The objective of this proposal is to investigate the viochemical mechanism of action of cyclic AMP-mediated hormones and insulin in a cultured cell line uniquely suited to these studies. In Section I we will analyze the mechanism by which adenylate cyclase becomes coupled to the Beta-adrenergic receptor during differentiation. We will systematically quantiate the Beta-adrenergic receptor (R), the guanine nucleotide regulatory protein (G/F) and the catalytic component of the cylcase throughout the differentiation program, establish the kinetics of appearance of catecholamine- and/or guanine nucleotide-stimulated adenylate cyclase activities and determine whether the expression of catecholamine-sensitive adenylate cyclase is directly coordinated with the accumulation of regulatory components (G/F and R). In Section II, we utilize the developmental acquisition of high affinity insulin receptors by the 3T3-Ll cells to study the structure and regulation of insulin receptor content using antibodies to the receptor, a cross-linking reagent that can selectively covalently label the receptor with [125I]-insulin, and a new procedure for reversibly removing cell surface insulin receptor activity with chymotrypsin. The information obtained will relate tot he molecular structure of the insulin receptor system during differentiation and the role of the """"""""latent"""""""" receptor pool in maintaining the complement of cell surface insulin receptors. In part III we investigate the effect of cAMP-mediated and insulin-promoted phosphorylation of a key lipogenic enzyme, ATP citrate lyase, that is found induced during 3T3-Ll differentiation. We will develop a site-specific assay for insulin-mediated protein phosphorylation, purify lyase-specific protein kinase and phosphatase and attempt to use the lyase in an in vitro system to study the biochemical mechanism of insulin action.
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