Calcium desensitization in Smooth Muscle

描述（由申请人提供）：平滑肌在各种生理过程中起着重要作用，尽管每种平滑肌的基本功能是相同的，即收缩和舒张，但平滑肌类型之间的机械特性和对激素、神经递质和药物的反应性差异很大。决定平滑肌收缩特性的因素包括质膜性质、信号转导蛋白的比例和互补性、收缩装置本身的组成。这些成分的正常混合物的改变被认为是涉及平滑肌的几种人类疾病的分子基础，包括高血压、支气管痉挛、性功能障碍、胃肠疾病和青光眼。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，将导致治疗这些疾病的更具选择性的疗法。最近完成的人类和小鼠基因组与先进的质谱技术相结合，提供了新的机会，探测细胞中的信号转导途径。在这个建议中，我们将采用蛋白质组学，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，以确定cGMP通过激活环GMP依赖性蛋白激酶（PKG）调节平滑肌松弛的分子机制。各种平滑肌的磷酸化蛋白质组的检查确定了PKG的早期蛋白质靶点的不同子集。在小鼠和人类基因组中鉴定了几种，包括CHASM，一种含有先前未鉴定的基序的新型蛋白质，该基序在平滑肌特异性蛋白质的smoothelin家族中高度保守。当添加到透化平滑肌中时，CHASM以磷酸化依赖性方式引起钙脱敏和松弛。在CHASM基序区域内插入的非保守氨基酸的序列差异程度表明CHASM和平滑蛋白可能是在介导cGMP/PKG的作用中重要的平滑肌特异性蛋白的较大家族的一部分。为了直接检验这一假设，我们已经删除了CHASM基因，并获得了CHASM无效小鼠。

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.