REGULATION OF RHO AND RAC BY MUSCARINIC RECEPTORS

这项研究将研究M3毒蕈碱乙酰胆碱受体（M3 mAChR）如何激活RhoA和Rac 1。虽然RhoA和Rac1参与重要的mAChR介导的过程，很少有人知道这些GTP酶的调节mAChR。我们建立了中国仓鼠卵巢（CHO）细胞稳定共表达人M3 mAChR和血凝素标记的RhoA或Rac1蛋白，具有野生型，组成型活性，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）或通过显微注射cDNA或SmgGDS（其是RhoA的鸟嘌呤核苷酸交换因子（GEF））而改变的缔合。基于这些和其他发现，我们假设M2 mAChR转导Galpha1和PKC依赖性信号，导致RhoA和Rac 1与负调节因子分离并与特定的GEF相关。这些事件引起GTP酶的易位和活化。这些假设将通过鉴定在M3 mAChR激活后与GTdR的相互作用发生改变的调节蛋白来检验。将用编码这些调节蛋白的显性负突变体的cDNA显微注射细胞，以确定它们参与M3 mAChR介导的RhoA和Rac 1活化（特异性Aim 1）。还将表征M3 mAChR活化对RhoA和Rac1的磷酸化、易位和GTdR活性的影响。将在特异性目的2中检查显性阴性Galphaq突变体或PKC拮抗剂抑制这些M3 mAChR介导的GTP酶变化的能力。M3 mAChR通过mAChR亚型特异性但非细胞类型特异性的信号传导激活GTP酶的假设将在特定目标3中进行检验。这将通过确定CHO细胞中M3 MaChR活化诱导的GTP酶变化是否类似地发生在CHO细胞中表达的M2 mAChR活化后或A7r5血管平滑肌细胞中表达的M3 mAChR活化后来实现。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.