Distinct internalization of M2 muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C

Although M-1-M-4 muscarinic acetylcholine receptors (mAChRs) in HEK-293 cells internalize on agonist stimulation, only M-1, M-3, and M-4 but not M-2 mAChRs recycle to the plasma membrane. To investigate the functional consequences of this phenomenon, we compared desensitization and resensitization of M-2 versus M-4 mAChRs. Treatment with 1 mM carbachol for 1 h at 37 degrees C reduced numbers of cell surface M-2 and M-4 mAChRs by 40-50% and M-2 and M-4 mAChR-mediated inhibition of adenylyl cyclase, intracellular Ca2+ concentration ([Ca2+](i)) increases, and phospholipase C (PLC) activation by 60-70%. Receptor-mediated inhibition of adenylyl cyclase and [Ca2+](i) increases significantly resensitized within 3 h. However, M-4 but not M-2 mAChR-mediated PLC activation resensitized. At 16 degrees C, M-2 mAChR-mediated [Ca2+](i) increases and PLC stimulation desensitized to a similar extent as at 37 degrees C. However, at 16 degrees C, where M-2 mAChR internalization is negligible, both M-2 mAChR responses resensitized, demonstrating that M-2 mAChR resensitization proceeds at the plasma membrane. Examination of M-2 mAChR responses following inactivation of cell surface mAChRs by quinuclidinyl benzilate revealed substantial receptor reserve for coupling to [Ca2+](i) increases but not to PLC. We conclude that M-2 mAChR internalization induces long-lasting PLC desensitization predominantly because receptor loss is not compensated for by receptor recycling or receptor reserve.