This research proposal is based on the recent recognition that retinal guanylate cyclases are coupled to three important signaling systems: (1) Phototransduction; (2) type A (ANF) and type C )CNP) natriuretic factors; and (3) nitric oxide. The first signal transduction system has direct implications in vision, the second system in regulation of the retinal vasculature tone and fluid secretion, and the third system in neurotransmission processes. The proposed research addresses (1) and (2), and is based on the following observations: (a) The complete identity of the ROS-GC is missing. The amino acid sequence of the four rod outer segment guanylate cyclase (ROS-GC) fragments has been determined. This sequence information is now being used to clone and express the ROS-GC; (b) ATP negatively regulates the bovine ROS-GC activity, but the mechanism for this negative regulation is now known; (c) the direct cloning studies show the presence of CNP receptor guanylate cyclase (CNP-RGC) and the presence of partial ANF receptor guanylate cyclase (ANF-RGC) in human retina cDNA library, indicating a natriuretic factor signaling network in the retina, which opens up the possibility of an autocrine and/or paracrine function of the natriuretic peptide hormones in this tissue; but the information regarding the cell- site synthesis of these hormones is missing; (d) The transmembrane migration of natriuretic factor signaling is allosterically regulated by an ATP-mediated event. ATP binds to an ATP regulatory module(s) (ARM) core sequence Gly-Xa-Xa-Xa-Gly of the cyclases, and amplifies the ANF- and CNP-dependent cyclase activities, but the critical ARM residue that acts as an ATP switch is not known: (e) in addition to the stimulatory(s)-ARM site, there also appears to be an inhibitory (i)-ARM site, which upon binding ATP causes inhibition of the cyclase signal. This required Mn2+ as a cofactor. The sequence of this i-ARM is not known. The proposed research is targeted to fill in these gaps. It will define the precise structural and functional components of the membrane guanylate cyclase-linked receptor network potentially related to the processes of phototransduction, neurovasculature regulation of blood flow, diuresis, and neurotransmission processes. The combined tools of biochemistry, immunology and genetic remodeling will be used to accomplish these goals. Although the proposed research is most basic in nature, its ramifications in understanding the disease processes related to vision, glaucoma, hypertension, and neurological disorders are obvious.
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