Role of the second disulfide bridge (Cys(18)-Cys(274)) in stabilizing the inactive AT₁ receptor.
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.
Martin
Renan Paulo
RP
Department of Biophysics, Federal University of São Paulo, São Paulo 04023-062, Brazil.
Rodrigues
Eliete da Silva
Eda S
Correa
Silvana Aparecida Alves
SA
Oliveira
Suzana Macedo
SM
Mortara
Renato Arruda
RA
Oliveira
Laerte
L
Nakaie
Clovis Ryuichi
CR
Shimuta
Suma Imura
SI
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*
D004220:Disulfides; D007295:Inositol Phosphates; D044140:Receptor, Angiotensin, Type 1; C516441:inositol trispyrophosphate; D003545:Cysteine