mutations lead to receptor loss-of-function. The Q680X leads to an inactive FGFR1, which lacks a major portion of the tyrosine kinase domain (TKD). The G237S mutation inhibits proper folding of D2 of the FGFR1 and likely leads to the loss of cell-surface expression of FGFR1. In contrast, the (P722H and N724K) double mutation causes structural perturbations in TKD, reducing the catalytic activity of TKD. We conclude that loss-of-function mutations in FGFR1 cause nIHH with normal MRI of the olfactory system. These mutations also account for some of the mixed pedigrees, thus challenging the current idea that KS and nIHH are distinct entities
— id: 68520, year: 2006, vol: 103, page: 6281, stat: Journal Article,

Mutations in fibroblast growth factor receptor 1 cause Kallmann syndrome with a wide spectrum of reproductive phenotypes
Pitteloud, Nelly; Meysing, Astrid; Quinton, Richard; Acierno, James S Jr; Dwyer, Andrew A; Plummer, Lacey; Fliers, Eric; Boepple, Paul; Hayes, Frances; Seminara, Stephanie; Hughes, Viriginia A; Ma, Jinghong; Bouloux, Pierre; Mohammadi, Moosa; Crowley, William F Jr
2006 Jul 25;254-255:60-69, Molecular & cellular endocrinology
BACKGROUND: Kallmann's syndrome (KS) is a clinically and genetically heterogeneous disorder consisting of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia. Mutations in KAL1 causing the X-linked form of KS have been identified in 10% of all KS patients and consistently result in a severe reproductive phenotype. KAL1 gene encodes for anosmin-1, a key protein involved in olfactory and GnRH neuronal migration through a putative interaction with FGFR1. Heterozygous mutations in the FGFR1 gene accompanied by a high frequency of cleft palate and other facial dysmorphisms were recently identified in 8% of a large KS cohort, yet the reproductive phenotype of KS patients harboring FGFR1 mutations has not been described. RESULTS: One hundred and fifty probands with KS (130 males and 20 females) were studied to determine the frequency and distribution of FGFR1 mutations and their detailed reproductive phenotypes. Fifteen heterozygous mutations in unrelated probands were identified. Twelve missense mutations (p.R78C, p.V102I, p.D224H, p.G237D, p.R254Q, p.V273M, p.E274G, p.Y339C, p.S346C, p.I538V, p.G703S and p.G703R) were distributed among the first, second and third immunoglobulin-like domains (D1-D3), as well as the tyrosine kinase domain (TKD). The mutations Y339C and S346C are located in exon 8B and code for the isoform FGFR1c. Additionally, two nonsense mutations (p.T585X and p.R622X) were documented in the TKD of the protein. A wide spectrum of reproductive function was observed among KS probands including: (1) a severe phenotype demonstrated by microphallus, cryptorchidism, no pubertal development, undetectable serum gonadotropins and low serum testosterone (T) and inhibin B; (2) partial pubertal development; (3) the fertile eunuch variant of IHH with normal testicular size and active spermatogenesis with a reversal of HH after T therapy. In addition, we found an even wider spectrum of reproductive function within pedigrees carrying an FGFR1 mutation ranging from IHH to delayed puberty to normal reproductive function (anosmia only or asymptomatic carriers). These observations strongly suggest a role for other genes that modify the phenotype of FGFR1 mutations. CONCLUSION: KS patients and family members carrying an FGFR1 mutation present a broad spectrum of pubertal development in contrast to the almost uniform severe clinical phenotype described in KS subjects with a KAL1 mutation. Additionally, this report implicates the isoform FGFR1c in the pathogenesis of KS
— id: 68519, year: 2006, vol: 254-255, page: 60, stat: Journal Article,

Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family
Zhang, Xiuqin; Ibrahimi, Omar A; Olsen, Shaun K; Umemori, Hisashi; Mohammadi, Moosa; Ornitz, David M
2006 Jun 9;281(23):15694-15700, Journal of biological chemistry
In mammals, fibroblast growth factors (FGFs) are encoded by 22 genes. FGFs bind and activate alternatively spliced forms of four tyrosine kinase FGF receptors (FGFRs 1-4). The spatial and temporal expression patterns of FGFs and FGFRs and the ability of specific ligand-receptor pairs to actively signal are important factors regulating FGF activity in a variety of biological processes. FGF signaling activity is regulated by the binding specificity of ligands and receptors and is modulated by extrinsic cofactors such as heparan sulfate proteoglycans. In previous studies, we have engineered BaF3 cell lines to express the seven principal FGFRs and used these cell lines to determine the receptor binding specificity of FGFs 1-9 by using relative mitogenic activity as the readout. Here we have extended these semiquantitative studies to assess the receptor binding specificity of the remaining FGFs 10-23. This study completes the mitogenesis-based comparison of receptor specificity of the entire FGF family under standard conditions and should help in interpreting and predicting in vivo biological activity
— id: 68521, year: 2006, vol: 281, page: 15694, stat: Journal Article,

Understanding the molecular basis of apert syndrome
Ibrahimi, Omar A; Chiu, Ernest S; McCarthy, Joseph G; Mohammadi, Moosa
2005 Jan;115(1):264-270, Plastic & reconstructive surgery
Apert syndrome, first described in 1906, is one of the most severe of the craniosynostosis syndromes and is further characterized by midface hypoplasia, syndactyly, and other visceral abnormalities. Affected individuals generally require lifelong management by a multidisciplinary team of health care specialists. Apert syndrome results almost exclusively from one or the other of two point mutations in fibroblast growth factor receptor 2. Tremendous scientific advances have been made recently in understanding the molecular basis for Apert syndrome through clinical genetic, biochemical, and structural approaches. In this review, the authors provide the clinician with a basic overview of these findings and their therapeutic implications
— id: 56112, year: 2005, vol: 115, page: 264, stat: Journal Article,

Analysis of mutations in fibroblast growth factor (FGF) and a pathogenic mutation in FGF receptor (FGFR) provides direct evidence for the symmetric two-end model for FGFR dimerization
Ibrahimi, Omar A; Yeh, Brian K; Eliseenkova, Anna V; Zhang, Fuming; Olsen, Shaun K; Igarashi, Makoto; Aaronson, Stuart A; Linhardt, Robert J; Mohammadi, Moosa
2005 Jan;25(2):671-684, Molecular & cellular biology
Two competing models for fibroblast growth factor (FGF) receptor (FGFR) dimerization have recently emerged based on ternary FGF-FGFR-heparin crystal structures. In the symmetric two-end model, heparin promotes dimerization of two FGF-FGFR complexes by stabilizing bivalent interactions of the ligand and receptor through primary and secondary sites and by stabilizing direct receptor-receptor contacts. In the asymmetric model, there are no protein-protein contacts between the two FGF-FGFR complexes, which are bridged solely by heparin. To identify the correct mode of FGFR dimerization, we abolished interactions at the secondary ligand-receptor interaction site, which are observed only in the symmetric two-end model, using site-directed mutagenesis. Cellular studies and real-time binding assays, as well as matrix-assisted laser desorption ionization-time of flight analysis, demonstrate that loss of secondary ligand-receptor interactions results in diminished FGFR activation due to decreased dimerization without affecting FGF-FGFR binding. Additionally, structural and biochemical analysis of an activating FGFR2 mutation resulting in Pfeiffer syndrome confirms the physiological significance of receptor-receptor contacts in the symmetric two-end model and provides a novel mechanism for FGFR gain of function in human skeletal disorders. Taken together, the data validate the symmetric two-end model of FGFR dimerization and argue against the asymmetric model of FGFR dimerization
— id: 50286, year: 2005, vol: 25, page: 671, stat: Journal Article,

