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

说明(申请人提供)：鸟苷酸环化酶(GC)-A和B是同源的、多肽激活的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标。然而，缺乏监管信息阻碍了这些酶在治疗心血管、骨骼和生殖疾病方面的最大治疗性利用。腺嘌呤核苷酸通过结合未知的细胞内高亲和力激活和低亲和力抑制部位来调节GC-A和GC-B。这两种受体在静息细胞中都高度磷酸化，去磷酸化的受体对利钠肽(NPs)没有反应。与NPs作用相反的激素会升高细胞内的钙，从而导致所有受体磷酸化位点的去磷酸化。相反，激活的蛋白激酶C(PKC)被假设为磷酸化保守的受体共识位点，减少单独的关键调控位点的磷酸化。此应用程序的长期目标是 确定激素、腺嘌呤核苷酸和磷酸化如何调节GC-A和GC-B。我们打算通过追求以下四个具体目标来实现这一目标：1)确定腺核苷酸如何调节GC-A和GC-B，2)确定PKC如何抑制GC-B，3)确定致病错义突变如何影响GC-B功能，以及4)确定激素如何抑制GC-B。第一个目标是测量结构独特的和活性的嘌呤对GC-A和GC-B的动力学性质和结合位置的影响。含有嘌呤结合位点突变的受体将决定催化域是对称的还是不对称的同源二聚体。第二个目标将通过siRNA敲除、体外激酶分析和拟磷化突变体确定所需的PKC异构体和磷酸化位点来确定PKC如何抑制GC-B。第三个目标将确定导致错义突变的12种侏儒症如何使GC-B失活，以及一种新发现的导致骨骼过度生长的突变如何结构性地激活GC-B。这些突变对125I-CNP结合、鸟苷酸环化酶活性、翻译后加工和细胞定位的影响将被确定。最后，第四个目标将研究荷尔蒙如何 通过评估对钙升高、蛋白激酶C激活和各种受体磷酸化位点的需求，灭活小鼠卵泡、颗粒细胞、软骨细胞和平滑肌细胞中的GC-B。这项拟议的研究具有重要意义，因为这些特定目标的成功完成将促进对GC-A和GC-B依赖激素、腺核苷酸和磷酸化调控的理解，并可能揭示新的治疗靶点。

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.