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

激素、ATP 和磷酸化对鸟苷酸环化酶 A 和 B 的调节

• 批准号: 8705541
• 负责人: Lincoln Ross Potter
• 金额: $ 28.37万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8705541
• 关键词: Adenine Nucleotides; Affect; Affinity; Allosteric Site; Atrial Natriuretic Factor; Binding; Binding Sites; Biological Assay; Blood Pressure; Blood Volume; Bone Growth; C-Type Natriuretic Peptide; Calcium; Cardiovascular Diseases; Cardiovascular system; Catalytic Domain; Cell physiology; Cells; Chemicals; Chondrocytes; Consensus; Cyclic GMP; Defect; Disease; Drug Targeting; Dwarfism; Dysplasia; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Extracellular Domain; Fibroblast Growth Factor; Growth; Growth Disorders; Guanosine Triphosphate; Guanylate Cyclase; Heart Hypertrophy; Heart failure; Hormones; Human; Hypertension; Immunofluorescence Immunologic; In Vitro; Individual; Infusion procedures; Interphase Cell; Kinetics; Knowledge; Lead; Luteinizing Hormone; Mass Spectrum Analysis; Measures; Meiosis; Missense Mutation; Mission; Molecular; Mus; Musculoskeletal Diseases; Mutation; Natriuretic Peptides; Nucleotides; Oocytes; Ovarian Follicle; Ovulation; Pathway interactions; Patients; Peptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Prevalence; Process; Property; Protein Dephosphorylation; Protein Isoforms; Protein Kinase C; Public Health; Purines; Recombinants; Regulation; Research; Site; Site-Directed Mutagenesis; Small Interfering RNA; Smooth Muscle Myocytes; Sterility; Therapeutic; Tissues; Vasopressins; Western Blotting; atrial natriuretic factor receptor A; atrial natriuretic factor receptor B; base; bone; crosslink; disability; disease-causing mutation; fibroblast growth factor receptor 3; glycosylation; granulosa cell; heart dimension/size; inhibitor/antagonist; inorganic phosphate; long bone; mutant; new therapeutic target; novel; novel therapeutics; oocyte maturation; peptide B; prevent; protein B; public health relevance; purine; receptor; reproductive; research study; skeletal; skeletal dysplasia; trafficking

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.

描述（由申请人提供）：鸟苷酸环化酶 (GC)-A 和 B 是同源的、肽激活的 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点。然而，缺乏监管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。腺嘌呤核苷酸通过未明确的机制结合未识别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。两种受体在静息细胞中都高度磷酸化，而去磷酸化的受体对利尿钠肽 (NP) 无反应。对抗 NP 作用的激素会升高细胞内钙，从而导致所有受体磷酸化位点去磷酸化。相比之下，活化蛋白激酶 C (PKC) 被假设可磷酸化保守的受体共有位点，从而减少单独的关键调节位点的磷酸化。此应用程序的长期目标 目的是确定激素、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）确定引起疾病的错义突变如何影响 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且具有反应性的嘌呤对 GC-A 和 GC-B 的动力学特性和结合位点的影响。嘌呤结合位点含有突变的受体将决定催化结构域是对称还是不对称同二聚体。第二个目标是通过使用 siRNA 敲低、体外激酶测定和磷酸化突变体鉴定必需的 PKC 同工型和磷酸化位点，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 种侏儒症中的每一种如何使 GC-B 失活，以及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 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。最后，第四个目标将研究激素如何 通过评估钙升高、PKC 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。拟议的研究意义重大，因为成功完成这些具体目标将促进对 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。