title: Identification of an intracellular tyrosine residue critical for muscarinic receptor-mediated stimulation of phosphatidylinositol hydrolysis.
abstract: Several lines of evidence suggest that the N-terminal portion of the third cytoplasmic loop (i3) of muscarinic and other G protein-coupled receptors is of pivotal importance for G protein recognition and activation. The present study was designed to identify specific amino acids within this domain required for muscarinic receptor-induced activation of G proteins mediating stimulation of phosphatidylinositol (PI) hydrolysis. Among the five mammalian muscarinic receptors (m1-m5), only the m1, m3, and m5 receptors are efficiently coupled to this second messenger pathway. Initially, we created a series of rat m3 receptor mutants in which short segments in the N terminus of the i3 loop were replaced with the corresponding m2 receptor sequences. The effect of these substitutions on m3 receptor-mediated stimulation of PI hydrolysis was studied in transiently transfected COS-7 cells. We found that a stretch of 4 amino acids (Arg252-Ile253-Tyr254-Lys255) located at the beginning of the i3 domain of the m3 muscarinic receptor is critically involved in receptor-mediated stimulation of PI hydrolysis. Further mutational analysis of this 4-amino acid segment by single amino acid substitutions demonstrated that only Tyr254 is essential for efficient activation of the PI pathway. This tyrosine residue is conserved among all PI-coupled muscarinic receptors as well as in many other biogenic amine and glycoprotein hormone receptors, suggesting that it may also play an important functional role in other G protein-coupled receptors.
authors.affiliation: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
authors.forename: K
authors.initials: K
authors.lastname: Blüml
authors.forename: E
authors.initials: E
authors.lastname: Mutschler
authors.forename: J
authors.initials: J
authors.lastname: Wess
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000032:analysis*
chemical_list: D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D014443:Tyrosine