REGULATION OF RHO AND RAC BY MUSCARINIC RECEPTORS

这项拟议的研究将调查M3毒碱样乙酰胆碱受体(M3mAChR)如何激活RhoA和rac1。虽然RhoA和rac1参与了重要的mAChR介导的过程，但对mAChR对这些GTP酶的调控知之甚少。我们建立了稳定共表达人M3mAChR和血凝素标记的RhoA或rac1蛋白的中国仓鼠卵巢(CHO)细胞，这些蛋白具有野生型、成分活性或显性负功能。M3mAChR的激活改变了通过激活M3mAChR或蛋白激酶C(PKC)，或通过微量注射cDNAs或SmgGDS而改变的关联，SmgGDS是RhoA的鸟嘌呤核苷酸交换因子(Egf)。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，导致RhoA和rac1从负调控因子中解离，并与特定的GEF相关联。这些事件导致GTP酶的易位和激活。这些假说将通过鉴定在M3mAChR激活后与GTPase相互作用发生变化的调节蛋白来检验。这些细胞将被显微注射编码这些调节蛋白的显性负突变的cDNA，以确定它们参与M3mAChR介导的RhoA和rac1的激活(特异性目标1)。M3mAChR激活对RhoA和rac1的磷酸化、易位和GTP酶活性的影响也将被表征。将在特定目标2中检验显性负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。将在特定目标3中检验M3 mAChR通过mAChR亚型特异性而不是细胞类型特异性的信号激活GTP酶的假设。这将通过确定M3 MaChR激活在CHO细胞中诱导的GTPase变化是否类似于在CHO细胞中表达的M2 mAChR的激活，或者在A7r5血管平滑肌细胞中表达的M3 mAChR的激活来实现。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.