A protein canyon in the FGF-FGF receptor dimer selects from an a la carte menu of heparan sulfate motifs
Mohammadi, Moosa; Olsen, Shaun K; Goetz, Regina
2005 Oct;15(5):506-516, Current opinion in structural biology
Heparan sulfate (HS) is an essential and dynamic regulator of fibroblast growth factor (FGF) signaling. Two fundamentally different crystallographic models have been proposed to explain, at the molecular level, how HS/heparin enables FGF and FGF receptor (FGFR) to assemble into a functional dimer on the cell surface. In the symmetric 'two-end' model, the heparin-binding sites of FGF and FGFR merge to form a basic canyon that recruits two HS for binding. Within this canyon, the HS molecules primarily act to orchestrate and fortify multivalent and cooperative protein-protein contacts within the dimer that are the foundations of dimerization. In contrast, in the asymmetric model, which mechanistically resembles the previously proposed trans FGF dimer model, a single heparin molecule facilitates dimerization by cross-linking two FGFs into a trans dimer that brings together the two FGFRs. Interestingly, the crystal structure upon which the asymmetric model is based contains a symmetric dimer reminiscent of the symmetric two-end model, suggesting that a different interpretation of the crystal structure has led to the postulation of the asymmetric model. Importantly, the symmetric two-end model provides an intriguing solution to the problem of how HS selectivity is achieved in FGF signaling. The model reveals that, within the canyon, FGF and FGFR no longer adhere to their individual HS binding specificities, but instead act in unison to search for a unique HS motif from a plethora of HS epitopes that are expressed in a tissue-specific and developmentally regulated fashion. Primary sequence differences within the heparin-binding sites of FGFs and FGFRs, together with ligand-induced changes in FGFR conformation, lead to the formation of distinct canyons with unique HS specificity for individual FGF-FGFR complexes
— id: 62599, year: 2005, vol: 15, page: 506, stat: Journal Article,

Structural basis for fibroblast growth factor receptor activation
Mohammadi, Moosa; Olsen, Shaun K; Ibrahimi, Omar A
2005 Apr;16(2):107-137, Cytokine & growth factor reviews
FGF signaling plays a ubiquitous role in human biology as a regulator of embryonic development, homeostasis and regenerative processes. In addition, aberrant FGF signaling leads to diverse human pathologies including skeletal, olfactory, and metabolic disorders as well as cancer. FGFs execute their pleiotropic biological actions by binding, dimerizing and activating cell surface FGF receptors (FGFRs). Proper regulation of FGF-FGFR binding specificity is essential for the regulation of FGF signaling and is achieved through primary sequence variations among the 18 FGFs and seven FGFRs. The severity of human skeletal syndromes arising from mutations that violate FGF-FGFR specificity is a testament to the importance of maintaining precision in FGF-FGFR specificity. The discovery that heparin/heparan sulfate (HS) proteoglycans are required for FGF signaling led to numerous models for FGFR dimerization and heralded one of the most controversial issues in FGF signaling. Recent crystallographic analyses have led to two fundamentally different models for FGFR dimerization. These models differ in both the stoichiometry and minimal length of heparin required for dimerization, the quaternary arrangement of FGF, FGFR and heparin in the dimer, and in the mechanism of 1:1 FGF-FGFR recognition and specificity. In this review, we provide an overview of recent structural and biochemical studies used to differentiate between the two crystallographic models. Interestingly, the structural and biophysical analyses of naturally occurring pathogenic FGFR mutations have provided the most compelling and unbiased evidences for the correct mechanisms for FGF-FGFR dimerization and binding specificity. The structural analyses of different FGF-FGFR complexes have also shed light on the intricate mechanisms determining FGF-FGFR binding specificity and promiscuity and also provide a plausible explanation for the molecular basis of a large number craniosynostosis mutations
— id: 56118, year: 2005, vol: 16, page: 107, stat: Journal Article,

Identification of phosphopeptides by MALDI Q-TOF mass spectrometry in positive and negative ion modes after methyl esterification
Xu, Chong-Feng; Lu, Yun; Ma, Jinghong; Mohammadi, Moosa; Neubert, Thomas A
2005 Jun;4(6):809-818, Molecular & cellular proteomics
We have developed an efficient, sensitive and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by collision-induced dissociation of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification prior to mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode, and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of ss-casein and a-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain, and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides
— id: 50627, year: 2005, vol: 4, page: 809, stat: Journal Article,

Analysis of the biochemical mechanisms for the endocrine actions of FGF23
Yu, X; Ibrahimi, OA; Goetz, R; Zhang, F; Davis, SI; Garringer, HJ; Linhardt, RJ; Ornitz, DM; Mohammadi, M; White, KE
2005 SEP ;20(9):S27-S27, Journal of bone & mineral research
— id: 62403, year: 2005, vol: 20, page: S27, stat: Journal Article,

Analysis of the biochemical mechanisms for the endocrine actions of fibroblast growth factor-23
Yu, Xijie; Ibrahimi, Omar A; Goetz, Regina; Zhang, Fuming; Davis, Siobhan I; Garringer, Holly J; Linhardt, Robert J; Ornitz, David M; Mohammadi, Moosa; White, Kenneth E
2005 Nov;146(11):4647-4656, Endocrinology
Fibroblast growth factor (FGF)-23 has emerged as an endocrine regulator of phosphate and of vitamin D metabolism. It is produced in bone and, unlike other FGFs, circulates in the bloodstream to ultimately regulate phosphate handling and vitamin D production in the kidney. Presently, it is unknown which of the seven principal FGF receptors (FGFRs) transmits FGF23 biological activity. Furthermore, the molecular basis for the endocrine mode of FGF23 action is unclear. Herein, we performed surface plasmon resonance and mitogenesis experiments to comprehensively characterize receptor binding specificity. Our data demonstrate that FGF23 binds and activates the c splice isoforms of FGFR1-3, as well as FGFR4, but not the b splice isoforms of FGFR1-3. Interestingly, highly sulfated and longer glycosaminoglycan (GAG) species were capable of promoting FGF23 mitogenic activity. We also show that FGF23 induces tyrosine phosphorylation and inhibits sodium-phosphate cotransporter Npt2a mRNA expression using opossum kidney cells, a model kidney proximal tubule cell line. Removal of cell surface GAGs abolishes the effects of FGF23, and exogenous highly sulfated GAG is capable of restoring FGF23 activity, suggesting that proximal tubule cells naturally express GAGs that are permissive for FGF23 action. We propose that FGF23 signals through multiple FGFRs and that the unique endocrine actions of FGF23 involve escape from FGF23-producing cells and circulation to the kidney, where highly sulfated GAGs most likely act as cofactors for FGF23 activity. Our biochemical findings provide important insights into the molecular mechanisms by which dysregulated FGF23 signaling leads to disorders of hyper- and hypophosphatemia
— id: 68523, year: 2005, vol: 146, page: 4647, stat: Journal Article,

Structure/function analysis of fibroblast growth factor homologous factors
Goldfarb, M; Shtraizent, N; Garbi, M; Schoorlemmer, J; Pardee, C; Olsen, S; Mohammadi, M
2004 AUG ;90(5):106-106, Journal of neurochemistry
— id: 46905, year: 2004, vol: 90, page: 106, stat: Journal Article,

Biochemical analysis of pathogenic ligand-dependent FGFR2 mutations suggests distinct pathophysiological mechanisms for craniofacial and limb abnormalities
Ibrahimi, Omar A; Zhang, Fuming; Eliseenkova, Anna V; Itoh, Nobuyuki; Linhardt, Robert J; Mohammadi, Moosa
2004 Oct 1;13(19):2313-2324, Human molecular genetics
Gain-of-function missense mutations in FGF receptor 2 (FGFR2) are responsible for a variety of craniosynostosis syndromes including Apert syndrome (AS), Pfeiffer syndrome (PS) and Crouzon syndrome (CS). Unlike the majority of FGFR2 mutations, S252W and P253R AS mutations and a D321A PS mutation retain ligand-dependency and are also associated with severe limb pathology. In addition, a recently identified ligand-dependent S252L/A315S double mutation in FGFR2 was shown to cause syndactyly in the absence of craniosynostosis. Here, we analyze the effect of the canonical AS mutations, the D321A PS mutation and the S252L/A315S double mutation on FGFR2 ligand binding affinity and specificity using surface plasmon resonance. Both AS mutations and the D321A PS mutation, but not the S252L/A315S double mutation, increase the binding affinity of FGFR2c to multiple FGFs expressed in the cranial suture. Additionally, all four pathogenic mutations also violate FGFR2c ligand binding specificity and enable this receptor to bind FGF10. Based on our data, we propose that an increase in mutant FGFR2c binding to multiple FGFs results in craniosynostosis, whereas binding of mutant FGFR2c to FGF10 results in severe limb pathology. Structural and biophysical analysis shows that AS mutations in FGFR2b also enhance and violate FGFR2b ligand binding affinity and specificity, respectively. We suggest that elevated AS mutant FGFR2b signaling may account for the dermatological manifestations of AS
— id: 48229, year: 2004, vol: 13, page: 2313, stat: Journal Article,

Proline to arginine mutations in FGF receptors 1 and 3 result in Pfeiffer and Muenke craniosynostosis syndromes through enhancement of FGF binding affinity
Ibrahimi, Omar A; Zhang, Fuming; Eliseenkova, Anna V; Linhardt, Robert J; Mohammadi, Moosa
2004 Jan 1;13(1):69-78, Human molecular genetics
Identical proline-->arginine gain-of-function mutations in fibroblast growth factor receptor (FGFR) 1 (Pro252Arg), FGFR2 (Pro253Arg) and FGFR3 (Pro250Arg), result in type I Pfeiffer, Apert and Muenke craniosynostosis syndromes, respectively. Here, we characterize the effects of proline-->arginine mutations in FGFR1c and FGFR3c on ligand binding using surface plasmon resonance and X-ray crystallography. Both Pro252Arg FGFR1c and Pro250Arg FGFR3c exhibit an enhancement in ligand binding in comparison to their respective wild-type receptors. Interestingly, binding of both mutant receptors to FGF9 was notably enhanced and implicates FGF9 as a potential pathophysiological ligand for mutant FGFRs in mediating craniosynostosis. The crystal structure, of Pro252Arg FGFR1c in complex with FGF2, demonstrates that the enhanced ligand binding is due to an additional set of receptor-ligand hydrogen bonds, similar to those gain-of-function interactions that occur in the Apert syndrome Pro253Arg FGFR2c-FGF2 crystal structure. However, unlike the Apert syndrome Pro253Arg FGFR2c mutant, neither the Pfeiffer syndrome Pro250Arg FGFR1c mutant nor the Muenke syndrome Pro250Arg FGFR3c mutant bound appreciably to FGF7 or FGF10. This observation provides a potential explanation for why the limb phenotypes, observed in type I Pfeiffer and Muenke syndromes, are less severe than the limb abnormalities observed in Apert syndrome. Hence, although analogous proline-->arginine mutations in FGFR1-3 act through a common structural mechanism to result in gain-of-function, differences in the primary sequence among FGFRs result in varying effects on ligand binding specificity
— id: 48181, year: 2004, vol: 13, page: 69, stat: Journal Article,

Kinetic model for FGF, FGFR, and proteoglycan signal transduction complex assembly
Ibrahimi, Omar A; Zhang, Fuming; Hrstka, Sybil C Lang; Mohammadi, Moosa; Linhardt, Robert J
2004 May 27;43(16):4724-4730, Biochemistry
The current working model for fibroblast growth factor receptor (FGFR) dimerization and activation requires the assembly of a ternary complex of fibroblast growth factor (FGF), FGFR, and heparin or heparan sulfate proteoglycan (HSPG) on the plasma membrane. The recent FGF2-FGFR1-heparin crystal structure provides a detailed but static view of the FGF-FGFR-heparin complex. However, the kinetics of ternary complex assembly has yet to be investigated. Here, we characterize FGF2, FGFR1, and heparin interactions using surface plasmon resonance (SPR). Binding constants for binary FGF2/FGFR1 (KD = 62 nM), FGF2/heparin (KD = 39 nM), and FGFR1/heparin (KD = 3.2 microM) interactions correlate to the magnitude of binding interface observed in the FGF2-FGFR1-heparin crystal structure. Interestingly, comparison of sensorgrams of sequential injections of FGF2 and FGFR1 and equimolar FGF2-FGFR1 injections onto a heparin neoproteoglycan surface demonstrates that FGF2 dramatically enhances the association of FGFR1 with heparin and leads us to propose a model for the stepwise assembly of a ternary FGF-FGFR-HSPG complex. The weak binding affinity of the FGFR1-heparin interaction suggests that in this model, FGFR and HSPG are unbound in the absence of FGF ligand. The availability of FGF results in formation of initial FGF-HSPG complexes, which promotes the rapid binding of FGFR and creates a ternary complex capable of undergoing dimerization and subsequent FGFR activation. In contrast, alternative models for the kinetic assembly of a ternary complex in which binary FGF-FGFR or FGFR-HSPG complexes are intermediates do not conform well with the experimental data
— id: 46104, year: 2004, vol: 43, page: 4724, stat: Journal Article,

