Cell proliferation is dependent on macromolecular components, termed growth factors, which are present in serum. The binding of these growth factors to specific cell surface receptors appears to stimulate a variety of biochemical and physiological responses which culminate in a mitogenic signal. An understanding of the mechanisms by which these receptor-coupled signaling systems operate is of increasing interest since impairments in the signal transfer events may have important implications regarding why cells become cancerous. It is now suspected that growth factors may trigger mitogenesis through three component (receptor, transducer, effector) systems analogous to those operating in the hormonal regulation of adenylate cyclase activity, or in vertebrate vision. We intend to test this hypothesis as well as address other aspects of the molecular basis of growth factor action, using recently developed reconstitution approaches. Well defined phospholipid vesicle systems containing the purified epidermal growth factor (EGF) receptor, or the purified insulin receptor, will form the basis for comparing the mechanisms of action of these two growth factor receptor/tyrosine kinases within a lipid milieu. The studies proposed here are divided into three specific aims: 1) Structure-function studies of the EGF receptor and the insulin receptor/tyrosine kinases, both in detergent solution and in lipid vesicles, using a combination of reconstitution, steady state kinetic (phosphorylation) and hydrodynamic approaches, 2) The characterization of growth factor-induced conformational changes (and/or receptor-receptor interactions) in the EGF receptor and the insulin receptor by fluorescence spectroscopic techniques, and 3) An examination of the capabilities of different heterotrimeric GTP binding transducer proteins, and the ras oncogenic proteins, to act as transducers in growth factor action. Among the specific questions which will be addressed in these studies include: a.) what is the nature of the growth factor-induced conformational changes in these receptor/tyrosine kinases, and can they be transmitted across a membrane bilayer from the growth factor binding domain to the tyrosine kinase domain via an intramolecular mechanism, b.) what roles do receptor-receptor interactions play in the induction of growth factor-dependent tyrosine kinase activity and does a lipid milieu influence these interactions, and c.) can either the EGF or insulin receptor directly regulate the activation-deactivation cycles of GTP binding proteins? The construction of reconstituted phospholipid vesicle systems containing these purified receptors should constitute an important step toward delineating the important protein-protein (protein-transducer) interactions involved in growth factor-coupled signal transduction.
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