title: 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.
authors: Department of Biophysics, Federal University of São Paulo, 04023-062 São Paulo, SP, Brazil.
authors: Renan P
authors: RP
authors: Martin
authors: Eliete S
authors: ES
authors: Rodrigues
authors: Nelson A S
authors: NA
authors: Pacheco
authors: Silvana A A
authors: SA
authors: Corrêa
authors: Suzana M
authors: SM
authors: Oliveira
authors: Laerte
authors: L
authors: Oliveira
authors: Clóvis R
authors: CR
authors: Nakaie
authors: Suma I
authors: SI
authors: Shimuta
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II; D003545:Cysteine