Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity.
The molecular basis for endothelin (ET) isopeptide selectivity between ETA and ETB receptors was studied by examining ligand binding to several site-specific mutants of the human ETA receptor. Based on a computer-built three-dimensional model of the ETA receptor, five non-conserved amino acids, clustered around the putative ligand binding site, were targeted for mutation to alanine. Expression of the wild-type and mutant ETA receptors in COS-7 cells revealed that the binding profile of one of the ETA mutants, Tyr129-->Ala, was characteristic of the ETB receptor. In the Tyr129-->Ala ETA receptor mutant the affinity of two ETB-selective agonists, endothelin-3 and sarafotoxin S6c, was increased 10-200-fold, whereas that for two ETA-selective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane region of the wild-type ETA receptor results in subtype conversion. In addition, these data represent the first example of peptide interactions with a transmembrane region of a G protein-coupled receptor and indicate that Tyr129, located in the second transmembrane region of the ETA receptor, is a critical component for determination of endothelin receptor subtype-selective ligand binding.
Krystek
S R
SR
Department of Macromolecular Modeling, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
Patel
P S
PS
Rose
P M
PM
Fisher
S M
SM
Kienzle
B K
BK
Lach
D A
DA
Liu
E C
EC
Lynch
J S
JS
Novotny
J
J
Webb
M L
ML
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D009842:Oligopeptides; D017466:Receptors, Endothelin; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins