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 M₂ or M₃ acetylcholine receptors (AChRs). We show that activation of these receptors leads to divergent growth responses: M₂ AChR activation causes an increase in DNA synthesis, whereas M₃ AChR activation causes a dramatic decrease in DNA synthesis. We have characterized the M₃ AChR-mediated growth inhibition and show that it involves a G₁ 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 M₃ AChR activation. This increase in protein expression leads to an increase in p21^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 M₂ and M₃ AChRs, and their potential involvement in mediating growth arrest by the M₃ AChR is discussed.