Distinct binding mode of 125I-AngII to AT1 receptor without the Cys18-Cys274 disulfide bridge

Previous studies on angiotensin II (AngII) AT1 receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys274 residue that supposedly makes a disulfide bond with N-terminal Cys18. As demonstrated by assays with Del(267–275)AT1, the role of the Cys18-Cys274 disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT1 receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S–S disulfide bond was disrupted in (C18S,C274S) AT1 receptor. The importance of the free N-terminal amino group of Asp1 and of the Arg2 guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp1 for Sar1 or Arg2 for Lys2, as ligands. This study showed that like AngII, [Sar1]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys2]-AngII binding is still more reduced. Interestingly, when 125I-AngII instead of 3H-AngII was used, no significant binding of this mutant was observed although wild type AT1 receptor was shown to bind all AngII analogues.