Calcium desensitization in Smooth Muscle

平滑肌钙脱敏

• 批准号: 7103794
• 负责人: TIMOTHY A HAYSTEAD
• 金额: $ 31.85万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7103794
• 关键词: biological signal transduction; cGMP dependent protein kinase; calcium flux; confocal scanning microscopy; cyclic GMP; electron microscopy; gene expression profiling; gene targeting; genetically modified animals; immunocytochemistry; laboratory mouse; laboratory rabbit; muscle function; muscle proteins; organ culture; phosphoproteins; phosphorylation; protein localization; protein protein interaction; protein structure function; proteomics; site directed mutagenesis; smooth muscle

DESCRIPTION (provided by applicant): Smooth muscle plays an essential role in a wide variety of physiological processes, and although the basic function of every smooth muscle is the same, to contract and relax, the mechanical properties and responsiveness to hormones, neurotransmitters and drugs varies greatly between smooth muscle types. Factors that dictate the contractile characteristics of smooth muscle include plasma membrane properties, ratio and compliment of signal transducing proteins, the composition of the contractile apparatus itself. Alteration in the normal blend of these components is thought to underlie the molecular basis of several human diseases that involve smooth muscle including hypertension, bronco spasm, sexual dysfunction, gastrointestinal disorders and glaucoma. It is our hypothesis that by studying the molecular processes by which individual smooth muscles normally respond to stimulation will lead to more selective therapies to treat these disorders. The recent completion of the human and mouse genomes in combination with advanced techniques in mass spectrometry affords new opportunities for probing signal transduction pathways in cells. In this proposal we will employ a unique combination of proteomics, muscle physiology, molecular biology, immuno-histochemistry and mouse genetics to determine the molecular mechanisms by which cGMP through the activation of cyclic GMP dependant protein kinase (PKG) regulates smooth muscle relaxation. Examination of phosphoproteomes of various smooth muscles identified a distinct subset of early protein targets for PKG. Several were identified in the mouse and human genome, including CHASM, a novel protein containing a previously unidentified motif that is highly conserved in the smoothelin family of smooth muscle specific proteins. When added to permeabilized smooth muscles, CHASM causes calcium desensitization and relaxation in a phosphorylation dependant manner. The degree of sequence divergence of the intervening non-conserved amino acids within the CHASM motif region suggests that CHASM and the smoothelins may be part of a larger family of smooth muscle specific proteins that are important in the mediating the actions of cGMP/PKG. To directly test this hypothesis we have deleted the CHASM gene and obtained CHASM null mice.

描述(申请人提供)：平滑肌在多种生理过程中扮演着重要的角色，虽然每种平滑肌的基本功能是相同的，但收缩和松弛的机械性能和对激素、神经递质和药物的反应在不同的平滑肌类型之间存在很大差异。决定平滑肌收缩特性的因素包括质膜特性、信号转导蛋白的比例和互补、收缩装置本身的组成。这些成分正常混合的改变被认为是几种人类疾病的分子基础，这些疾病涉及到平滑肌，包括高血压、支气管痉挛、性功能障碍、胃肠道疾病和青光眼。我们的假设是，通过研究个体平滑肌对刺激的正常反应的分子过程，将导致更多的选择性疗法来治疗这些疾病。最近人类和小鼠基因组的完成与先进的质谱学技术相结合，为探索细胞中的信号转导途径提供了新的机会。在这项研究中，我们将运用蛋白质组学、肌肉生理学、分子生物学、免疫组织化学和小鼠遗传学的独特组合来确定cGMP通过激活cGMP依赖的蛋白激酶(PKG)来调节平滑肌松弛的分子机制。对不同平滑肌的磷酸蛋白质组的检查确定了PKG的早期蛋白靶标的一个独特的子集。在小鼠和人类基因组中发现了几种蛋白质，包括Chasm，一种新的蛋白质，包含一个以前未确定的基序，在平滑蛋白家族的平滑肌特异性蛋白质中高度保守。当加入到通透性的平滑肌肉中时，裂隙以一种磷酸化依赖的方式引起钙脱敏和松弛。裂隙基序区域内相互作用的非保守氨基酸的序列差异程度表明，裂隙和平滑蛋白可能是一个更大的平滑肌特异蛋白家族的一部分，这些蛋白质在介导cGMP/PKG的作用中起重要作用。为了直接验证这一假说，我们删除了该基因，并获得了缺失该基因的小鼠。