Growth inhibition by the muscarinic M(3) acetylcholine receptor: evidence for p21(Cip1/Waf1) involvement in G(1) 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) AChR 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 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 M(2) and M(3) AChRs, and their potential involvement in mediating growth arrest by the M(3) AChR is discussed.
Department of Cell Physiology and Pharmacology, University of Leicester School of Medicine, Medical Sciences Building, University Road, P.O. Box 138, Leicester LE1 9HN, U.K.
Drew
D
Burdon
Rajnikant
R
Patel
R A John
RA
Challiss
Jonathan L
JL
Blank
D000818:Animals; D042846:CDC2-CDC28 Kinases; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D018679:Cholinergic Agonists / Q000494:pharmacology; D006224:Cricetinae; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism
C493956:CDKN1A protein, human; D018679:Cholinergic Agonists; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D002217:Carbachol; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases