REGULATION OF RHO AND RAC BY MUSCARINIC RECEPTORS

拟研究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，但关于这些gtpase受mAChR调控的了解甚少。我们建立了中国仓鼠卵巢（CHO）细胞，稳定地共表达人M3 mAChR和血凝素标记的RhoA或Rac1蛋白，这些蛋白具有野生型、组成活性或显性负功能。M3 mAChR的激活改变了通过激活M3 mAChR或蛋白激酶C (PKC)，或通过微注射cDNA或SmgGDS （RhoA的鸟嘌呤核苷酸交换因子（GEF））改变的关联。基于这些和其他发现，我们假设M2 mAChR转导Galpha1和pkc依赖的信号，这些信号导致RhoA和Rac1与负调节因子分离并与特定的gef相关联。这些事件导致gtpase的易位和激活。这些假设将通过鉴定在M3 mAChR激活后与GTPase表现出改变的相互作用的调节蛋白来验证。将这些细胞微注射编码这些调节蛋白显性阴性突变体的cdna，以确定它们参与M3 machr介导的RhoA和Rac1的激活（Specific Aim 1）。M3 mAChR激活对RhoA和Rac1的磷酸化、易位和GTPase活性的影响也将被表征。显性阴性Galphaq突变体或PKC拮抗剂抑制M3 machr介导的gtpase变化的能力将在Specific Aim 2中进行研究。M3 mAChR通过信号通路激活gtpase的假设是mAChR亚型特异性的，而不是细胞类型特异性的，将在Specific Aim 3中进行验证。这将通过确定CHO细胞中M3 MaChR激活诱导的gtpase的变化是否同样发生在CHO细胞中表达的M2 MaChR激活时，还是在A7r5血管平滑肌细胞中表达的M3 MaChR激活时。这些研究将有助于确定M3 mAChR如何激活Rho家族成员来调节重要的肺和心血管功能。

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.