IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF A M5 MUSCARINIC RECEPTOR IN A2058 HUMAN-MELANOMA CELLS - COUPLING TO INHIBITION OF ADENYLYL-CYCLASE AND STIMULATION OF PHOSPHOLIPASE-A2

We report the identification and biochemical characterization of an en dogenous m5 muscarinic acetylcholine receptor (mAChR) in the A2058 hum an melanoma cell line. This is the first demonstration of a m5AChR out side the central nervous system. The unusual effector coupling of this endogenous m5AChR is presented. The coding region amplified by polyme rase chain reaction was identical to the known m5AChR sequence. Bindin g studies indicated a K-d of 99 +/- 6 pM and a B-max of 45 +/- 4 fmol/ mg membrane protein. This m5AChR coupled to stimulation of arachidonic acid release and to a 50% inhibition of forskolin-stimulated cAMP acc umulation. The inhibition of cAMP production was insensitive to pertus sis toxin treatment, but was dependent upon extracellular calcium. In contrast to the odd mAChR pattern, no cAMP was produced in response 60 carbachol (CC) stimulation. Moreover, no release of inositol phosphat es could be measured after CC treatment despite the presence of at lea st 2 phospholipase C isoforms in A2058 cells. CC-stimulated arachidoni c acid release (EC(50) = 17.8 +/- 0.1 mu M) was dependent upon externa l Ca2+, with marked reduction after coincubation with EGTA, Co2+, Or h igh doses of verapamil (IC50 = 166 mu M) or diltiazem (IC50 = 243 mu M ). Brief exposure to phorbol la-myristate 13-acetate augmented CC-stim ulated arachidonic acid release, whereas prolonged phorbol 12-myristat e 13-acetate treatment resulted in down-regulation of release. Activat ion of the m5AChR resulted in Ca2+ influx that was attenuated by musca rinic antagonism and removal of extracellular Ca2+. A2058 cells expose d to CC had no alteration of cell shape or growth potential in monolay er culture, however, a statistically significant reduction in density- independent growth was observed over the range of CC concentrations fr om 0.1 to 100 mu M. This endogenous m5AChR has a novel signal transduc tion coupling profile and receptor activation reduces clonogenic poten tial.