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Identifiction of the natriuretic peptide receptor kinase

利钠肽受体激酶的鉴定

基本信息

批准号：
7663267
负责人：
Lincoln Ross Potter
金额：
$ 18.19万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7663267
关键词：
Adult American Atrial Natriuretic Factor Binding Biological Assay Blood Circulation Blood Pressure Bone Growth Brain natriuretic peptide C-Type Natriuretic Peptide Cardiac Cardiovascular Diseases Cardiovascular system Catalytic Domain Cell Extracts Cells Cessation of life Chimeric Proteins Congestive Congestive Heart Failure Coupled Cyclic GMP Dimerization Disease Drug Design Economic Burden End stage renal failure Endocrine system Enzymes Family Figs - dietary Future Gel Glutathione S-Transferase Goals Grant Guanylate Cyclase Health Heart Heart Atrium Heart Diseases Heart Hypertrophy Heart failure Hela Cells Hormones Human Hypertension Incidence Lead Libraries Ligand Binding Domain Ligands MALDI-TOF Mass Spectrometry Mass Spectrum Analysis Mediating Membrane Modeling Molecular Morbidity - disease rate Natriuretic Peptides Peptide Receptor Peptides Phosphorylation Phosphotransferases Protein Dephosphorylation Protein Kinase Proteins Receptor Activation Receptor Signaling Serine Signal Transduction Signaling Molecule Small Interfering RNA Stroke Techniques Threonine United States Vasodilation analog atrial natriuretic factor receptor A atrial natriuretic factor receptor B base combat cost crosslink desensitization extracellular hypertension treatment inorganic phosphate long bone loss of function mutation mortality novel paracrine peptide B prevent public health relevance receptor response skeletal disorder social therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Forty percent of all deaths in the United States result from cardiovascular disease at an estimated annual cost of over 400 billion dollars. Endogenous hormones called natriuretic peptides combat cardiovascular disease. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are released from the heart in response to elevated blood pressure. They bind natriuretic peptide receptor-A (NPR-A), which decreases blood pressure and inhibits cardiac hypertrophy by synthesizing the intracellular signaling molecule cGMP. C-type natriuretic peptide (CNP) is a paracrine factor that stimulates long bone growth and inhibits vasorelaxation by activating natriuretic peptide receptor-B (NPR-B). The guanylyl cyclase activity of NPR-A and NPR-B is controlled not only by natriuretic peptide binding but also by enzymes that regulate the amount of phosphate covalently attached to highly conserved serines and threonine residues within their intracellular domains. Importantly, phosphorylation is required for receptor activation and dephosphorylation mediates receptor inactivation. Our long-term goal is to understand the molecular basis of natriuretic peptide receptor control in health and disease. The objective of this application is to identify the kinase that phosphorylates NPR-A. Our central hypothesis is that receptor phosphorylation is required for ligand-dependent activation of NPR-A, and therefore, is essential for cardiac natriuretic peptide signal transduction. Each specific aim describes a unique approach to identify the natriuretic peptide receptor kinase. We have shown that a glutathione-S-transferase (GST) protein fused to the intracellular domain of NPR-A stably associates with a molecule that phosphorylates NPR-A. The first Specific Aim of this application is to identify the proteins that associate with GST-NPR-A by MALDI-TOF mass spectrometry. The second Specific Aim is to perform a siRNA screen against all known human protein kinases using reductions in ANP-dependent cGMP elevations in Hela cells as an indicator of reduced NPR-A phosphorylation. The third Specific Aim uses an in-gel kinase assay coupled with mass spectrometry to identify the NPR-A kinase. Finally, the fourth Specific Aim takes advantage of a novel bifunctional ATP analog to cross- link NPR-A to its cognate kinase. The identification of the NPR-A kinase is anticipated to provide the framework for future therapeutic targeting of this molecule for treatment of hypertension, congestive heart disease and skeletal disorders. PUBLIC HEALTH RELEVANCE: Natriuretic peptides inhibit blood pressure and stimulate long bone growth in humans. Unfortunately, one of the key regulators of this endocrine system, the natriuretic peptide receptor kinase, is unknown. The goal of this grant is to identify this critical molecule so that it can be targeted by novel drugs designed to treat cardiovascular and skeletal diseases.

