A deletion mutation in the third cytoplasmic loop of the mouse m1 muscarinic acetylcholine receptor unmasks cryptic G-protein binding sites.
Mutations were introduced in the highly conserved carboxyl-terminal region of the third cytoplasmic loop of the mouse m1 muscarinic acetylcholine receptor (mAChR) gene by site-directed mutagenesis. The effects of these mutations on ligand binding and on mAChR coupling to phosphoinositide turnover have been examined following expression in mouse Y1 adrenal carcinoma cells, Chinese hamster ovary (CHO) cells, and Rat-2 fibroblasts. Point mutations in the region proximal to the sixth transmembrane domain had no effect on antagonist binding but did result in decreased agonist affinity. A deletion of four amino acids in the same region effectively uncoupled the receptor from phosphoinositol turnover in Y1 adrenal carcinoma cells but had no effect in either CHO cells or Rat-2 fibroblasts. Differential sensitivity to pertussis toxin indicates that the m1 mAChR can interact with multiple G-proteins in CHO cells and Rat-2 cells via distinct recognition sites on the receptor. These data demonstrate multiple G-proteins can interact with an individual receptor, that the same receptor may couple to different second messenger pathways, and that these responses can vary in a cell type-specific manner.
Shapiro
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RA
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Palmer
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D
Cislo
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010716:Phosphatidylinositols / Q000378:metabolism; D017354:Point Mutation; D051381:Rats; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D008024:Ligands; D009838:Oligodeoxyribonucleotides; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins