Tyr-129 is Important to the Peptide Ligand Affinity and Selectivity of Human Endothelin Type A Receptor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 15; pp. 7164 - 7168
• Main Authors: Lee, Jonathan A., Elliott, John D., Sutiphong, Joyce A., Friesen, Westley J., Ohlstein, Eliot H., Stadel, Jeffrey M., Gleason, John G., Peishoff, Catherine E.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 19.07.1994; National Acad Sciences; National Academy of Sciences
• Subjects: Agonists; Animals; Binding sites; Binding, Competitive; Biochemistry; Cattle; Cell Line; Cell membranes; Chimeras; Computer Graphics; Coordinate systems; Endothelins; Endothelins - metabolism; Humans; Ligands; Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; Receptor, Endothelin A; Receptor, Endothelin B; Receptors; Receptors, Endothelin - chemistry; Receptors, Endothelin - genetics; Receptors, Endothelin - metabolism; Recombinant Fusion Proteins - metabolism; Tyrosine - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.15.7164

Molecular modeling and protein engineering techniques have been used to study residues within G-proteincoupled receptors that are potentially 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 (hETA) was targeted on the basis of differences between the hETAand type B receptor (hETB) sequences and the position of the residue on ET receptor models built using the coordinates of bacteriorhodopsin. Replacement of Tyr-129 of hETAby alanine, glutamine, asparagine, histidine, lysine, serine, or phenylalanine results in receptor variants with enhanced ET-3 and sarafotoxin 6C affinities but with unchanged ET-1 and ET-2 affinities. Except for Tyr-129 → Phe hETA, these hETAvariants have two to three orders of magnitude lower binding affinity for the ETA-selective antagonist BQ123. Replacement of His-150, the residue in hETBthat is analogous in sequence to Tyr-129 of hETA, by either tyrosine or alanine does not affect the affinity of peptide ligands. These results indicate that although transmembrane domain 2 is important in ligand selectivity for hETA, it does not play a significant role in the lack of ligand selectivity shown by hETB. Chimeric receptors have been constructed that further support these conclusions and indicate that at least two hETAregions contribute to ligand selectivity. Additionally, the data support an overlap in the binding site in hETAof agonists ET-3 and sarafotoxin 6C with that of the antagonist BQ123.