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 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.
Kohn
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EC
Signal Transduction and Prevention Unit, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Alessandro
R
R
Probst
J
J
Jacobs
W
W
Brilley
E
E
Felder
C C
CC
D000067956:Adenylyl Cyclase Inhibitors; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008545:Melanoma; D008969:Molecular Sequence Data; D019832:N-Methylscopolamine; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D043642:Receptor, Muscarinic M5; D011976:Receptors, Muscarinic / Q000032:analysis / Q000096:biosynthesis / Q000502:physiology*; D012602:Scopolamine Derivatives / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000067956:Adenylyl Cyclase Inhibitors; D017931:DNA Primers; D043642:Receptor, Muscarinic M5; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D005576:Colforsin; D016718:Arachidonic Acid; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D000242:Cyclic AMP; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium; D019832:N-Methylscopolamine