Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620-derived photoprobe.
On the basis of the structure of IRL-1620, a specific agonist of the endothelin-B receptor subtype (ET(B)), a few photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing the photoreactive amino acid, p-benzoyl-l-phenylalanine in position 5 showed, as assessed with endothelin-A (ET(A)) and ET(B) receptor paradigms, pharmacological properties very similar to those of IRL-1620. The binding capacity of the probe was also evaluated on transfected Chinese hamster ovary (CHO) cells overexpressing the human ET(B) receptor. Data showed that binding of the radiolabeled peptide was inhibited by ET-1 and IRL-1620. Therefore, this photolabile probe was used to label the ET(B) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 49 kDa. An excess of ET-1 or IRL-1620 completely abolished the formation of the complex, showing the selectivity of the photoprobe. Digestions of the [Bpa(5),Tyr((125)I)(6)]IRL-1620-ET(B) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacts. Results showed that Endo Lys-C digestion gave a 3.8-kDa fragment corresponding to the Asp(274)-Lys(303) segment, whereas migration after V8 digestion revealed a fragment of 4.6 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp(275)-Asp(313) stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.6 kDa, corresponding to Gln(267)-Met(296). Thus, the combined cleavage data strongly suggested that the agonist binding domain of ET(B) includes a portion of the fifth transmembrane domain, between residues Trp(275) and Met(296).
Boivin
Stéphane
S
Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, 245 boul. Hymus, Pointe-Claire, Québec, Canada, H9R 1G6.
Tessier
Sophie
S
Aubin
Jacinthe
J
Lampron
Philipe
P
Detheux
Michel
M
Fournier
Alain
A
D000595:Amino Acid Sequence; D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D016232:Endothelins / Q000138:chemical synthesis / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D044023:Receptor, Endothelin B / Q000819:agonists / Q000378:metabolism*; D014162:Transfection
D016232:Endothelins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D044023:Receptor, Endothelin B; C074308:sovateltide; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase