Calcium desensitization in Smooth Muscle

描述（由申请人提供）：平滑肌在多种生理过程中起着至关重要的作用，尽管每一种平滑肌的基本功能都是相同的，即收缩和放松，但不同类型的平滑肌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特性、信号转导蛋白的比例和补充、收缩装置本身的组成。这些成分正常混合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、野马痉挛、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治疗这些疾病。最近完成的人类和小鼠基因组与先进的质谱技术相结合，为探测细胞中的信号转导途径提供了新的机会。在这个提议中，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来确定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。对各种平滑肌磷酸化蛋白组的研究发现了PKG早期蛋白靶点的不同亚群，其中一些在小鼠和人类基因组中被发现，包括CHASM，这是一种新的蛋白，包含一个先前未被识别的基板，在平滑肌特异性蛋白的平滑蛋白家族中高度保守。当加入到渗透平滑肌时，CHASM以磷酸化依赖的方式引起钙脱敏和松弛。CHASM基序区域内非保守氨基酸的序列差异程度表明，CHASM和平滑蛋白可能是平滑肌特异性蛋白大家族的一部分，在介导cGMP/PKG的作用中起重要作用。为了直接验证这一假设，我们删除了CHASM基因，获得了CHASM null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.