Navegando por Palavras-chave "Inositol trisphosphate"

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    Identificação de diferentes modos de ligação do domínio N-terminal da angiotensina II com receptores AT, selvagem e mutantes
    (Universidade Federal de São Paulo (UNIFESP), 2009-08-26) Martin, Renan Paulo [UNIFESP]; Shimuta, Suma Imura [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Purpose: Our aim was to assess: a) the relative expression of endogenous and exogenous AT1 receptor in a rabbit smooth muscle cell line either expressing only the endogenous AT1 receptor or in addition the exogenous rat AT1 receptor; b) the binding affinity of the wild AT1 receptor and of the receptor bearing the Cys18Ser mutation (C18S); c) the intracellular predominance of the mutated receptor by using specific antagonists to revert the constitutive internalization and d) a possible defect in the maturation of the receptor by mutation. Methods: The real time PCR was carried out to determine the expression level of both, endogenous and exogenous AT1 receptor. The competitive binding test was used to evaluate the binding affinity of the mutant and of the wild AT1 receptor using AngII, [Lys2]-AngII or [Sar1]-AngII as ligands and 3H-AngII and 125I-AngII as radioactive tracers to find out the reason of the predominant intracellular localization of the mutant receptor. The hypothesis for constitutive internalization was tested, pre-treating the cells with the specific AT1 receptor antagonists, DuP753 or [Sar1Leu8]-AngII and the IP3 production and the intracellular calcium concentration [Ca2+]i were determined. The hypothesis that a defective maturation was also tested, using calnexin, a endoplasmic reticulum marker. Cells expressing the wild or mutant AT1 receptor conjugated with green fluorescent protein (GFP) were targeted to the anti-calnexin primary antibody and the Texas Red labeled anti-rabbit secondary antibody and then merged images were analysed under confocal microscopy. Results and conclusions: It was found that the expression of the exogenous rat AT1 receptor without the 3’,5’-UTR was much higher (about 200.000 fold) than that of the endogenous receptor expression in the rabbit arterial smooth muscle cell line, which could be responsible for the tachyphylatic effect to [Lys2]-AngII. The high expression of the recombinant receptor was probably due to the ubiquitin used as expression vector of the exogenous receptor, which is known to be a strong vector. In another approach, it was verified from the competitive binding profiles using 3H-AngII, that the affinity of C18S AT1 receptor mutant was higher to [Sar1]-AngII than to AngII whereas the binding to [Lys2]-AngII was prevented. On the other hand, when the 125IAngII was used as radiolabel, the binding to the mutant but not to the wild receptor was completed blocked. These results from binding assays showed that the lack of the second S-S bridge induced the receptor to a more unfavorable condition to bind AngII rather than [Sar1]-AngII whereas the mutation completely blocked the receptor to bind [Lys2]-AngII. Furthermore the mutation led the AT1 receptor to acquire a conformation causing a different binding mode that drastically affected the binding of 125I-AngII, probably due to the monoiodinated Tyr4 side chain in its molecule. Concerning the low expression of the C18S mutant on the plasma membrane, it can be concluded that it was due to its internalization rather than to an impaired transport of the mutant from the endoplasmic reticulum to the cell membrane surface, since the pre-treatment with AT1 receptor antagonist was capable to revert the basal activity of the receptor. Indeed, the low basal levels of IP3 and [Ca2+]i were recovered and the responses to AngII were increased to the level of that found in the WT receptor. The hypothesis for a lack of maturation of the mutant receptor was ruled out since calnexin, the endoplasmic reticulum marker was poorly co-localized with C18S receptor. Therefore, our results suggest that the mutant receptor assumed a conformational structure similar to that of active mode of the AT1 receptor, favouring its internalization even in the absence of the agonist.