Transmembrane domain v of the endothelin-A receptor is a binding domain of ETA-selective TTA-386-derived photoprobes

摘要

On the basis of the structure of TTA-386, a specific antagonist of the endothelin-A receptor subtype (ETA), 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-benzoylphenylalanine showed pharmacological properties very similar to those of TTA-386. Affinity of the probes were also evaluated on transfected CHO cells overexpressing the human ETA 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 ETA 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 [I-125-Tyr(5), D-or L-Bpa(6)]TTA-386-ETA 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 Asp(256)-LyS(299) 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 Trp(257)-Glu(281) stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.9 kDa corresponding to Glu(249)-Met(278). Thus, the combined cleavage data strongly suggested that the binding domain of ETA includes a portion of the fifth transmembrane domain, between residues Trp(257) and Met(278).