A diverse set of agents including platelet-derived growth factor, fibroblast-derived factor, vasopression, phorbolesters, Ca++ ionophore A23187, and catecholamines have all been shown to inhibit insulin and epidermal growth factor (EGF) receptor binding activities. These agents in effect, desensitize the cells to the physiological actions of insulin and EGF. It is not surprising that hormones such as catecholamines inhibit insulin binding because of the direct antagonistic nature of these hormones with respect to the cell's metabolic responses. Thus, if one considers the binding of ligand to a cell surface receptor as the commitment step in a metabolic pathway, then the regulation of receptor binding activities should be expected. Further, if one assumes the ligand to be an allosteric regulator then a change in the intrinsic signalling mechanism of the receptors may also be regulated via modification of the ligand-receptor interaction. Our long term goal is to define the molecular mechanism by which these growth factor (insulin and EGF) receptor binding activities are coordinately regulated and determine what effect this has on their transmembrane signalling mechanisms. All the known actions of catecholamines, which are Beta-adrenergic receptor agonists are mediated thru cyclic AMP-dependent protein kinase (A kinase). Phorbolesters are known activators (substituting for diacylglycerol) of the Ca++, phospholipid-dependent protein kinase (C kinase). Vasopressin, although in some systems causing the elevation of cyclic AMP, primarily stimulates and increase in the intracellular Ca++ concentration. Treatment of isolated membranes or intact cells with platelet-derived growth factor leads to a dramatic increase in the phosphorylation state of membranes. Thus, for all the agents known to inhibit insulin and EGF receptor binding activities a circumstantial case can be made for the involvement of a kinase cascade. We plan to purify the insulin and EGF receptors both by conventional chromatography and by immunoprecipitation. We will then examine what effects these agents have on the phosphyrylation states of the receptors both in vivo and in vitro. The in vitro studies will involve purification of the A kinase and the C kinase and direct phosphorylation of the purified receptors. The effects of both the in vivo and in vitro phosphorylations will be examined with respect to receptor binding activities and intrinsic tyrosine kinase activities.
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