Role of the second disulfide bridge (Cys18-Cys274) in stabilizing the inactive AT1 receptor

Abstract

AbstractPrevious research showed that disruption of the Cys18-Cys274bond in the angiotensin II (AngII) AT1receptor 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 AT1receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT1receptor 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 AT1receptor leads to a conformational structure similar to that of the active mode of the AT1receptor, favoring its internalization in the absence of the agonist.