IDENTIFICATION OF A REGION REQUIRED FOR SUBTYPE-SPECIFIC AGONIST-INDUCED SEQUESTRATION OF THE M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR

When the m1 and m2 muscarinic acetylcholine receptors are transiently expressed in JEG-3 cells, the m2, but not the mi, receptor undergoes a gonist-induced sequestration. Both receptors exhibit internalization w hen expressed in Y1 cells. These results suggest that the m1 and m2 re ceptors use distinct cellular mechanisms or pathways for agonist-induc ed internalization and that JEG-3 cells are deficient in the mechanism or pathway used by the m1 receptor. Transfection experiments with chi meric receptors indicate that the specificity for agonist-induced inte rnalization for the m2 receptor lies in the carboxyl-terminal fifth of the receptor. The intracellular carboxyl-terminal tail of the m2 rece ptor is neither sufficient nor required for the m2-specific sequestrat ion. Site-directed mutagenesis demonstrates that two amino acids in th e carboxyl-terminal end of the third cytoplasmic loop of the m2 recept or are required for sequestration in JEG-3 cells. In addition, the six th transmembrane domain, which is adjacent to this cytoplasmic domain, is also required. Thus, m2-specific agonist-induced sequestration req uires sequences both in the carboxyl-terminal end of the third cytopla smic loop and the adjacent transmembrane domain.