REGULATION OF RHO AND RAC BY MUSCARINIC RECEPTORS

毒蕈碱受体对 RHO 和 RAC 的调节

• 批准号: 6491705
• 负责人: Carol Lucille Williams
• 金额: $ 8.29万
• 依托单位: GUTHRIE FOUNDATION FOR EDUCATION AND RES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-29 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6491705
• 关键词: CHO cells; complementary DNA; enzyme activity; enzyme induction /repression; guanine nucleotide binding protein; guanine nucleotide exchange factors; guanosinetriphosphatases; microinjections; muscarinic receptor; protein kinase C; receptor expression; vascular smooth muscle; western blottings

The proposed research will investigate how M3 muscarinic acetylcholine receptors (M3 mAChR) activate RhoA and Rac1. Although RhoA and Rac1 participate in vital mAChR-mediated processes, very little is known about the regulation of these GTPases by mAChR. We established Chinese hamster ovary (CHO) cells stably co-expressing human M3 mAChR and hemagglutinin-tagged RhoA or Rac1 proteins which have either wildtype, constitutively active, or dominant negative functions. Activation of M3 mAChR alters the association altered by activating M3 mAChR or protein kinase C (PKC), or by microinjecting cDNA or SmgGDS, which is a guanine nucleotide exchange factor (GEF) for RhoA. Based on these and other findings, we hypothesize that M2 mAChR transduce Galpha1- and PKC-dependent signals which cause RhoA and Rac1 to dissociate from negative regulators and associate with specific GEFs. These events cause the translocation and activation of the GTPases. These hypothesis will be tested by identifying regulatory proteins which exhibit altered interactions with the GTPase after M3 mAChR activation. The cells will be microinjected with cDNAs coding for dominant negative mutants of these regulatory proteins, to determine their participation in the M3 mAChR-mediated activation of RhoA and Rac1 (Specific Aim 1). The effects of M3 mAChR activation on the phosphorylation, translocation, and GTPase activities of RhoA and Rac1 will also be characterized. The ability of dominant negative Galphaq mutants or PKC antagonists to inhibit these M3 mAChR-mediated changes in the GTPases will be examined in Specific Aim 2. The hypothesis that M3 mAChR activate the GTPases through signaling which are mAChR subtype-specific, but not cell type-specific will be tested in Specific Aim 3. This will be accomplished by determining whether the changes in the GTPases induced by M3 MaChR activation in CHO cells similarly occur upon activation of M2 mAChR expressed in CHO cells, or upon activation of M3 mAChR expressed in A7r5 vascular smooth muscle cells. These studies will help determine how M3 mAChR activate Rho family members to regulate vital pulmonary and cardiovascular functions.

拟议的研究将研究M3毒蕈碱乙酰胆碱受体（M3 MACHR）如何激活RhoA和Rac1。尽管Rhoa和Rac1参与了重要的MACHR介导的过程，但对MACHR对这些GTPASE的调节知之甚少。我们建立了稳定地共同表达人M3 MACHR和黑绿丁质蛋白标记的RHOA或RAC1蛋白的中国仓鼠卵巢（CHO）细胞，它们具有野生型，构成活性或显性负功能。 M3 MACHR的激活通过激活M3 MACHR或蛋白激酶C（PKC）或微注射cDNA或SMGGD来改变了缔合，这是RhoA的鸟嘌呤核苷酸交换因子（GEF）。基于这些发现和其他发现，我们假设M2 MACHR会导致galpha1-和PKC依赖性信号，这些信号导致RhoA和Rac1与负调节器分离并与特定的GEF相关。这些事件导致GTPases的易位和激活。这些假设将通过鉴定调节蛋白来检验，这些调节蛋白在M3 MACHR激活后与GTPase表现出改变的相互作用。这些细胞将与编码这些调节蛋白的显性阴性突变体的cDNA进行微注射，以确定其参与M3 MACHR介导的RhoA和Rac1的激活（特定的AIM 1）。 M3 MACHR激活对RhoA和Rac1的磷酸化，易位和GTPase活性的影响也将得到表征。在特定的目的2中，将检查主要的负阴性Galphaq突变体或PKC拮抗剂在GTPase中抑制这些M3 MACHR介导的变化的能力。假设M3 MACHR通过MACHR亚型特异性，而不是通过特定的目标进行了测试的MACHR亚型特异性，而在该目标中可以通过MACHR亚型特异性进行测试。在CHO细胞中表达的M2 MACHR激活或在A7R5血管平滑肌细胞中表达的M3 MACHR激活时，CHO细胞中的激活也会发生。这些研究将有助于确定M3 MACHR如何激活Rho家族成员，以调节重要的肺部和心血管功能。