Mapping of single amino acid residues required for selective activation of Gq/11 by the m3 muscarinic acetylcholine receptor.
Each G protein-coupled receptor can interact only with a limited number of the many structurally similar G proteins expressed within a cell. This study was undertaken to identify single amino acids required for selectively coupling the m3 muscarinic acetylcholine receptor to G proteins of the Gq/11 family. To this goal, distinct intracellular segments/amino acids of the m3 receptor were systematically substituted into the structurally closely related m2 muscarinic receptor, which couples to Gi/o proteins, not Gq/11 proteins. The resultant mutant receptors were expressed in COS-7 cells and studied for their ability to induce agonist-dependent stimulation of phosphatidylinositol hydrolysis, a response known to be mediated by G proteins of the Gq/11 class. Using this approach, we were able to identify four amino acids in the second intracellular loop and four amino acids at the C terminus of the third intracellular loop of the m3 muscarinic receptor that are essential for efficient Gq/11 activation. We could demonstrate that these amino acids, together with a short segment at the N terminus of the third intracellular loop, fully account for the G protein coupling preference of the m3 muscarinic receptor. Taken together, our data strongly suggest that only a limited number of amino acids, located on different intracellular regions, are required to determine the functional profile of a given G protein-coupled receptor.
Blin
N
N
Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Yun
J
J
Wess
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D016415:Sequence Alignment
D000596:Amino Acids; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D002217:Carbachol; D019204:GTP-Binding Proteins