The goals of this proposal are to identify the biochemical steps involved in vasopressin action at the level of (a) adenylate cyclase activation, and (b) actin filament network organization. We will establish the role for calcium-regulatory proteins and/or protein phosphorylation at these sites of hormone activity. Hormone receptor-adenylate cyclase interactions will be studied in the pig kidney cell line, LLC-PK1. We plan to determine 1) the functional and structural association of vasopressin receptors and adenylate cyclase subunits utilizing techniques of target size determination by radiation inactivation as well as receptor enzyme kinetic analysis; 2) the site of activation of vasopressin-sensitive adenylate cyclase by calmodulin utilizing solubilized and reconstituted adenylate cyclase subunits. In addition, we will determine if toad bladder epithelium contain calmodulin-regulated adenylate cyclase and whether this is the site of inhibition of the vasopressin-stimulated enzyme by prostaglandins. We have identified a class of regulatory proteins in toad bladder epithelial cells which modulate actin filament network organization. These proteins, in turn, are regulated by calcium (gelsolin, villin) and cAMP-dependent protein phosphorylation (actin binding protein). We now propose to purify these regulatory proteins, make antibodies to them, and define the in vivo changes in the organization of the actin filament network induced by vasopressen. We will determine whether actin binding protein is phosphorylated in intact toad bladder epithelium in response to the hormone. Once this is established, we will evaluate the functional and structural interactions of actin and phosphorylated actin binding protein in vitro. While the focus of these studies is on vasopressin action, the questions addressed are of widespread scientific interest. Success in this project will result in a better definition of hormone-receptor adenylate cyclase interactions, one of the hormone - signalling mechanism. In addition, the role calcium and cAMP in the most important regulation of the cell cytoskeleton has relevance to a variety of cell functions ranging from cell mobility to epithelial transport.
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