Understanding the processes which regulate cellular growth and proliferation is important not only for a rational approach to the treatment of cancer, but for a host of other disease processes wherein cell proliferation contributes to the pathophysiologic process or complicates its therapy. The best studied model of growth regulation involves the stimulation of the ubiquitous Ras/mitogen activated protein (MAP) kinase pathway via activation of peptide growth factor receptors with intrinsic tyrosine kinase activity. Recently, it has been increasingly appreciated that receptors which transduce signals via heterotrimeric guanine nucleotide-binding (G) proteins possess to ability to activate this pathway. Among these are receptors for potently mitogenic substances produced by platelets and vascular endothelium, such as lysophosphatidic acid, alpha-thrombin and endothelin, which may play a role in the proliferative complications of diabetes mellitus or vascular smooth muscle proliferation in heart disease or post-angioplasty. The mechanisms whereby heterotrimeric G proteins stimulate mitogenesis are poorly understood but appear to involve distinct functions performed by G protein G-alpha and G-beta-gamma subunits. Evidence also exists that some tyrosine kinase growth factor receptors, among them the insulin receptor, may mediate some of their mitogenic effects through heterotrimeric G protein-dependent mechanisms. This project tests the hypothesis that G protein G-alpha and G-beta-gamma subunits play distinct roles in the stimulation of the Ras/MAP kinase pathway through G protein-coupled receptors. Using stable and transient expression and permeabilized whole cell model systems the initial aims of the project will be to distinguish the contributions of G-alpha and G-beta-gamma subunits using specific G- beta-gamma-antagonist peptides and to identify the specific step(s) in the Ras/MAP kinase pathway that are modulated by each subunit. A parallel aim is to determine the function of the putative G-beta-gamma subunit-binding pleckstrin homology (PH) domains found in several proteins involved in Ras activation, both by studying the effects of PH domain peptides on G protein-mediated Ras activation and by characterizing the proteins which bind PH domains using a two-hybrid yeast expression system. Another aim is to determine the extent of cross-talk between the G protein-mediated and tyrosine kinase receptor-mediated Ras-activation pathways using antagonists of G protein G-alpha and G-beta-gamma subunits. The characterization of specific peptide antagonists of G protein-coupled receptor-mediated mitogenesis may help to define strategies for the prevention of the proliferative complications of diseases such as diabetes mellitus. The candidate for this award is an endocrinology fellow and Medical Scientist Training Program graduate with three years of clinical internal medicine training and two years of postdoctoral research experience. The candidate seeks this award to evolve into an independent investigator of signal transduction relevant to the pathophysiology and complications of diabetes. The environment for this award the laboratory of Dr. Robert J. Lefkowitz, has pioneered the study of G protein coupled receptors and their regulation and has a strong record of developing independent clinician scientists.
