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

描述（由申请方提供）：鸟苷酸环化酶（GC）-A和B是同源的肽活化cGMP合成酶，可调节血压、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，缺乏监管信息阻止了这些酶用于治疗心血管、骨骼和生殖疾病的最大治疗利用。腺嘌呤核苷酸通过未确定的机制结合未鉴定的细胞内高亲和力激活和低亲和力抑制位点来调节GC-A和GC-B。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（NP）无反应。对抗NP作用的激素升高细胞内钙，这导致所有受体磷酸化位点的去磷酸化。相反，活化的蛋白激酶C（PKC）被假设为磷酸化一个保守的受体共识网站，减少磷酸化的一个单独的关键调控位点。本申请的长期目标是 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）确定腺嘌呤核苷酸如何调节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和反应性嘌呤对GC-A和GC-B的动力学性质和结合位点的影响。在嘌呤结合位点中含有突变的受体将决定催化结构域是对称的还是不对称的同二聚体。第二个目标将确定PKC如何抑制GC-B，通过使用siRNA敲除、体外激酶测定和磷酸化模拟突变体鉴定必需的PKC亚型和磷酸化位点。第三个目标将确定12个导致侏儒症的错义突变中的每一个如何激活GC-B，以及一个新发现的导致骨骼过度生长的突变如何组成性激活GC-B。将确定这些突变对125 I-CNP结合、鸟苷酸环化酶活性、翻译后加工和细胞定位的影响。最后，第四个目标将研究激素如何 通过评估对钙升高、PKC活化和各种受体磷酸化位点的需求，在小鼠卵泡、颗粒细胞、软骨细胞和平滑肌细胞中检测GC- B。拟议的研究是重要的，因为这些特定目标的成功完成将促进对GC-A和GC-B的激素，腺嘌呤核苷酸和磷酸化依赖性调节的理解，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.