Regulation of guanylyl cyclase A and B by hormones, ATP and phosphorylation

描述（由申请人提供）：关酰环化酶(GC)-A和B是同源的，肽激活的，cGMP合成酶，调节血压，心脏大小，长骨生长和卵母细胞成熟。因此，它们是理想的药物靶点。然而，缺乏调控信息阻碍了这些酶在心血管、骨骼和生殖疾病治疗中的最大治疗利用。腺嘌呤核苷酸通过结合细胞内未识别的高亲和力激活位点和低亲和力抑制位点来调节GC-A和GC-B，其机制尚未明确。这两种受体在静息细胞中高度磷酸化，去磷酸化受体对利钠肽（NPs）无反应。与NPs作用相反的激素升高细胞内钙，导致所有受体磷酸化位点的去磷酸化。相比之下，活化的蛋白激酶C （PKC）被假设磷酸化一个保守的受体共识位点，从而减少一个单独的关键调控位点的磷酸化。此应用程序的长期目标

DESCRIPTION (provided by applicant): Guanylyl cyclase (GC)-A and B are homologous, peptide-activated, cGMP synthesizing enzymes that regulate blood pressure, heart size, long bone growth and oocyte maturation. Hence, they are desirable drug targets. However, lack of regulatory information prevents maximal therapeutic utilization of these enzymes for the treatment of cardiovascular, skeletal and reproductive diseases. Adenine nucleotides regulate GC-A and GC-B by binding unidentified intracellular high affinity activation and low affinity inhibitory sites through undefined mechanisms. Both receptors are highly phosphorylated in resting cells and dephosphorylated receptors are unresponsive to natriuretic peptides (NPs). Hormones that oppose the actions of NPs elevate intracellular calcium, which causes the dephosphorylation of all receptor phosphorylation sites. In contrast, activated protein kinase C (PKC) is hypothesized to phosphorylate a conserved receptor consensus site that reduces phosphorylation of a separate critical regulatory site. The long-term objective of this application is to determine how hormones, adenine nucleotides and phosphorylation regulate GC-A and GC-B. We intend to accomplish this objective by pursuing the following four specific aims: 1) Determine how adenine nucleotides regulate GC- A and GC-B, 2) Identify how PKC inhibits GC-B, 3) Determine how disease-causing missense mutations affect GC-B function, and 4) Identify how hormones inhibit GC-B. The first aim will measure the effects of structurally unique and reactive purines on the kinetic properties and binding sites of GC-A and GC-B. Receptors containing mutations in purine binding sites will determine whether the catalytic domains are symmetric or asymmetric homodimers. The second aim will determine how PKC inhibits GC-B by identifying the requisite PKC isoform and phosphorylation sites using siRNA knockdown, in vitro kinase assays and phosphomimetic mutants. The third aim will determine how each of the twelve dwarfism causing missense mutations inactivate GC-B as well as how a newly discovered mutation that leads to skeletal overgrowth constitutively activates GC-B. Effects of these mutations on 125I-CNP binding, guanylyl cyclase activity, post-translational processing and cellular localization will be determined. Finally, the fourth aim will investigate how hormones inactivate GC- B in mouse follicles, granulosa cells, chondrocytes and smooth muscle cells by assessing the requirements for calcium elevations, PKC activation, and various receptor phosphorylation sites. The proposed research is significant because the successful completion of these specific aims will advance understanding of hormone-, adenine nucleotide- and phosphorylation-dependent regulation of GC-A and GC-B and may reveal new therapeutic targets.