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.
Burdon
Drew
D
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.
Patel
Rajnikant
R
Challiss
R A John
RA
Blank
Jonathan L
JL
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