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Elevated Rac1 Activity Changes the M₃ Muscarinic Acetylcholine Receptor-Mediated Inhibition of Proliferation to Induction of Cell Death

Molecular Pharmacology Laboratory, Guthrie Research Institute, Sayre, Pennsylvania

Although muscarinic acetylcholine receptors (mAChRs) regulate proliferation in many cell types, the signaling pathways involved are unclear. The participation of the small GTPases Rac1 and RhoA in M₃ mAChR-mediated inhibition of proliferation was investigated by activating M₃ mAChRs stably transfected in Chinese hamster ovary cells stably coexpressing hemagglutinin (HA)-tagged wild-type or mutant Rac1 or RhoA proteins. Activation of M₃ mAChRs activates both Rac1 and RhoA and inhibits cell proliferation in all cell lines tested. mAChR-mediated inhibition of proliferation is diminished in cells expressing dominant-negative HA-Rac1^Asn17 (m3DNRac) but is enhanced in cells expressing HA-Rac1 (m3WTRac) or constitutively active HA-Rac^Val12 (m3CARac). The activation of mAChRs in m3WTRac and m3CARac cells also induces apoptosis. Expression of wild-type or mutant RhoA proteins does not alter mAChR-mediated inhibition of proliferation. mAChR-induced inhibition of proliferation is abrogated in all cell lines when G_q/11 signaling is terminated by transient expression of the COOH-terminal fragment of phospholipase C (PLC-1ct), the NH₂-terminal fragment of G protein-coupled receptor kinase, or the regulator of G protein signaling 2. Pretreatment of all cells expressing wild-type or mutant Rac1 proteins with edelfosine, a phosphatidylinositol-specific PLC inhibitor, or Go 6976, which inhibits conventional protein kinase C (PKC) isoforms, diminishes the M₃ mAChR's ability to inhibit proliferation. Our results identify G_q/11, PLC, and PKC as participants in the M₃ mAChR-mediated inhibition of cell proliferation. These findings indicate that in the context of high Rac1 activity, but not RhoA activity, M₃ mAChR-mediated activation of these participants triggers cell death.

Address correspondence to: Carol L. Williams, Ph.D., Molecular Pharmacology Laboratory, One Guthrie Square, Sayre, PA 18840. E-mail: Williams_carol{at}guthrie.org