Structural basis for FGF receptor activation
Mohammadi, M
2004 AUG ;90(5):106-106, Journal of neurochemistry
— id: 46904, year: 2004, vol: 90, page: 106, stat: Journal Article,

Insights into the molecular basis for fibroblast growth factor receptor autoinhibition and ligand-binding promiscuity
Olsen, Shaun K; Ibrahimi, Omar A; Raucci, Angela; Zhang, Fuming; Eliseenkova, Anna V; Yayon, Avner; Basilico, Claudio; Linhardt, Robert J; Schlessinger, Joseph; Mohammadi, Moosa
2004 Jan 27;101(4):935-940, Proceedings of the National Academy of Sciences of the United States of America
The prototypical fibroblast growth factor receptor (FGFR) extracellular domain consists of three Ig domains (D1-D3) of which the two membrane-proximal D2 and D3 domains and the interconnecting D2-D3 linker bear the determinants of ligand binding and specificity. In contrast, D1 and the D1-D2 linker are thought to play autoinhibitory roles in FGFR regulation. Here, we report the crystal structure of the three-Ig form of FGFR3c in complex with FGF1, an FGF that binds promiscuously to each of the seven principal FGFRs. In this structure, D1 and the D1-D2 linker are completely disordered, demonstrating that these regions are dispensable for FGF binding. Real-time binding experiments using surface plasmon resonance show that relative to two-Ig form, the three-Ig form of FGFR3c exhibits lower affinity for both FGF1 and heparin. Importantly, we demonstrate that this autoinhibition is mediated by intramolecular interactions of D1 and the D1-D2 linker with the minimal FGF and heparin-binding D2-D3 region. As in the FGF1-FGFR2c structure, but not the FGF1-FGFR1c structure, the alternatively spliced betaC'-betaE loop is ordered and interacts with FGF1 in the FGF1-FGFR3c structure. However, in contrast to the FGF1-FGFR2c structure in which the betaC'-betaE loop interacts with the beta-trefoil core region of FGF1, in the FGF1-FGFR3c structure, this loop interacts extensively with the N-terminal region of FGF1, underscoring the importance of the FGF1 N terminus in conferring receptor-binding affinity and promiscuity. Importantly, comparison of the three FGF1-FGFR structures shows that the flexibility of the betaC'-betaE loop is a major determinant of ligand-binding specificity and promiscuity
— id: 42613, year: 2004, vol: 101, page: 935, stat: Journal Article,

Targeting the ectodomain of FGFR3 in multiple meloma
Green, D; Olsen, S; Pinnell, J; Mohammadi, M
2003 NOV 16 ;102(11):681A-681A, Blood
— id: 42497, year: 2003, vol: 102, page: 681A, stat: Journal Article,

Fibroblast growth factor (FGF) homologous factors share structural but not functional homology with FGFs
Olsen, Shaun K; Garbi, Meirav; Zampieri, Niccolo; Eliseenkova, Anna V; Ornitz, David M; Goldfarb, Mitchell; Mohammadi, Moosa
2003 Sep 5;278(36):34226-34236, Journal of biological chemistry
Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1-FHF4; also known as FGF11-FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-A resolution. The FHF1b core domain assumes a beta-trefoil fold consisting of 12 antiparallel beta strands (beta 1 through beta 12). The FHF1b beta-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the beta 4-beta 5 loop and Val95 in the beta 9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets
— id: 39191, year: 2003, vol: 278, page: 34226, stat: Journal Article,

Structure-based mutational analyses in FGF7 identify new residues involved in specific interaction with FGFR2IIIb
Sher, Ifat; Yeh, Brian K; Mohammadi, Moosa; Adir, Noam; Ron, Dina
2003 Sep 25;552(2-3):150-154, FEBS letters
Receptor binding specificity is an essential element in regulating the diverse activities of fibroblast growth factors (FGFs). FGF7 is ideal to study how this specificity is conferred at the structural level, as it interacts exclusively with one isoform of the FGF-receptor (FGFR) family, known as FGFR2IIIb. Previous mutational analysis suggested the importance of the beta4/beta5 loop of FGF7 in specific receptor recognition. Here a theoretical model of FGFR2IIIb/FGF7 complex showed that this loop interacts with the FGFR2IIIb unique exon. In addition, the model revealed new residues that either directly interact with the FGFR2IIIb unique exon (Asp63, Leu142) or facilitate this interaction (Arg65). Mutations in these residues reduced both receptor binding affinity and biological activity of FGF7. Altogether, these results provide the basis for understanding how receptor-binding specificity of FGF7 is conferred at the structural level
— id: 68524, year: 2003, vol: 552, page: 150, stat: Journal Article,

Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors
Yeh, Brian K; Igarashi, Makoto; Eliseenkova, Anna V; Plotnikov, Alexander N; Sher, Ifat; Ron, Dina; Aaronson, Stuart A; Mohammadi, Moosa
2003 Mar 4;100(5):2266-2271, Proceedings of the National Academy of Sciences of the United States of America
Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb ('b') and IIIc ('c'), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes
— id: 39297, year: 2003, vol: 100, page: 2266, stat: Journal Article,

Structural basis for fibroblast growth factor (FGF) receptor activation
Mohammadi, M
2002 Mar 20;16(4):A523-A523, FASEB journal
— id: 27502, year: 2002, vol: 16, page: A523, stat: Journal Article,

Synthesis of sulfosucrose derivatives for evaluation as regulators of fibroblast growth factor activity
Polat, T; Mohammadi, M; Linhardt, RJ
2002 NOV 4 ;43(45):8047-8049, Tetrahedron letters
Based oil X-ray crystallographic studies on sucrose octasulfate in complex with fibroblast growth factor and its receptor, three analogs of sucrose octasulfate were regioselectively synthesized for biological evaluation as regulations of cell proliferation and differentiation. (C) 2002 Elsevier Science Ltd. All rights reserved
— id: 55278, year: 2002, vol: 43, page: 8047, stat: Journal Article,

