Many important cellular signaling cascades are initiated at the cell surface by the binding of a polypeptide ligand to a transmembrane receptor possessing intrinsic tyrosine kinase activity in its cytoplasmic domain. The receptor tyrosine kinase (RTK) family includes, among others, the insulin receptor, insulin-like growth factor-1 (IGF1) receptor, epidermal growth factor receptor, fibroblast growth factor receptor and MuSK, the receptor for agrin. Ligand binding induces receptor oligomerization (growth factor receptors) or a conformational change within the receptor (insulin/IGF1 receptor), leading to autophosphorylation of specific tyrosine residues in the cytoplasmic domains of the receptors. Tyrosine autophosphorylation stimulates receptor catalytic activity and generates recruitment sites for downstream signaling proteins. RTKs are critical components in signal transduction pathways that mediate cell proliferation, differentiation, migration and metabolism, and are active during organismal development and adult homeostasis. RTKs also play primary roles in the onset or progression of pathological conditions such as diabetic retinopathy, atherosclerosis and cancer.

Using x-ray crystallography as our primary experimental technique, we are attempting to understand the molecular basis for insulin receptor activation and for recruitment of downstream signaling proteins to the activated (phosphorylated) insulin receptor. Several cytoplasmic adapter proteins bind to the activated insulin receptor, including insulin receptor substrate (IRS) proteins and APS, which are positive factors in insulin signaling pathways culminating in glucose uptake. The insulin receptor is downregulated by the adapter proteins Grb10 and Grb14 as well as the tyrosine phosphatase PTP1B. We are determining crystal structures of complexes between these proteins and the insulin receptor kinase domain to elucidate the modes of interaction and the determinants of specificity.

Another project concerns the IGF1 receptor, which is highly related in sequence and structure to the insulin receptor, but has distinct biological functions, one of which is cell survival. Therefore, this RTK is a potential target for inhibition in tumor cells. In collaboration with Dr. Todd Miller at SUNY-Stony Brook, we have determined the three-dimensional structure of the IGF1 receptor kinase domain using x-ray crystallography. Several amino acid differences between the IGF1 receptor and the insulin receptor near the ATP binding cleft might be exploited by small-molecule inhibitors to gain selectivity for the IGF1 receptor over the insulin receptor. To this end, structural studies of inhibitors bound to the IGF1 receptor kinase are being pursued.

Another RTK of interest is MuSK, or muscle-specific kinase, which is expressed exclusively in muscle cells and plays an essential role in the formation of neuromuscular synapses, by promoting clustering of acetylcholine receptors. Activation of MuSK by agrin results in autophosphorylation of several tyrosines in the cytoplasmic domain of MuSK. In a collaboration with Dr. Steven Burden at the NYU School of Medicine, we have determined the crystal structure of the cytoplasmic (tyrosine kinase-containing) domain of MuSK to understand how kinase activity is regulated in this receptor. The structure reveals that MuSK is strongly autoinhibited by the kinase activation loop and suggests that an additional in vivo component might contribute to negative regulation by binding to the juxtamembrane region of MuSK.