The metabolic and growth-stimulatory effects of the hormone insulin are mediated through the insulin receptor (IR), a multi-subunit transmembrane glycoprotein with intrinsic tyrosine kinase activity in the cytoplasmic domains. Insulin binding to the extracellular domains of the IR activates the receptor by inducing a conformational change that facilitates autophosphorylation of specific tyrosine residues in the cytoplasmic domains. Tyrosine autophosphorylation stimulates receptor catalytic activity and creates recruitment sites for downstream signaling proteins. This proposal focuses on the structural and biochemical aspects of the tyrosine kinase domain of the IR (IRK).
The specific aims of this proposal are: I) Structural characterization of the interaction between IRK and negative regulatory proteins. II) Structural and biochemical characterization of the enzymological properties of IRK. III) Characterization of the insulin-induced structural rearrangement in the cytoplasmic domains of the IR. The primary experimental techniques that will be used to accomplish these aims are x-ray crystallography, site-directed mutagenesis, steady-state kinetics, and transient transfection studies. The results obtained from this work should contribute significantly to our understanding of the molecular mechanisms by which the IR is activated and downregulated, and facilitate efforts to design small molecule, intracellular agonists of the IR for potential use as anti-diabetic therapeutics. In addition, knowledge gained on the structural and enzymological properties of IRK will yield insights into the biochemistry of related receptor tyrosine kinases, such as the insulin-like growth factor 1 receptor, the fibroblast growth factor receptors, and the epidermal growth factor receptor. These and other receptor tyrosine kinases have been implicated in the onset or progression of many human cancers, and the proposed high resolution structural work on IRK should prove valuable in the design of potent and specific tyrosine kinase inhibitors.
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