MUSCARINIC ACETYLCHOLINE-RECEPTOR DOWN-REGULATION LIMITS THE EXTENT OF INHIBITION OF CELL-CYCLE PROGRESSION IN CHINESE-HAMSTER OVARY CELLS

Cellular desensitization is believed to be important for growth contro l but direct evidence is lacking. In the current study we compared eff ects of wild-type and down-regulation-resistant mutant m3 muscarinic r eceptors on Chinese hamster ovary (CHO-K1) cell desensitization, proli feration, and transformation. We found that down-regulation of m3 musc arinic acetylcholine receptors was the principal mechanism of desensit ization of receptor-activated inositol phosphate phospholipid hydrolys is in these cells. Activation of wild-type and mutant receptors inhibi ted anchorage-independent growth as assayed by colony formation in aga r. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of e ither receptor also initially inhibited anchorage-dependent cell proli feration in randomly growing populations. Rates of DNA synthesis and c ell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameter s indicated that cell cycle progression was inhibited at transitions f rom G(1) to S and G(2)/M to G(1) phases. However, mutant receptor effe cts on anchorage-dependent growth were sustained, whereas wild-type re ceptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor bl ocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-depend ent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these poin ts were eliminated by wild-type receptor down-regulation while the oth er was not. These results directly demonstrate that desensitization me chanisms can act as principal determinants of cellular growth response s.