Abstract
We report the identification and biochemical characterization of an endogenous m5 muscarinic acetylcholine receptor (mAChR) in the A2058 human melanoma cell line. This is the first demonstration of a m5AChR outside the central nervous system. The unusual effector coupling of this endogenous m5AChR is presented. The coding region amplified by polymerase chain reaction was identical to the known m5AChR sequence. Binding studies indicated a Kd of 99 +/- 6 pM and a Bmax 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 accumulation. The inhibition of cAMP production was insensitive to pertussis toxin treatment, but was dependent upon extracellular calcium. In contrast to the odd mAChR pattern, no cAMP was produced in response to carbachol (CC) stimulation. Moreover, no release of inositol phosphates could be measured after CC treatment despite the presence of at least 2 phospholipase C isoforms in A2058 cells. CC-stimulated arachidonic acid release (EC50 = 17.8 +/- 0.1 microM) was dependent upon external Ca2+, with marked reduction after coincubation with EGTA, Co2+, or high doses of verapamil (IC50 = 166 microM) or diltiazem (IC50 = 243 microM). Brief exposure to phorbol 12-myristate 13-acetate augmented CC-stimulated arachidonic acid release, whereas prolonged phorbol 12-myristate 13-acetate treatment resulted in down-regulation of release. Activation of the m5AChR resulted in Ca2+ influx that was attenuated by muscarinic antagonism and removal of extracellular Ca2+. A2058 cells exposed to CC had no alteration of cell shape or growth potential in monolayer culture, however, a statistically significant reduction in density-independent growth was observed over the range of CC concentrations from 0.1 to 100 microM. This endogenous m5AChR has a novel signal transduction coupling profile and receptor activation reduces clonogenic potential.
Highlights
From the Signal Transduction and Prevention Unit, Laboratory of Pathology, National Cancer Institute and the §Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892
The full coding length of the A2058 AChR has been amplified, subcloned, and sequenced and was found to be homologous to that of the hu-m5AChR. These results demonstrate that the muscarinic acetylcholine receptor (mAChR) on the A2058 cells is genetically a m5 muscarinic receptor
Removal of extracellular calcium from the reaction mixture resulted in abrogation of 50% of forskolin-stimulated cAMP production. While this effect was observed independently of concomitant exposure to CC, it abrogated the CCsensitive component (Fig. 3B). These results suggest that the CC-mediated inhibition of cAMP production may be due to mAChR-stimulated influx of calcium and that there may be multiple isotypes of adenylyl cyclase activated by forskolin in these cells
Summary
Vol 271, No 29, Issue of July 19, pp. 17476 –17484, 1996 Printed in U.S.A. Elise C. The even-numbered receptors (m2 and m4) are associated with inhibition of receptor-mediated adenylyl cyclase activation, augmentation of arachidonic acid release in response to stimulation of other receptors [6, 16, 17], and stimulation of inositol phosphate release [18] These patterns of signal transduction of the receptor family subtypes are consistent across tissues, species, and state of malignant transformation [1, 5, 6, 17]. This m5 receptor is present in extremely low abundance and mediates the inhibition of anchorage independent proliferation It is functionally associated with a unique combination of signal transduction pathway coupling including calcium mobilization, release of arachidonic acid, inhibition of the generation of cAMP, and lack of inositol polyphosphate production
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
