MAPPING THE LIGAND INTERACTION OF THE AT1 –RECEPTOR WITH PHOTOPROBES IN ALL POSITIONS OF ANGIOTENSIN II

We have combined a photoaffinity labelling approach to study the molecular structure of the liganded human angiotensin II (AngII) type 1 receptor (hAT1). Systematic incorporation of the photoprobes p‐benzoyl‐L‐phenylalanine (Bpa) or p–[3–(trifluoromethyl)–3H–diazirin–3–yl]–L–phenylalanine (Tdf) into each of the eight positions of the AngII sequence was carried out. Since the constitutively active mutant [N111G]‐hAT1 (hAT1‐CAM) had a more permissive structure‐activity relationship, photoaffinity labelling studies were carried out on this CAM‐receptor. The AngII‐photoprobe analogues substituted in position 1, 2, 3 and 5 displayed AngII‐like properties on hAT1‐CAM. Analogues substituted in position 7 and 8 were shown to be low nM affinity neutral antagonists whereas analogues substituted in positions 4 and 6 had too low affinities. Receptor labelling experiments evidence localisation of the peptide N‐terminus in the extracellular space of the receptor through contact with most extracellular elements of hAT1 A single contact with ECL2 was found at position 3 of AngII. The middle sequence elements are still ill‐defined but the AngII C‐terminal is in close contact with the trans‐membrane bundle formed by TMD3, 5, 6 and 7, in accord with previous results (Clement, JBC. 2005; 280:27121). This study again confirms the extended ligand structure in the hAT1 receptor.