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 m1, receptor undergoes agonist-induced sequestration. Both receptors exhibit internalization when expressed in Y1 cells. These results suggest that the m1 and m2 receptors use distinct cellular mechanisms or pathways for agonist-induced internalization and that JEG-3 cells are deficient in the mechanism or pathway used by the m1 receptor. Transfection experiments with chimeric receptors indicate that the specificity for agonist-induced internalization for the m2 receptor lies in the carboxyl-terminal fifth of the receptor. The intracellular carboxyl-terminal tail of the m2 receptor is neither sufficient nor required for the m2-specific sequestration. Site-directed mutagenesis demonstrates that two amino acids in the carboxyl-terminal end of the third cytoplasmic loop of the m2 receptor are required for sequestration in JEG-3 cells. In addition, the sixth transmembrane domain, which is adjacent to this cytoplasmic domain, is also required. Thus, m2-specific agonist-induced sequestration requires sequences both in the carboxyl-terminal end of the third cytoplasmic loop and the adjacent transmembrane domain.
Goldman
P S
PS
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750, USA.
Schlador
M L
ML
Shapiro
R A
RA
Nathanson
N M
NM
D000595:Amino Acid Sequence; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D018721:Muscarinic Agonists / Q000494:pharmacology*; D019832:N-Methylscopolamine; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D015183:Restriction Mapping; D012602:Scopolamine Derivatives / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D018721:Muscarinic Agonists; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D012602:Scopolamine Derivatives; D005576:Colforsin; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D008156:Luciferases; D019832:N-Methylscopolamine