This is a revised proposal to investigate the involvement of a protein called the guanylate cyclase regulatory protein (GCRP) in the regulation of ANF-stimulated guanylate cyclase activity. This protein which was recently cloned and expressed in the applicant's laboratory has been shown to amplify ANF-dependent cyclase activity in cotransfection experiments. The applicant hypothesizes that; (1) guanylate cyclase exists in an active and inactive form and that the presence of ANF and ATP promotes the development of the former; (2) the inactive form is suppressed by an autoinhibitory site present in the kinase-like domain of the cyclase and (3) in the presence of ANF and ATP, GCRP, and/or its splice variant GCRP-2, associates with the cyclase and induces a conformational change which releases the inhibitory constraint imposed on the active site. To evaluate this hypothesis the applicant proposes to: (1) locate the autoinhibitory site on the guanylate cyclase molecule using site-directed mutagenesis and to determine whether ANF and ATP activate the cyclase by releasing the inhibitory constraint, (2) investigate whether ANF induces the association of GCRP and GCRP-2 with guanylate cyclase and (3) examine whether limited proteolysis or binding at the autoinhibitory site induces a conformational change at the active site of the cyclase.
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