PMID- 8226735 OWN - NLM STAT- MEDLINE DCOM- 19931201 LR - 20210210 IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 268 IP - 30 DP - 1993 Oct 25 TI - Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling. PG - 22273-6 AB - Signal transduction of the heptahelical G protein-coupled receptors (GPCRs) involves multiple receptor domains, but a universal consensus domain for coupling has not yet been defined. Alanine mutagenesis scanning was performed on the intracellular loops and the COOH tail of the human muscarinic cholinergic receptor (Hm1) to identify coupling domains. Stimulation of phosphatidylinositol (PI) turnover was determined after transfection of the alanine mutants into U293 human embryonic kidney cells. Alanine substitutions in four regions (loops i1, i2, and NH2 and COOH junctions of i3) impaired coupling efficiency by approximately 50% or more, but the strongest reduction (> 80%) resulted from alanine replacement of a single amino acid, leucine 131. This residue is located in the middle of the second intracellular loop (i2), within the highly conserved GPCR motif (DRYXXV(I)XXPL). The position equivalent to Leu-131 in Hm1 contains a bulky hydrophobic amino acid (L, I, V, M, or F) in nearly all cloned GPCRs. Substitution of Leu-131 with polar amino acids (aspartate and asparagine) also resulted in strongly defective coupling, whereas phenylalanine (found in the equivalent position in the beta 2 adrenoceptor) can replace leucine without losing PI coupling ability of Hm1. Alanine substitution of the corresponding amino acid in the Hm3 receptor (L174A) also inhibited agonist-stimulated PI turnover, while replacing Phe-139 with alanine in the beta 2 adrenoceptor suppressed stimulation of adenylyl cyclase. We propose that a bulky hydrophobic amino acid in the middle of the i2 loop serves as a general site relevant to G protein coupling, whereas coupling selectivity is governed by other receptor domains. FAU - Moro, O AU - Moro O AD - Department of Pharmacy, University of California, San Francisco 94143-0446. FAU - Lameh, J AU - Lameh J FAU - Hogger, P AU - Hogger P FAU - Sadee, W AU - Sadee W LA - eng GR - GM43102/GM/NIGMS NIH HHS/United States GR - MH00996/MH/NIMH NIH HHS/United States PT - Comparative Study PT - Journal Article PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Phosphatidylinositols) RN - 0 (Receptors, Cell Surface) RN - 0 (Receptors, Muscarinic) RN - 0 (Recombinant Proteins) RN - 8Y164V895Y (Carbachol) RN - E0399OZS9N (Cyclic AMP) RN - EC 3.6.1.- (GTP-Binding Proteins) RN - GMW67QNF9C (Leucine) RN - L628TT009W (Isoproterenol) SB - IM MH - Amino Acid Sequence MH - Binding Sites MH - Carbachol/pharmacology MH - Cell Line MH - Consensus Sequence MH - Cyclic AMP/metabolism MH - Embryo, Mammalian MH - GTP-Binding Proteins/*chemistry/*metabolism MH - Humans MH - Isoproterenol/pharmacology MH - Kidney MH - Kinetics MH - Leucine MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Phosphatidylinositols/metabolism MH - Point Mutation MH - *Protein Structure, Secondary MH - Receptors, Cell Surface/*chemistry/metabolism MH - Receptors, Muscarinic/biosynthesis/*chemistry/*metabolism MH - Recombinant Proteins/biosynthesis/chemistry/metabolism MH - Sequence Homology, Amino Acid MH - Transfection EDAT- 1993/10/25 00:00 MHDA- 1993/10/25 00:01 CRDT- 1993/10/25 00:00 PHST- 1993/10/25 00:00 [pubmed] PHST- 1993/10/25 00:01 [medline] PHST- 1993/10/25 00:00 [entrez] AID - S0021-9258(18)41524-4 [pii] PST - ppublish SO - J Biol Chem. 1993 Oct 25;268(30):22273-6.