描述（由申请人提供）：美国 40% 的死亡是由心血管疾病造成的，估计每年造成的损失超过 4000 亿美元。称为利尿钠肽的内源性激素可对抗心血管疾病。当血压升高时，心脏会释放心房钠尿肽 (ANP) 和 B 型钠尿肽 (BNP)。它们与利尿钠肽受体-A (NPR-A) 结合，通过合成细胞内信号分子 cGMP 来降低血压并抑制心脏肥大。 C 型利钠肽 (CNP) 是一种旁分泌因子，通过激活利钠肽受体 B (NPR-B) 刺激长骨生长并抑制血管舒张。 NPR-A 和 NPR-B 的鸟苷酸环化酶活性不仅受到利尿钠肽结合的控制，还受到调节共价连接到其胞内结构域内高度保守的丝氨酸和苏氨酸残基上的磷酸盐量的酶的控制。重要的是，磷酸化是受体激活所必需的，而去磷酸化则介导受体失活。我们的长期目标是了解利钠肽受体控制健康和疾病的分子基础。本应用的目的是鉴定磷酸化 NPR-A 的激酶。我们的中心假设是受体磷酸化是 NPR-A 配体依赖性激活所必需的，因此对于心脏利尿钠肽信号转导至关重要。每个具体目标都描述了识别利尿钠肽受体激酶的独特方法。我们已经证明，与 NPR-A 胞内结构域融合的谷胱甘肽-S-转移酶 (GST) 蛋白与磷酸化 NPR-A 的分子稳定结合。该应用的第一个具体目标是通过 MALDI-TOF 质谱法鉴定与 GST-NPR-A 相关的蛋白质。第二个具体目标是使用 Hela 细胞中 ANP 依赖性 cGMP 升高的减少作为 NPR-A 磷酸化减少的指标，对所有已知的人类蛋白激酶进行 siRNA 筛选。第三个具体目标使用凝胶内激酶测定结合质谱法来鉴定 NPR-A 激酶。最后，第四个具体目标利用新型双功能 ATP 类似物将 NPR-A 与其同源激酶交联。 NPR-A 激酶的鉴定预计将为该分子未来治疗高血压、充血性心脏病和骨骼疾病的治疗靶向提供框架。公共健康相关性：利尿钠肽可抑制血压并刺激人体长骨生长。不幸的是，这种内分泌系统的关键调节因子之一——利尿钠肽受体激酶——尚不清楚。这笔赠款的目标是识别这种关键分子，以便它可以成为治疗心血管和骨骼疾病的新药物的目标。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Guanylyl cyclase structure, function and regulation.

DOI：
10.1016/j.cellsig.2011.09.001
发表时间：
2011-12
期刊：
Cellular signalling
影响因子：
4.8
作者：
Potter LR
通讯作者：
Potter LR

A functional screen provides evidence for a conserved, regulatory, juxtamembrane phosphorylation site in guanylyl cyclase a and B.

功能筛选为鸟苷酸环化酶 a 和 B 中保守的、调节性的近膜磷酸化位点提供了证据。

DOI：
10.1371/journal.pone.0036747
发表时间：
2012
期刊：
PloS one
影响因子：
3.7
作者：
Yoder,AndreaR;Robinson,JeridW;Dickey,DeborahM;Andersland,Joshua;Rose,BethA;Stone,MatthewD;Griffin,TimothyJ;Potter,LincolnR
通讯作者：
Potter,LincolnR

Regulation and therapeutic targeting of peptide-activated receptor guanylyl cyclases.