Growth inhibition by the muscarinic M3 acetylcholine receptor:: evidence for p21Cip1/Waf1 involvement in G1 arrest

We have assessed the growth response of Chinese-hamster ovary (CHO) cells to activation of recombinantly expressed G-protein-coupled muscarinic M-2 or M-3 acetylcholine receptors (AChRs). We show that activation of these receptors leads to divergent growth responses: M-2 ACbR activation causes an increase in DNA synthesis, whereas M-3 AChR activation causes a dramatic decrease in DNA synthesis. We have characterized the M-3 AChR-mediated growth inhibition and show that it involves a G(1) phase cell-cycle arrest. Further analysis of this arrest indicates that it involves an increase in expression of the cyclin-dependent kinase (CDK) inhibitor, p21(Cip1/Waf1) (where Cip1 is CDK-interacting protein 1 and Waf1 is wild-type p53-associated fragment 1), in response to M-3 AChR activation. This increase in protein expression leads to an increase in p2(CiP1/Waf1) association with CDK2, a decrease in CDK2 activity and an accumulation of hypophosphorylated retinoblastoma protein. The increased p21(Cip1/Waf1) expression is due, at least in part, to an increase in p21(Cip1/Waf1) mRNA, and receptor-mediated changes in phosphorylation of c-Jun provide a mechanism to account for this p21(Cip1/Waf1) transcriptional regulation. Evaluation of the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase activities has shown striking differences in the profiles of activation of these mitogen-activated protein kinases by the M-2 and M-3 AChRs, and their potential involvement in mediating growth arrest by the M-3 AChR is discussed.