Identification of ET_A and ET_B binding domains using ET-derived photoprobes

摘要

Using the structure of ET-1 as a template, a series of photosensitive analogs were developed to investigate the binding domain of ET_A and ET_B 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-Bpa~7, Tyr(~(125)I)~(13)]hET-l) or 12 ([Me~7, L-Bpa~(12), Tyr(~(125)I)~(13)]hET-l) 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 overexpress-ing either ET_A or ET_B receptors. Subsequently, these photoprobes were used to label ET_A and ET_B 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 Phe~(147)-Lys~(166), covering the second segment of EC I and the top part of TM III, contains a contact point for [Nle~7, L-Bpa~(12), Tyr(l25I)~(13)]hET-l on ET_A receptors whereas Ile~(292)-Trp~(319), 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-Bpa~7, Tyr(~(125)I)~(13)]hET-l. Moreover, upon binding of [Nle~7, L-Bpa~(12), Tyr(~(125)I)~(13)]hET-l, it was observed that Thr~(263)-Met~(266) (EC II) of the ET_B receptor would come close with the ligand.