title: Role of the second disulfide bridge (Cys(18)-Cys(274)) in stabilizing the inactive AT₁ receptor.
abstract: Previous research showed that disruption of the Cys(18)-Cys(274) bond in the angiotensin II (AngII) AT₁ receptor mutant (C18S), expressed in CHO cells, causes an increase in the basal activity and attenuation of the maximum response to AngII. In addition, this mutant was mostly intracellularly distributed. Our aim was to investigate whether the intracellular presence of the mutant was due to a constitutive internalization or to a defective maturation of the receptor. The first hypothesis was assessed by pretreating the cells with losartan or [Sar¹Leu⁸]-AngII, specific AT₁ receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT₁ receptor antagonists causes an increase in the binding ability of the mutant to AngII. Furthermore, whereas the maximum effect is increased, it reduces the enhanced basal levels of IP₃. The hypothesis for a lack of maturation of the mutant receptor was ruled out because calnexin was poorly colocalized with the intracellular C18S receptor. Our results suggest that the mutation of the AT₁ receptor leads to a conformational structure similar to that of the active mode of the AT₁ receptor, favoring its internalization in the absence of the agonist.
authors: Department of Biophysics, Federal University of São Paulo, São Paulo 04023-062, Brazil.
authors: Renan Paulo
authors: RP
authors: Martin
authors: Eliete da Silva
authors: Eda S
authors: Rodrigues
authors: Silvana Aparecida Alves
authors: SA
authors: Correa
authors: Suzana Macedo
authors: SM
authors: Oliveira
authors: Renato Arruda
authors: RA
authors: Mortara
authors: Laerte
authors: L
authors: Oliveira
authors: Clovis Ryuichi
authors: CR
authors: Nakaie
authors: Suma Imura
authors: SI
authors: Shimuta
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005786:Gene Expression Regulation; D007295:Inositol Phosphates / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability; D021381:Protein Transport; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D007295:Inositol Phosphates; D044140:Receptor, Angiotensin, Type 1; C516441:inositol trispyrophosphate; D003545:Cysteine