HYDROPHOBIC AMINO-ACID IN THE I2-LOOP PLAYS A KEY ROLE IN RECEPTOR-G-PROTEIN COUPLING

Signal transduction of the heptahelical G protein-coupled receptors (G PCRs) involves multiple receptor domains, but a universal consensus do main for coupling has not yet been defined. Alanine mutagenesis scanni ng was performed on the intracellular loops and the COOH tail of the h uman muscarinic cholinergic receptor (Hm1) to identify coupling domain s. Stimulation of phosphatidylinositol (PI) turnover was determined af ter transfection of the alanine mutants into U293 human embryonic kidn ey cells. Alanine substitutions in four regions (loops i1, i2, and NH2 and COOH junctions of i3) impaired coupling efficiency by approximate ly 50% or more, but the strongest reduction (>80%) resulted from alani ne replacement of a single amino acid, leucine 131. This residue is lo cated in the middle of the second intracellular loop (i2), within the highly conserved GPCR motif (DRYXXV(I)XXPL). The position equivalent t o 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 a mino acids (aspartate and asparagine) also resulted in strongly defect ive coupling, whereas phenylalanine (found in the equivalent position in the beta2 adrenoceptor) can replace leucine without losing PI coupl ing ability of Hm1. Alanine substitution of the corresponding amino ac id in the Hm3 receptor (L174A) also inhibited agonist-stimulated PI tu rnover, while replacing Phe-139 with alanine in the beta2 adrenoceptor suppressed stimulation of adenylyl cyclase. We propose that a bulky h ydrophobic 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.