PROJECT SUMMARY Cyclic cGMP (cGMP) enables phototransduction in vertebrate rods and cones. The cGMP synthesis by retinal guanylyl (guanylate) cyclase (RetGC), one of the most essential processes in the photoreceptor physiology, is controlled by calcium, guanylyl cyclase activating proteins (GCAPs), and retinal degeneration 3 (RD3) protein. The abnormalities in cGMP signaling cause photoreceptor dysfunction and death. Among them, deficiency in RetGC activity and regulation lead to a variety of recessive and dominant forms of congenital blindness. The basic principles of the RetGC regulation and its fundamental importance for the photoreceptor signaling and survival have been established, and the first clinical trials for RetGC-linked blindness now have begun, evolved from the molecular studies. However, some key molecular and cellular aspects of RetGC regulation still remain insufficiently understood, including the molecular structure and the interactions of RetGC with the regulatory proteins that define its biological function in photoreceptors. This proposal, conforming to the NEI mission to support research with respect to blinding eye diseases, visual disorders and mechanisms of visual function, is built on recent advancements in understanding of how RetGC enables the photoreceptor function: (i) identification of new mutations that affect RetGC interactions with GCAPs and RD3; (ii) establishing the structure on RD3 and identification of its RetGC-binding interface; (iii) development of new mouse genetic models for studying mechanisms of signal transduction and their abnormalities caused by mutations in RetGC1, GCAP1, and RD3; (iv) establishing the complex physiological role of RD3 in photoreceptor function and survival. We here propose a diversified study designed to evaluate, by integrating protein biochemistry, molecular biology, and molecular genetics, new hypotheses and to acquire better mechanistic understanding of the regulatory processes that control cGMP synthesis in photoreceptors. Aim 1 will address the mechanistic interactions of RD3 that control the levels of RetGC in photoreceptors. Aim 2 will determine, by using new transgenic mouse models, structural and functional relations that define RetGC regulation in complexes with GCAPs and RD3. Aim 3 will seek developing new biochemical approaches designed to pave the road for future structural studies of RetGC1. By completing these specific aims, we expect to achieve deeper and more reliable understanding of mechanistic interactions that define the fundamental role of RetGC in photoreceptor biology and underlie inherited physiological abnormalities of the retina.