The two key enzymes involved in regulation of cGMP are photoreceptor-specific phosphodiesterase (PDE) and guanylate cyclase (GC). PDE is a multisubunit enzyme (putative stoichiometry PDE-alpha/beta/gamma2/delta2) and peripherally membrane-associated, while GC is a single subunit integral membrane protein. GC1 is regulated by [Ca2+] via one or two Ca2+-binding regulators (guanylate cyclase activating proteins, GCAP1 and GCAP2). This application is directed at the characterization of these components by techniques of molecular biology.
In specific aim 1, PDE subunits will be co-expressed to establish the essential components for biological activity. Photoreceptor PDE is the only mammalian PDE that has not been functionally expressed in unicellular systems. As a subaim, the novel PDE subunit PDE-delta will be tested for function using gene expression replacement techniques in mice whose wild-type PDE-delta gene has been targeted for disruption (PDE-delta """"""""gene knock-out"""""""").
In specific aim 2, GCAP1 expression and mutagenesis studies will seek to identify domains interacting with GC1. A subaim here, as a collaborative effort, is to knock out the genes encoding either or both GCAPs.
Specific aim 3 addresses expression and mutagenesis of GC1. The applicant is particularly interested in the rd chicken, an animal model for early onset recessive autosomal retinitis pigmentosa (Leber's congenital amaurosis). Preliminary data indicate that the gene encoding GC1 in the rd chicken retina is not expressed, and this proposal aims to identify the specific gene defect. Collectively, this research will enhance fundamental knowledge of structure/function relationships of components participating in the regulation of cGMP and Ca2+, two important second messengers of phototransduction.
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