Transmembrane domain V of the endothelin-A receptor is a binding domain of ETA-selective TTA-386-derived photoprobes.
On the basis of the structure of TTA-386, a specific antagonist of the endothelin-A receptor subtype (ET(A)), photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing, in position 6, the photoreactive amino acid D- or L-p-benzoyl-phenylalanine showed pharmacological properties very similar to those of TTA-386. Affinity of the probes were also evaluated on transfected CHO cells overexpressing the human ET(A) receptor. Data showed that binding of the radiolabeled peptides were inhibited by ET-1 and BQ-610. Therefore, these photolabile probes were used to label the ET(A) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 66 kDa. An excess of ET-1 or BQ-610 completely abolished the formation of the complex showing the selectivity of the photoprobes. Digestions of the [125I-Tyr5, D- or L-Bpa6]TTA-386-ET(A) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacted. Results showed that Endo Lys-C digestion gave a 4.8 kDa fragment corresponding to the Asp256-Lys299 segment, whereas migration after V8 digestion revealed a fragment of 2.9 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp257-Glu281 stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.9 kDa corresponding to Glu249-Met278. Thus, the combined cleavage data strongly suggested that the binding domain of ET(A) includes a portion of the fifth transmembrane domain, between residues Trp257 and Met278.
Tessier
Sophie
S
Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, 245 boulevard Hymus, Pointe-Claire (Montréal), Québec, Canada, H9R 1G6.
Boivin
Stéphane
S
Aubin
Jacinthe
J
Lampron
Philipe
P
Detheux
Michel
M
Fournier
Alain
A
D000595:Amino Acid Sequence; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D065130:Endothelin A Receptor Antagonists; D019332:Endothelin-1 / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D019920:Photoaffinity Labels / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D044022:Receptor, Endothelin A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D065130:Endothelin A Receptor Antagonists; D019332:Endothelin-1; D008565:Membrane Proteins; D009842:Oligopeptides; D019920:Photoaffinity Labels; D044022:Receptor, Endothelin A; C108845:TTA 386