Identifiction of the natriuretic peptide receptor kinase

描述(申请人提供)：在美国，40%的死亡是由心血管疾病引起的，估计每年的成本超过4000亿美元。被称为利钠肽的内源性激素可以对抗心血管疾病。心钠素(ANP)和B型利钠肽(BNP)是在血压升高时从心脏释放出来的。它们结合钠尿肽受体-A(NPR-A)，通过合成细胞内信号分子cGMP来降低血压和抑制心肌肥厚。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，抑制血管松弛。NPR-A和NPR-B的鸟苷酸环化酶活性不仅受利钠肽结合的控制，还受调节胞内高度保守的丝氨酸和苏氨酸残基上共价结合的磷酸盐的酶的控制。重要的是，磷酸化是受体激活所必需的，去磷酸化是受体失活的中介。我们的长期目标是了解钠尿肽受体在健康和疾病中控制的分子基础。这项应用的目的是确定使NPR-A磷酸化的激酶。我们的中心假设是，受体磷酸化是依赖配体的NPR-A激活所必需的，因此，对于心钠素信号转导是必不可少的。每个特定的目的都描述了一种识别利钠肽受体激酶的独特方法。我们已经证明，融合到NPR-A胞内结构域的谷胱甘肽-S转移酶(GST)蛋白与磷酸化NPR-A的分子稳定地结合在一起。这项应用的第一个特定目标是通过MALDI-TOF质谱学鉴定与GST-NPR-A相关的蛋白质。第二个具体目的是对所有已知的人类蛋白激酶进行siRNA筛选，使用Hela细胞中ANP依赖的cGMP升高的减少作为NPR-A磷酸化减少的指标。第三个特定目的是使用凝胶内激酶分析结合质谱学来鉴定NPR-A激酶。最后，第四个特定目的是利用一种新的双功能ATP类似物将NPR-A与其同源激酶交联。NPR-A激酶的鉴定有望为该分子未来治疗高血压、充血性心脏病和骨骼疾病的靶向治疗提供框架。与公共健康相关：利钠肽可抑制人体血压并刺激长骨生长。不幸的是，这一内分泌系统的关键调节因子之一--利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，以便将其作为治疗心血管和骨骼疾病的新药的靶点。

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.