Identification of different binding modes of Angiotensin II N-terminal domain with wild-type and mutant AT1 receptors

Abstract

Previous study reported that in CHO cell line transfected with the AT1 receptor an unespexted tachyphylaxis phenomenon to [Lys2]-AII was showed. This effect was ascribed to the lack of the 3 and 5 untranslated regions (3, 5-UTR) in the cDNA for AT1 receptor. Furthermore whereas the tachyphylaxis was specific to AII in rabbit arterial smooth muscle cell line that expresses endogenous AT1 receptor, it became tachyphylactic to [Lys2]-AII when it was just cotransfected with the exogenous gene for AT1 receptor without the 3, 5-UTR. Therefore it will be of interest to investigate whether the recombinant receptor would be overexpressed in the transfected cell line or the tachyphylaxis was due to the predominant effect of the exogenous AT1 receptor without the 3, 5-UTR. In another approach on the study of AT1 receptor we found recently that AT1 receptor bearing the Ser instead of Cys at position 18, resulted in a mutant which was expressed preferably in the perinuclear region of the cells, leading to the hypothesis that it was due to a constitutive internalization. To investigate whether the treatment with specific AT1 antagonists [Sar1Leu8]-AII or Dup753 would be able to reverse such internalization, binding and IP3 production assays will be determined using the fluorescence polarization technology in a specific microplate reader, in addition to the determination of [Ca2+]i levels using the fluo-3 to label the Ca2+ with confocal microscopy. Besides, the colocalization of the wild and mutant AT1 receptor with the calnexin, an endoplasmic reticulum chaperone, the protein responsible for the correct protein maturation will be assessed detecting the fluorescence of the secundary antibody anti-rabbit labelled with Texas Red using the confocal microscopy with and without the antagonist treatment to reverte the possible constitutive internalization.