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, p21Cip1/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 p21Cip1/Waf1 association with CDK2, a decrease in CDK2 activity and an accumulation of hypophosphorylated retinoblastoma protein. The increased p21Cip1/Waf1 expression is due, at least in part, to an increase in p21Cip1/Waf1 mRNA, and receptor-mediated changes in phosphorylation of c-Jun provide a mechanism to account for this p21Cip1/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.
Growth inhibition by the muscarinic M3 acetylcholine receptor: evidence for p21Cip1/Waf1 involvement in G1 arrest
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Drew BURDON, Rajnikant PATEL, R.A. John CHALLISS, Jonathan L. BLANK; Growth inhibition by the muscarinic M3 acetylcholine receptor: evidence for p21Cip1/Waf1 involvement in G1 arrest. Biochem J 15 October 2002; 367 (2): 549–559. doi: https://doi.org/10.1042/bj20020446
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