Identification of ETA and ETB binding domains using ET-derived photoprobes.
Using the structure of ET-1 as a template, a series of photosensitive analogs were developed to investigate the binding domain of ETA and ETB receptors. Accordingly, a p-benzoyl-l-phenylalanine (Bpa) residue was introduced into the peptide chain following a pattern aiming at scanning N- to C-terminal portions of the molecule. Among the analogs, those containing a Bpa amino acid in position 7 ([L-Bpa7, Tyr(125I)13]hET-1) or 12 ([Nle7, L-Bpa12, Tyr(125I)13]hET-1) exhibited the capacity to activate both receptors, thus showing that residues Met-7 and Val-12 of ET-1 do not play a key role in the activation process. The binding capacity of the probes was also evaluated on transfected CHO cells overexpressing either ETA or ETB receptors. Subsequently, these photoprobes were used to label ETA and ETB receptors overexpressed in transfected CHO cells. Enzymatic digestions and chemical cleavages were then performed on ligand-receptor complexes and fragments produced by the lysis were analyzed to point out putative interaction areas on the receptors. Results showed that Phe147-Lys166, covering the second segment of EC I and the top part of TM III, contains a contact point for [Nle7, L-Bpa12, Tyr(125I)13]hET-1 on ETA receptors whereas Ile292-Trp319, spanning from the second half of the intracellular loop III up to the middle turns of TM VI, includes a residue that can interact with [L-Bpa7, Tyr(125I)13]hET-1. Moreover, upon binding of [Nle7, L-Bpa12, Tyr(125I)13]hET-1, it was observed that Thr263-Met266 (EC II) of the ETB receptor would come close with the ligand.
Aubin
Jacinthe
J
INRS - Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Laval, QC H7V 1B7, Canada.
Létourneau
Myriam
M
Francoeur
Eugène
E
Burgeon
Emmanuel
E
Fournier
Alain
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019332:Endothelin-1 / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008024:Ligands; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D044022:Receptor, Endothelin A / Q000737:chemistry* / Q000378:metabolism; D044023:Receptor, Endothelin B / Q000737:chemistry* / Q000378:metabolism
D019332:Endothelin-1; D008024:Ligands; D010455:Peptides; D019920:Photoaffinity Labels; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B