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The applicant notes that receptor guanylyl cyclases are a family of proteins which regulate important (patho)physiological processes, in particular cardiovascular and intestinal fluid and electrolyte homeostasis. The most important ligands for these receptors are atrial natriuretic peptide (ANP) and the E.coli heat-stable enterotoxin (ST). Binding of ANP to guanylyl cyclase A (G Cyclase-A) produces vascular smooth muscle relaxation, natriuresis, and diuresis, responses directed at maintaining blood pressure and circulating fluid volume. Binding of ST to guanylyl cyclase C (G Cyclase-C) results in the secretion of fluid and electrolytes in the intestine and is a principal cause of diarrheal disease in animals and humans. Because these receptors mediate key (patho)physiological processes, it is important to understand the regulation of this signaling mechanism. Recently, a novel nucleotide-dependent pathway was identified in this laboratory which modulates G Cyclase-C in intestinal cells. Indeed, 2-substituted adenine nucleotides prevent the activation of G Cyclase-C by ST in an allosteric and guanine nucleotide-dependent fashion. Furthermore, this pathway was successfully employed to block electrolyte transport induced by ST in intact human intestinal cells, which is central to induction of diarrhea. Thus, allosteric nucleotide inhibition represents a unique regulatory mechanism which may be a general characteristic of the family of receptor guanylyl cyclases. This pathway could function as a type of "gain control", regulating the responsiveness of receptor guanylyl cyclases to their ligands in vivo. Alternatively, this pathway may play a role in acute and/or chronic desensitization mechanisms which regulate the activity of receptor guanylyl cyclases in normal and pathological processes. Moreover, this inhibitory pathway could be exploited to interrupt key (patho)physiological processes mediated by receptor guanylyl cyclases in intact tissues and animals, such as vascular smooth muscle relaxation and intestinal allosteric nucleotide inhibition and the applicability of that pathway to modulating receptor guanylyl cyclase signaling in intact tissues.

The present proposal seeks to: (1) define the sensitivity to this allosteric nucleotide inhibitory pathway of the 6 known members of the receptor guanylyl cyclase family; (2) examine the mechanisms mediating this inhibition, including the role of accessory regulatory proteins, the domains and sequences of guanylyl cyclases mediating inhibition, and endogenous agonists which induce this inhibitory pathway; and (3) determine the utility of this pathway to inhibit physiological responses mediated by receptor guanylyl cyclases in intact tissue, including natriuretic peptide-dependent vascular smooth muscle relaxation. The applicant hopes that the results will establish a foundation for examining the role of this pathway in the physiological regulation of receptor guanylyl cyclases, the molecular components and their interactions mediating this regulation, and the potential utility of this pathway to interrupt guanylyl cyclase signaling in vivo.
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