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.