CYCLIC GMP IN PHOTORECEPTORS

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DESCRIPTION (Adapted from applicant's abstract): The goal of the proposed study is to define new mechanisms regulating the phototransduction and adaptation processes in visual receptors. The major focus is to determine the physiological role of a novel Ca2+-binding protein, p24 (GCAP-2), and two photoreceptor gluanylyl cyclase (GCs) in synthesis of cGMP, a second messenger in the photoresponse. This proposal is based on the recent finding that two different membrane GCs, RetGC-1 and RetGC-2, are present in photoreceptor cells and two different Ca2+-binding proteins can regulate activity of both cyclases as a function of free Ca2+ concentration. This finding demonstrates the diversity of regulatory elements providing feedback mechanisms for resynthesis of cGMP after its hydrolysis caused by photoexcitation. The first aim of this study is to define, by using directed mutagenesis, what parts of GCAP-2 activate GC. GCAP-1 and GCAP-2 are homologs and RetGC-1 and RetGC-2 are also homologs. Therefore, the second aim of this proposal is to assess their localization, as determined by immunocytochemical analysis. The relative efficiency of RetGC-1 and RetGC-2 regulation by GCAP-1 or GCAP-2 will also be measured in vitro. These experiments will indicate whether either of the two GCAPs specifically interacts with a certain cyclase and they will also provide characteristics of such interactions. The third aim is to evaluate the relative importance of GCAP-1 and -2 as well as RetGC-1 and -2 for the regulation of cGMP synthesis in truncated outer segments. The long-term goal of this project is to identify all structural determinants of GCAP-2 involved in GC regulation. The experiments proposed here are relevant to understanding the intricate feedback controls that regulate photoreceptor activity and that might be perturbed in several inherited retinal diseases.