Structural basis for activation of fibroblast growth factor signaling by sucrose octasulfate
Yeh, Brian K; Eliseenkova, Anna V; Plotnikov, Alexander N; Green, David; Pinnell, Jared; Polat, Tulay; Gritli-Linde, Amel; Linhardt, Robert J; Mohammadi, Moosa
2002 Oct;22(20):7184-7192, Molecular & cellular biology
Sucrose octasulfate (SOS) is believed to stimulate fibroblast growth factor (FGF) signaling by binding and stabilizing FGFs. In this report, we show that SOS induces FGF-dependent dimerization of FGF receptors (FGFRs). The crystal structure of the dimeric FGF2-FGFR1-SOS complex at 2.6-A resolution reveals a symmetric assemblage of two 1:1:1 FGF2-FGFR1-SOS ternary complexes. Within each ternary complex SOS binds to FGF and FGFR and thereby increases FGF-FGFR affinity. SOS also interacts with the adjoining FGFR and thereby promotes protein-protein interactions that stabilize dimerization. This structural finding is supported by the inability of selectively desulfated SOS molecules to promote receptor dimerization. Thus, we propose that SOS potentiates FGF signaling by imitating the dual role of heparin in increasing FGF-FGFR affinity and promoting receptor dimerization. Hence, the dimeric FGF-FGFR-SOS structure substantiates the recently proposed 'two-end' model, by which heparin induces FGF-FGFR dimerization. Moreover, the FGF-FGFR-SOS structure provides an attractive template for the development of easily synthesized SOS-related heparin agonists and antagonists that may hold therapeutic potential
— id: 39589, year: 2002, vol: 22, page: 7184, stat: Journal Article,

Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis
Bellosta P; Iwahori A; Plotnikov AN; Eliseenkova AV; Basilico C; Mohammadi M
2001 Sep;21(17):5946-5957, Molecular & cellular biology
Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity
— id: 26713, year: 2001, vol: 21, page: 5946, stat: Journal Article,

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome
Ibrahimi OA; Eliseenkova AV; Plotnikov AN; Yu K; Ornitz DM; Mohammadi M
2001 Jun 19;98(13):7182-7187, Proceedings of the National Academy of Sciences of the United States of America
Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 --> Trp and Pro-253 --> Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2). These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2-FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 --> Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 --> Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients
— id: 48159, year: 2001, vol: 98, page: 7182, stat: Journal Article,

Crystal structure of fibroblast growth factor 9 reveals regions implicated in dimerization and autoinhibition
Plotnikov AN; Eliseenkova AV; Ibrahimi OA; Shriver Z; Sasisekharan R; Lemmon MA; Mohammadi M
2001 Feb 9;276(6):4322-4329, Journal of biological chemistry
Fibroblast growth factors (FGFs) constitute a large family of heparin-binding growth factors with diverse biological activities. FGF9 was originally described as glia-activating factor and is expressed in the nervous system as a potent mitogen for glia cells. Unlike most FGFs, FGF9 forms dimers in solution with a K(d) of 680 nm. To elucidate the molecular mechanism of FGF9 dimerization, the crystal structure of FGF9 was determined at 2.2 A resolution. FGF9 adopts a beta-trefoil fold similar to other FGFs. However, unlike other FGFs, the N- and C-terminal regions outside the beta-trefoil core in FGF9 are ordered and involved in the formation of a 2-fold crystallographic dimer. A significant surface area (>2000 A(2)) is buried in the dimer interface that occludes a major receptor binding site of FGF9. Thus, we propose an autoinhibitory mechanism for FGF9 that is dependent on sequences outside of the beta-trefoil core. Moreover, a model is presented providing a molecular basis for the preferential affinity of FGF9 toward FGFR3
— id: 21265, year: 2001, vol: 276, page: 4322, stat: Journal Article,

SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors
Laird AD; Vajkoczy P; Shawver LK; Thurnher A; Liang C; Mohammadi M; Schlessinger J; Ullrich A; Hubbard SR; Blake RA; Fong TA; Strawn LM; Sun L; Tang C; Hawtin R; Tang F; Shenoy N; Hirth KP; McMahon G; Cherrington
2000 Aug 1;60(15):4152-4160, Cancer research
Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials
— id: 20814, year: 2000, vol: 60, page: 4152, stat: Journal Article,

Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity
Plotnikov AN; Hubbard SR; Schlessinger J; Mohammadi M
2000 May 12;101(4):413-424, Cell
To elucidate the structural determinants governing specificity in fibroblast growth factor (FGF) signaling, we have determined the crystal structures of FGF1 and FGF2 complexed with the ligand binding domains (immunoglobulin-like domains 2 [D2] and 3 [D3]) of FGF receptor 1 (FGFR1) and FGFR2, respectively. Highly conserved FGF-D2 and FGF-linker (between D2-D3) interfaces define a general binding site for all FGF-FGFR complexes. Specificity is achieved through interactions between the N-terminal and central regions of FGFs and two loop regions in D3 that are subject to alternative splicing. These structures provide a molecular basis for FGF1 as a universal FGFR ligand and for modulation of FGF-FGFR specificity through primary sequence variations and alternative splicing
— id: 11676, year: 2000, vol: 101, page: 413, stat: Journal Article,

Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization
Schlessinger J; Plotnikov AN; Ibrahimi OA; Eliseenkova AV; Yeh BK; Yayon A; Linhardt RJ; Mohammadi M
2000 Sep;6(3):743-750, Molecular cell
The crystal structure of a dimeric 2:2:2 FGF:FGFR:heparin ternary complex at 3 A resolution has been determined. Within each 1:1 FGF:FGFR complex, heparin makes numerous contacts with both FGF and FGFR, thereby augmenting FGF-FGFR binding. Heparin also interacts with FGFR in the adjoining 1:1 FGF:FGFR complex to promote FGFR dimerization. The 6-O-sulfate group of heparin plays a pivotal role in mediating both interactions. The unexpected stoichiometry of heparin binding in the structure led us to propose a revised model for FGFR dimerization. Biochemical data in support of this model are also presented. This model provides a structural basis for FGFR activation by small molecule heparin analogs and may facilitate the design of heparin mimetics capable of modulating FGF signaling
— id: 39538, year: 2000, vol: 6, page: 743, stat: Journal Article,

Structural basis for FGF receptor dimerization and activation
Plotnikov AN; Schlessinger J; Hubbard SR; Mohammadi M
1999 Sep 3;98(5):641-650, Cell
The crystal structure of FGF2 bound to a naturally occurring variant of FGF receptor 1 (FGFR1) consisting of immunoglobulin-like domains 2 (D2) and 3 (D3) has been determined at 2.8 A resolution. Two FGF2:FGFR1 complexes form a 2-fold symmetric dimer. Within each complex, FGF2 interacts extensively with D2 and D3 as well as with the linker between the two domains. The dimer is stabilized by interactions between FGF2 and D2 of the adjoining complex and by a direct interaction between D2 of each receptor. A positively charged canyon formed by a cluster of exposed basic residues likely represents the heparin-binding site. A general model for FGF- and heparin-induced FGFR dimerization is inferred from the crystal structure, unifying a wealth of biochemical data
— id: 8352, year: 1999, vol: 98, page: 641, stat: Journal Article,

