Nitric Oxide Signaling Mechanisms in Vascular Cells

描述（由申请人提供）：本竞争性更新申请探索了通过一氧化氮/cGMP/cGMP依赖性蛋白激酶（PKG）途径调节血管平滑肌细胞（VSMC）舒张的分子机制。血管平滑肌细胞的收缩状态是由控制放线肌球蛋白收缩的调节性肌球蛋白轻链（MLC）磷酸化动态调节的。[Ca 2 +] in增加激活Ca 2 +/钙调素依赖性MLC激酶（MLCK），使MLC磷酸化，引起VSMC收缩。相反，肌球蛋白磷酸酶（PP1M）活性的增加，例如响应于内源性血管扩张剂一氧化氮（NO）而发生的，使MLC去磷酸化，引起松弛。在过去的十年中，我们一直致力于通过识别新的PKGI α靶点并测试其功能作用来了解调节血管舒张的分子机制。PKGI通过多种机制诱导VSMC松弛，所述机制涉及直接调节（i）放线肌球蛋白收缩应力纤维松弛和（ii）GPCR介导的[Ca +2]动员的抑制的底物的磷酸化。我们已经表征了几种在VSMC松弛中重要的PKG1 α-靶蛋白相互作用，包括与（a）PP1 M，（B）G蛋白信号传导2（RGS2）的调节剂，和（c）含同源结构域的蛋白1型，或FHOD 1，一种刺激VSMC应力纤维形成的多功能蛋白的相互作用。所有这3种途径均由PKG1 α通过其N-末端亮氨酸拉链（LZ）相互作用结构域结合/激活这些靶点介导，支持PKG1 α通过PKG1 α LZ结构域介导的PKG1 α-靶蛋白相互作用在正常VSMC张力和生物学调节中发挥核心作用的假设。为了探索这一假设，我们现在已经创建了表达蛋白质-蛋白质相互作用缺陷的LZ突变体（LZM）PKG1 α但在其他方面与野生型PKG1 α相同的小鼠。LZM "基因敲入"小鼠具有支持上述假设的令人兴奋的表型：LZM VSMC具有过量的放线菌球蛋白应力纤维; LZM小鼠血管异常松弛;并且完整的PKGI alpha LZM小鼠是高血压的，这为探索本申请的中心假设提供了令人兴奋的工具。我们建议调查：PKGIa抑制VSMC放线肌球蛋白应激纤维形成和收缩的分子机制，重点是FHOD1-PKG LZ结构域相互作用（SA 1）; PKGIa抑制GPCR介导的[Ca +2]动员，重点是LZ介导的相互作用调节（i）RGS2-GPCR-IP 3途径和（ii）血栓素受体（SA 2）;以及PKG1 α LZ靶向结构域在完整血管和动物中的血管调节中的功能作用，使用WT和LZM小鼠（SA3）。这些研究有望增加我们对血管张力调节的理解，并有可能促进人类心血管疾病的诊断和治疗。

DESCRIPTION (provided by applicant): This competitive renewal application explores molecular mechanisms regulating vascular smooth muscle cell (VSMC) relaxation by the nitric oxide/cGMP/cGMP-dependent protein kinase (PKG) pathway. VSMC contractile state is dynamically regulated by phosphorylation of the regulatory myosin light (MLC) chains controlling actinomyosin contraction. Increases in [Ca2+]in activate the Ca 2+/calmodulin-dependent MLC kinase (MLCK), which phosphorylates MLC, causing VSMC contraction. Conversely, increases in myosin phosphatase (PP1M) activity, such as occurs in response to the endogenous vasodilator nitric oxide (NO), dephosphorylate MLC, causing relaxation. Over the past decade, we have focused on understanding molecular mechanisms regulating vascular relaxation by identifying new PKGIalpha targets and testing their functional role. PKGI induces VSMC relaxation by multiple mechanisms involving phosphorylation of substrates that directly regulate (i) actinomyosin contractile stress fiber relaxation and (ii) inhibition of GPCR- mediated [Ca+2]in mobilization. We have characterized several PKGIa-target protein interactions important in VSMC relaxation, including interactions with (a) PP1M, (b) the regulator of G protein signaling 2 (RGS2), and (c) the formin homology domain-containing protein type 1, or FHOD1, a multi-functional protein that stimulates VSMC stress fiber formation. All 3 of these pathways are mediated by PKGIalpha binding/activation of these targets via its N-terminal leucine zipper (LZ) interaction domain, supporting the hypothesis that PKGIalpha has a central role in the regulation of normal VSMC tone and biology via PKGIalpha-target protein interactions mediated by the PKGIalpha LZ domain. To explore this hypothesis, we now have created mice expressing a LZ mutant (LZM) PKGIalpha defective for protein-protein interactions, but otherwise identical to wild-type PKGIa. LZM 'knock-in' mice have an exciting phenotype supporting the above hypothesis: LZM VSMC have excessive actinomyosin stress fibers; LZM mouse blood vessels relax abnormally; and intact PKGIalpha LZM mice are hypertensive, providing exciting tools with which to explore the central hypothesis of this application. We propose to investigate: the molecular mechanisms by which PKGIa inhibits VSMC actinomyosin stress fiber formation and contraction, with a focus on the FHOD1-PKG LZ domain interaction (SA1); PKGIa inhibition of GPCR-mediated [Ca+2]in mobilization, with a focus on LZ-mediated interactions regulating the (i) RGS2-GPCR-IP3 pathway and (ii) thromboxane receptor (SA2); and the functional role of the PKGIalpha LZ targeting domain in vascular regulation in intact blood vessels and animals, using WT and LZM mice (SA3). These studies are expected to increase our understanding of vascular tone regulation, with the potential to advance the diagnosis and therapy of human cardiovascular diseases.