The broad long-term objectives of this proposal are to understand the mechanisms involved in signaling through the cyclic GMP pathway, the normal biological role of PP5, a protein-serine phosphatase that is phosphorylated and activated by cyclic GMP-dependent protein kinase (PKG), and the role of tetratricopeptide repeat (TPR) domains in subcellular targeting.
The specific aims are to: i) identify the mechanisms regulating the binding of PP5 to the atrial natriuretic peptide (ANP) receptor/guanylyl cyclase (GC-A); PP5 is targeted to GC-A and appears to mediate GC-A dephosphorylation and desensitization; ii) identify the residues in PP5 that are phosphorylated by PKG and test whether the amino-terminal regulatory domain of PP5 is autoinhibitory; test whether PKG activates PP5 by relieving autoinhibition; iii) determine the structural requirements for PP5 targeting to GC-A. The health relatedness of the project lies in gaining a better understanding of (1) the mechanisms of action of natriuretic peptides that are potential therapeutic agents, and the mechanisms by which responsiveness to these peptides is lost in cardiovascular disease states; (2) of the PKG signaling pathway, which mediates diverse hormonal responses. The experimental plan is: i) to use metabolic labeling and immunoprecipitation to test whether PP5 is phosphorylated by PKG and PKC in intact cells. To perform co-immunoprecipitation experiments to test whether treatments with ANP, TPA, or 8-bromo cyclic GMP increase association of PP5 with GC-A. To use in vitro membrane-binding assays and surface plasmon resonance assays to test effects of PP5 phosphorylation on its binding to the kinase-domain of GC-A; ii) to use phosphoamino acid analysis, phosphopeptide mapping, and peptide sequencing to identify sites on PP5 phosphorylated by PKG. To use deletion mutagenesis and phosphatase assays to test whether the amino terminal regulatory domain of PP5 contains autoinhibitory sequences; iii) to use deletion mutagenesis and in vitro binding assays to identify the minimal structural requirements for binding of the PP5 TPR domain to the GC-A kinase-like domain, to use site directed mutagenesis of full-length PP5 and GC-A, in conjunction with co-immunoprecipitation and desensitization assays, to test the in vivo importance of residues identified as important for binding in vitro.
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