TYR-129 IS IMPORTANT TO THE PEPTIDE LIGAND AFFINITY AND SELECTIVITY OF HUMAN ENDOTHELIN TYPE-A RECEPTOR

Molecular modeling and protein engineering techniques have been used t o study residues within G-protein-coupled receptors that are potential ly important to ligand binding and selectivity. In this study, Tyr-129 located in transmembrane domain 2 of the human endothelin (ET) type A receptor A (hET(A)) was targeted on the basis of differences between the hET(A) and type B receptor (hET(B)) sequences and the position of the residue on ET receptor models built using the coordinates of bacte riorhodopsin. Replacement of Tyr-129 of hET(A) by alanine, glutamine, asparagine, histidine, lysine, serine, or phenylalanine results in rec eptor variants with enhanced ET-3 and sarafotoxin 6C affinities but wi th unchanged ET-1 and ET-2 affinities. Except for Tyr-129 --> Phe hET( A), these hET(A) variants have two to three orders of magnitude lower binding affinity for the ET(A)-selective antagonist BQ123. Replacement of His-150, the residue in hET(B) that is analogous in sequence to Ty r-129 of hET(A), by either tyrosine or alanine does not affect the aff inity of peptide ligands. These results indicate that although transme mbrane domain 2 is important in ligand selectivity for hET(A), it does not play a significant role in the lack of ligand selectivity shown b y hET(B). Chimeric receptors have been constructed that further suppor t these conclusions and indicate that at least two hET(A) regions cont ribute to ligand selectivity. Additionally, the data support an overla p in the binding site in hET(A) of agonists ET-3 and sarafotoxin 6C wi th that of the antagonist BQ123.