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 ET(A) and ET(B) receptors was studied by examining ligand binding to several site-specific mutants of the human ET(A) receptor. Based on a computer-built three-dimensional model of the ET(A) receptor, five non -conserved amino acids, clustered around the putative ligand binding s ite, were targeted for mutation to alanine. Expression of the wild-typ e and mutant ET(A) receptors in COS-7 cells revealed that the binding profile of one of the ET(A) mutants, Tyr(129) --> Ala, was characteris tic of the ET(B) receptor. In the Tyr(129) --> Ala ET(A) receptor muta nt the affinity of two ET(B)-selective agonists, endothelin-3 and sara fotoxin S6c, was increased 10-200-fold, whereas that for two ET(A)-sel ective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane reg ion of the wild-type ET(A) receptor results in subtype conversion. In addition, these data represent the first example of peptide interactio ns with a transmembrane region of a G protein-coupled receptor and ind icate that Tyr(129), located in the second transmembrane region of the ET(A) receptor, is a critical component for determination of endothel in receptor subtype-selective ligand binding.