Autoregulatory mechanisms in protein-tyrosine kinases
Hubbard SR; Mohammadi M; Schlessinger J
1998 May 15;273(20):11987-11990, Journal of biological chemistry
— id: 7610, year: 1998, vol: 273, page: 11987, stat: Journal Article,

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain
Mohammadi M; Froum S; Hamby JM; Schroeder MC; Panek RL; Lu GH; Eliseenkova AV; Green D; Schlessinger J; Hubbard SR
1998 Oct 15;17(20):5896-5904, EMBO journal
Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation
— id: 7692, year: 1998, vol: 17, page: 5896, stat: Journal Article,

Differential pre- and postsynaptic modulation of chemical transmission in the squid giant synapse by tyrosine phosphorylation
Llinas R; Moreno H; Sugimori M; Mohammadi M; Schlessinger J
1997 Mar 4;94(5):1990-1994, Proceedings of the National Academy of Sciences of the United States of America
To assess the role of tyrosine phosphorylation/dephosphorylation balance in synaptic transmission, a set of studies was implemented at the squid giant synapse. Presynaptic induction of tyrosine phosphorylation, following administration of the tyrosine phosphatase inhibitor pervanadate, produced a sizable increase in presynaptic calcium current and a concomitant and paradoxical decrement of the postsynaptic potential amplitude. Presynaptic microinjection of an active protein tyrosine kinase dramatically increased calcium currents and incremented postsynaptic potential amplitude. By contrast, the same procedure at the postsynaptic terminal reduced the size of the postsynaptic potential. This differential effect may be prodromic to long-term plasticity, as postsynaptic sensitivity is momentarily deemphasized, whereas presynaptic second messenger cascades triggered by increased calcium currents are accentuated
— id: 9884, year: 1997, vol: 94, page: 1990, stat: Journal Article,

Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors
Mohammadi M; McMahon G; Sun L; Tang C; Hirth P; Yeh BK; Hubbard SR; Schlessinger J
1997 May 9;276(5314):955-960, Science
A new class of protein tyrosine kinase inhibitors was identified that is based on an oxindole core (indolinones). Two compounds from this class inhibited the kinase activity of fibroblast growth factor receptor 1 (FGFR1) and showed differential specificity toward other receptor tyrosine kinases. Crystal structures of the tyrosine kinase domain of FGFR1 in complex with the two compounds were determined. The oxindole occupies the site in which the adenine of adenosine triphosphate binds, whereas the moieties that extend from the oxindole contact residues in the hinge region between the two kinase lobes. The more specific inhibitor of FGFR1 induces a conformational change in the nucleotide-binding loop. This structural information will facilitate the design of new inhibitors for use in the treatment of cancer and other diseases in which cell signaling by tyrosine kinases plays a crucial role in disease pathogenesis
— id: 8448, year: 1997, vol: 276, page: 955, stat: Journal Article,

Structural studies of receptor tyrosine kinases
Hubbard, SR; Mohammadi, M; Schlessinger, J; Wei, L; Ellis, L; Hendrickson, WA
1996 APR 30 ;10(6):P2-P2, FASEB journal
— id: 52902, year: 1996, vol: 10, page: P2, stat: Journal Article,

Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction
Mohammadi M; Dikic I; Sorokin A; Burgess WH; Jaye M; Schlessinger J
1996 Mar;16(3):977-989, Molecular & cellular biology
Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation
— id: 6941, year: 1996, vol: 16, page: 977, stat: Journal Article,

Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism
Mohammadi M; Schlessinger J; Hubbard SR
1996 Aug 23;86(4):577-587, Cell
The crystal structure of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1K) has been determined in its unliganded form to 2.0 angstroms resolution and in complex with with an ATP analog to 2.3 angstrosms A resolution. Several features distinguish the structure of FGFR1K from that of the tyrosine kinase domain of the insulin receptor. Residues in the activation loop of FGFR1K appear to interfere with substrate peptide binding but not with ATP binding, revealing a second and perhaps more general autoinhibitory mechanism for receptor tyrosine kinases. In addition, a dimeric form of FGFR1K observed in the crystal structure may provide insights into the molecular mechanisms by which FGF receptors are activated. Finally, the structure provides a basis for rationalizing the effects of kinase mutations in FGF receptors that lead to developmental disorders in nematodes and humans
— id: 7021, year: 1996, vol: 86, page: 577, stat: Journal Article,

Induction of urokinase-type plasminogen activator by fibroblast growth factor (FGF)-2 is dependent on expression of FGF receptors and does not require activation of phospholipase Cgamma1
Roghani M; Mohammadi M; Schlessinger J; Moscatelli D
1996 Dec 6;271(49):31154-31159, Journal of biological chemistry
The roles of heparan sulfate proteoglycans and tyrosine kinase fibroblast growth factor (FGF) receptors in mediating the induction of plasminogen activator (PA) by FGF-2 were investigated using L6 myoblast cells that normally do not express detectable FGF receptors. PA was induced by FGF-2 in a dose-dependent manner in L6 cells expressing transfected FGF receptor-1 but not in nontransfected cells or cells transfected with the vector alone. The PA produced in these cells was characterized as urokinase-type PA (uPA). Thus, expression of a tyrosine kinase FGF receptor was required for induction of uPA. Internalization of FGF through heparan sulfates does not seem to be involved in this response as soluble heparin and suramin at concentrations which inhibited FGF-2 binding to heparan sulfates but not receptors did not affect the induction of uPA by FGF-2. Mutant receptors in which the tyrosine kinase was inactivated were not able to respond to FGF-2. In contrast, mutation of the site of phospholipase Cgamma1 (PLCgamma) binding in the receptor, which causes loss of PLCgamma activation, had no effect on uPA induction by FGF-2. These results suggest that PLCgamma activation is not required for induction of uPA by FGF-2
— id: 8518, year: 1996, vol: 271, page: 31154, stat: Journal Article,

Reduced activation of RAF-1 and MAP kinase by a fibroblast growth factor receptor mutant deficient in stimulation of phosphatidylinositol hydrolysis
Huang J; Mohammadi M; Rodrigues GA; Schlessinger J
1995 Mar 10;270(10):5065-5072, Journal of biological chemistry
Signaling via the fibroblast growth factor receptor 1 (FGFR1, flg) was analyzed in Ba/F3 hematopoietic cells expressing either wild-type or a mutant FGF receptor (Y766F) unable to activate phospholipase C-gamma (PLC-gamma) and stimulate phosphatidylinositol (PI) hydrolysis. Stimulation of cells expressing wild-type or mutant FGFR with acidic FGF (aFGF) caused similar activation of Ras. However, an approximately 3-fold reduced activation of Raf-1 and MAP kinase was observed in aFGF-stimulated cells expressing mutant receptors as compared to cells expressing wild-type FGF receptors. Comparison of phosphopeptide maps of Raf-1 immunoprecipitated from the two cell types activated by either aFGF or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) suggests that Raf-1 is phosphorylated by both Ras-dependent and PLC-gamma-dependent mechanisms. In spite of the differential effect on Raf-1 and MAP kinase activation, aFGF stimulated similar proliferation of cells expressing wild-type or mutant receptors indicating that Ras-dependent activation of Raf-1 and MAP kinase is sufficient for transduction of FGFR mitogenic signals. Ras may also activate signal transduction pathways that are complementary or parallel to the MAP kinase pathway to stimulate cell proliferation
— id: 6645, year: 1995, vol: 270, page: 5065, stat: Journal Article,

Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes
Mainiero F; Pepe A; Wary KK; Spinardi L; Mohammadi M; Schlessinger J; Giancotti FG
1995 Sep 15;14(18):4470-4481, EMBO journal
We have examined the mechanism of signal transduction by the hemidesmosomal integrin alpha 6 beta 4, a laminin receptor involved in morphogenesis and tumor progression. Immunoprecipitation and immune complex kinase assays indicated that antibody- or laminin-induced ligation of alpha 6 beta 4 causes tyrosine phosphorylation of the beta 4 subunit in intact cells and that this event is mediated by a protein kinase(s) physically associated with the integrin. Co-immunoprecipitation and GST fusion protein binding experiments showed that the adaptor protein Shc forms a complex with the tyrosine-phosphorylated beta 4 subunit. Shc is then phosphorylated on tyrosine residues and recruits the adaptor Grb2, thereby potentially linking alpha 6 beta 4 to the ras pathway. The beta 4 subunit was found to be phosphorylated at multiple tyrosine residues in vivo, including a tyrosine-based activation motif (TAM) resembling those found in T and B cell receptors. Phenylalanine substitutions at the beta 4 TAM disrupted association of alpha 6 beta 4 with hemidesmosomes, but did not interfere with tyrosine phosphorylation of Shc and recruitment of Grb2. These results indicate that signal transduction by the alpha 6 beta 4 integrin is mediated by an associated tyrosine kinase and that phosphorylation of distinct sites in the beta 4 tail mediates assembly of the hemidesmosomal cytoskeleton and recruitment of Shc/Grb2
— id: 7928, year: 1995, vol: 14, page: 4470, stat: Journal Article,

CATALYTIC SPECIFICITY OF PROTEIN-TYROSINE KINASES IS CRITICAL FOR SELECTIVE SIGNALING
SONGYANG, Z; CARRAWAY, KL; ECK, MJ; HARRISON, SC; FELDMAN, RA; MOHAMMADI, M; SCHLESSINGER, J; HUBBARD, SR; SMITH, DP; ENG, C; LORENZO, MJ; PONDER, BAJ; MAYER, BJ; CANTLEY, LC
1995 FEB 9 ;373(6514):536-539, Nature
How do distinct protein-tyrosine kinases activate specific downstream events? Src-homology-2 (SH2) domains on tyrosine kinases or targets of tyrosine kinases recognize phosphotyrosine in a specific sequence context and thereby provide some specificity(1-3). The role of the catalytic site of tyrosine kinases in determining target specificity has not been fully investigated. Here we use a degenerate peptide library to show that each of nine tyrosine kinases investigated has a unique optimal peptide substrate. We find that the cytosolic tyrosine kinases preferentially phosphorylate peptides recognized by their own SH2 domains or closely related SH2 domains (group I; ref. 3), whereas receptor tyrosine kinases preferentially phosphorylate peptides recognized by subsets of group In SH2 domains(3). The importance of these findings for human disease is underscored by our observation that a point mutation in the RET receptor-type tyrosine kinase, which causes multiple endocrine neoplasia type 2B, results in a shift in peptide substrate specificity
— id: 87442, year: 1995, vol: 373, page: 536, stat: Journal Article,

Internalization of fibroblast growth factor receptor is inhibited by a point mutation at tyrosine 766
Sorokin A; Mohammadi M; Huang J; Schlessinger J
1994 Jun 24;269(25):17056-17061, Journal of biological chemistry
Binding of fibroblast growth factor (FGF) to the fibroblast growth factor receptor leads to autophosphorylation of the receptor on several tyrosine residues. Wild-type FGF receptor 1 (flg) and a mutated receptor (Y766F), in which an autophosphorylation site (Tyr-766) was mutated to phenylalanine, were expressed in rat myoblasts and in hematopoietic Ba/F3 cells. It was found that the point mutation at Tyr-766 resulted in a decrease in FGF receptor internalization, as well as a reduction in both ligand-induced FGF receptor down-regulation and degradation. It has been shown previously that phosphorylation of Tyr-766 is essential for interaction with phospholipase C gamma and that the Y766F FGF receptor mutant is unable to stimulate phosphatidylinositol hydrolysis and Ca2+ release from internal stores. The results presented in this report indicate that Tyr-766 is also essential for cellular trafficking of FGF receptor
— id: 6525, year: 1994, vol: 269, page: 17056, stat: Journal Article,

Point mutation in the fibroblast growth factor receptor eliminates phosphatidylinositol hydrolysis without affecting neuronal differentiation of PC12 cells
Spivak-Kroizman T; Mohammadi M; Hu P; Jaye M; Schlessinger J; Lax I
1994 May 20;269(20):14419-14423, Journal of biological chemistry
Fibroblast growth factors (FGF) stimulate growth arrest and differentiation in rat pheochromocytoma PC12 cells. We examined the role of phosphatidylinositol (PI) hydrolysis in FGF-induced differentiation of PC12 cells by exploring the biological and biochemical activity of a mutant FGF receptor 1 (flg) defective in stimulation of PI hydrolysis. We show that point mutation at Tyr-766 (Y766F) of the FGF receptor prevents tyrosine phosphorylation of phospholipase C gamma and eliminates acidic FGF (aFGF)-induced stimulation of PI hydrolysis in PC12 cells. Treatment of PC12 cells expressing either wild-type or the Y766F mutant with aFGF led to tyrosine phosphorylation of Shc, the association of Shc with GRB2, a shift in the electrophoretic mobility of the Ras guanine nucleotide-releasing factor, Sos (son of sevenless), and enhancement in mitogen-activated protein kinase phosphorylation. Moreover, stimulation with aFGF led to a typical neurite outgrowth of PC12 cells expressing either wild-type or the Y766F FGF receptor mutant. These experiments indicate that PI hydrolysis is not essential for FGF-induced neuronal differentiation of PC12 cells. Moreover, the aFGF-induced Ras signaling pathway, which is essential for PC12 cell differentiation, is not affected by elimination of PI hydrolysis
— id: 6527, year: 1994, vol: 269, page: 14419, stat: Journal Article,

Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase
Mohammadi M; Honegger A; Sorokin A; Ullrich A; Schlessinger J; Hurwitz DR
1993 Aug 31;32(34):8742-8748, Biochemistry
Various agents are able to stimulate the EGF receptor protein tyrosine kinase in the absence of ligand binding. To characterize their mechanism of action, we investigated their effects on the kinase activity of the intracellular domain of the EGF receptor (EGFR-IC). EGFR-IC (67 kDa) lacking the extracellular domain and transmembrane segment of the EGF receptor, but retaining kinase and autophosphorylation domains, was produced and purified as a soluble, cytoplasmic protein from Sf9 insect cells infected with a recombinant baculovirus. EGFR-IC was able to undergo autophosphorylation in a manner similar to full-length EGFR. Synthetic substrate peptides showed similar affinity to EGFR-IC as to the full-length receptor. The activity of the EGFR-IC was found to be dependent on divalent cations, Mn2+ being a more potent activator than Mg2+. Agents capable of aggregating the kinase by direct interaction (cross-linking antibodies, polycations) or through altering the surrounding solvent structure and thereby decreasing protein solubility [ammonium sulfate, poly(ethylene glycol), 2-methyl-2,4-pentanediol] activated the kinase in a manner which correlated with their ability to precipitate the EGFR intracellular domain. The widely different chemical nature of these agents suggests that they do not act by direct interaction with specific allosteric regulatory sites, but rather by facilitating the interactions between kinase molecules. These results support the hypothesis that full-length receptor aggregation itself, induced by ligand binding to the extracellular domain, results in intracellular domain interactions and the activation of kinase activity
— id: 13090, year: 1993, vol: 32, page: 8742, stat: Journal Article,

The function of GRB2 in linking the insulin receptor to Ras signaling pathways
Skolnik EY; Batzer A; Li N; Lee CH; Lowenstein E; Mohammadi M; Margolis B; Schlessinger J
1993 Jun 25;260(5116):1953-1955, Science
Insulin-induced activation of extracellular signal-regulated kinases [ERKs, also known as mitogen-activated protein (MAP) kinases] is mediated by Ras. Insulin activates Ras primarily by increasing the rate of guanine nucleotide-releasing activity. Here, we show that insulin-induced activation of ERKs was enhanced by stable overexpression of growth factor receptor-bound protein 2 (GRB2) but not by overexpression of GRB2 proteins with point mutations in the Src homology 2 and 3 domains. Moreover, a dominant negative form of Ras (with Ser17 substituted with Asn) blocked insulin-induced activation of ERKs in cells that overexpressed GRB2. GRB2 overexpression led to increased formation of a complex between the guanine nucleotide-releasing factor Sos (the product of the mammalian homolog of son of sevenless gene) and GRB2. In response to insulin stimulation, this complex bound to tyrosine-phosphorylated IRS-1 (insulin receptor substrate-1) and Shc. In contrast to the activated epidermal growth factor receptor that binds the GRB2-Sos complex directly, activation of the insulin receptor results in the interaction of GRB2-Sos with IRS-1 and Shc, thus linking the insulin receptor to Ras signaling pathways
— id: 13127, year: 1993, vol: 260, page: 1953, stat: Journal Article,

Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis
Mohammadi M; Dionne CA; Li W; Li N; Spivak T; Honegger AM; Jaye M; Schlessinger J
1992 Aug 20;358(6388):681-684, Nature
Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis
— id: 13475, year: 1992, vol: 358, page: 681, stat: Journal Article,

SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma
Rotin D; Margolis B; Mohammadi M; Daly RJ; Daum G; Li N; Fischer EH; Burgess WH; Ullrich A; Schlessinger J
1992 Feb;11(2):559-567, EMBO journal
Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 13703, year: 1992, vol: 11, page: 559, stat: Journal Article,

Role of SH2-containing proteins in cellular signaling by receptor tyrosine kinases
Schlessinger J; Mohammadi M; Margolis B; Ullrich A
1992 ;57:67-74, Cold Spring Harbor symposia on quantitative biology
— id: 56585, year: 1992, vol: 57, page: 67, stat: Journal Article,

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1
Mohammadi M; Honegger AM; Rotin D; Fischer R; Bellot F; Li W; Dionne CA; Jaye M; Rubinstein M; Schlessinger J
1991 Oct;11(10):5068-5078, Molecular & cellular biology
Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown
— id: 13894, year: 1991, vol: 11, page: 5068, stat: Journal Article,

Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases
Skolnik EY; Margolis B; Mohammadi M; Lowenstein E; Fischer R; Drepps A; Ullrich A; Schlessinger J
1991 Apr 5;65(1):83-90, Cell
A novel method has been developed to allow cloning of protein targets for receptors with tyrosine kinase activity. By utilizing the carboxy-terminal tail of EGF receptor (EGFR) as a probe to screen lambda gt11 expression libraries, several EGFR-binding proteins have been cloned; two have been analyzed and contain unique SH2 and SH3 domains. One gene (GRB-1) has been fully sequenced, is expressed in various tissues and cell lines, and has a molecular mass of 85 kd. Interestingly, GRB-1 encodes the human counterpart of the PI3 kinase-associated protein p85. Advantages of this technique include the ease of cloning tyrosine kinase receptor targets present at low levels and the ability to identify proteins that are related in their capacity to bind activated receptors but contain no significant DNA sequence homology. This method, termed CORT (for cloning of receptor targets), offers a general approach for the identification and cloning of various receptor targets
— id: 57500, year: 1991, vol: 65, page: 83, stat: Journal Article,

The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma
Margolis B; Li N; Koch A; Mohammadi M; Hurwitz DR; Zilberstein A; Ullrich A; Pawson T; Schlessinger J
1990 Dec;9(13):4375-4380, EMBO journal
Phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP) are substrates of EGF, PDGF and other growth factor receptors. Since either PLC-gamma or GAP also bind to the activated receptors it was suggested that their SH2 domains are mediating this association. We attempted to delineate the specific region of the EGF receptor that is responsible for the binding, utilizing EGF receptor mutants, PLC-gamma, and a bacterially expressed TRP E fusion protein containing the SH2 domains of GAP. As previously shown, tyrosine autophosphorylation of the wild-type receptor wsa crucial in mediating the association and in agreement, a kinase negative EGF receptor could bind PLC-gamma or TRP E GAP SH2, but only when cross tyrosine phosphorylated by an active EGF receptor kinase. The importance of autophosphorylation for association was confirmed by demonstrating that a carboxy-terminal deletion of the EGFR missing four autophosphorylation sites bound these proteins poorly. To study the role of EGF receptor autophosphorylation further, a 203 amino acid EGF receptor fragment was generated with cyanogen bromide that contained all known tyrosine autophosphorylation sites. This fragment bound both TRP E GAP SH2 and PLC-gamma but only when tyrosine phosphorylated. This data localizes a major binding site for SH2 domain containing proteins to the carboxy-terminus of the EGF receptor and points to the importance of tyrosine phosphorylation in mediating this association
— id: 57499, year: 1990, vol: 9, page: 4375, stat: Journal Article,