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

Abstract Previous studies on angiotensin II (AngII) AT 1 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 Cys 274 residue that supposedly makes a disulfide bond with N-terminal Cys 18 . As demonstrated by assays with Del(267–275)AT 1 , the role of the Cys 18 -Cys 274 disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT 1 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) AT 1 receptor. The importance of the free N-terminal amino group of Asp 1 and of the Arg 2 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 Asp 1 for Sar 1 or Arg 2 for Lys 2 , as ligands. This study showed that like AngII, [Sar 1 ]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys 2 ]-AngII binding is still more reduced. Interestingly, when 125 I-AngII instead of 3 H-AngII was used, no significant binding of this mutant was observed although wild type AT 1 receptor was shown to bind all AngII analogues.