ANALYSES OF GCAP IN NORMAL AND RD MUTANT RETINA

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Our long-range goal is to understand the biochemical processes within photoreceptor cells, that if disrupted, lead to cell dysfunction and degeneration. The retinal degeneration (rd) chicken, the only animal model of inherited retinal disease that possesses a cone-dominant retina, is the focus of the proposed studies. Retinas of chicks homozygous for the rd mutation are fully differentiated and ultrastructurally indistinguishable from normal chick retina at hatch but do not produce scotopic or photopic ERG responses following light stimulation. Within 7- 10 days of hatch, the photoreceptors begin to degenerate. The molecular defect underlying this phenotype is not known; however, levels of cyclic GMP in the photoreceptor cells of the mutant retina are significantly reduced prior to their degeneration suggesting that the rd gene may encode a protein directly involved in photoreceptor cGMP metabolism. Mammalian photoreceptors express at least two guanylate cyclase activating proteins (GCAPs), GCAP1 and GCAP2 (p24). Western blot analyses show that normal chick retina contains a third GCAP protein that is antigenically similar to GCAP1. Western blot analyses of this protein in the rd mutant retina shows that it is not expressed in this retina. The aims of the proposed studies are (1) to clone and characterize the GCAP1 variant present in normal chicken retina that is not expressed in the mutant retina and (2) to identify possible mutations in the GCAP1 candidate gene that would disable expression of this variant in the mutant retina. GCAP cDNAs will be amplified from first-strand cDNA using the PCR and/or isolated from our normal and mutant chick retina cDNA expression libraries using antibody and cDNA probes. The clones will be analyzed by sequencing, northern and Southern blot. Anti-peptide antibodies will be generated to allow western blot and immunocytochemical analyses of the proteins encoded by these clones. Mutations found within the GCAP candidate cDNAs will be verified through gene analyses, and pedigree analyses will be carried out to confirm association of the mutation with the rd mutant phenotype. Eventually, mutant GCAP will be expressed in vitro and tested for its competence to activate guanylate cyclase. The results of these studies will improve our understanding of the cGMP metabolism and how a defect in cGMP synthesis leads to retinal disease.