Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling.

O Moro,J Lameh,P Högger,W Sadée

doi:10.1016/s0021-9258(18)41524-4

O Moro, J Lameh + Show 2 more

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https://doi.org/10.1016/s0021-9258(18)41524-4

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Abstract

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.

Highlights

Kidney cells.Alanine substitutionsin four regions impaired cou- ring the mutated p2 gene, cut with XbaI, back to pCDNAIneo
We propose that a bulkyhy- curves with carbachol, to determine the carbachol IC5, binding value, drophobic amino acidin the middle ofthe i2 loopserves were camed out in intact cells using 0.1 nM L3H1NMS
Mutant 7 involves changes in the COOH-terminalloio3pjunction, which has been implicated in coupling [2, 3]

Summary

Introduction

The position equivalent to Leu-131 in Hml contains a described before(9,lO).Transient expression yields were -900fmoYmg bulky hydrophobic amino acid (IL, V, , M, or F)in nearly protein for Hml, whereas some Hml mutants yieldedconsiderably all cloned GPCRs. Substitution ofLeu-131 with polar lower expression. Amino acid in the Hm3 receptor (L174A) inhibited Receptor Binding Assays-Receptor expression of Hml, Hm3 and agonist-stimulatedPI turnover,while replacing Phe-139 their respective mutants was determined in intact cell monolayers as with alanine in the p2 adrenoceptor suppressed stimu- described previously [9, 10] using 2 nM f3H1NMS.Binding competition lation of adenylyl cyclase.

Results

Conclusion