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Mutational analysis and binding mode between AngII and AT1 receptor: cross talk interactions between AngII and kinin receptors in mice deficient in B1 or B2 receptors

Abstract

Many studies performed with site-directed mutagenesis of angiotensin II (AII) AT1 receptor, have identified several amino acid residues important to binding, activation and G protein coupling of this receptor in response to the stimulation with AII. It was recently found that AT1 receptor bearing the Ser instead of Cys at position 18, resulted in a mutant which was expressed preferably in the interior, in the perinuclear region of the cells, leading to the hypothesis that it was due to a constitutive internalization of the receptor. To investigate this hypothesis, specific AT1 antagonists such as [Sar1Leu8]-AII or Dup753 will be incubated with the mutant receptor. These AT1 receptor antagonists will function as inverse agonists, a method usually used to take back the receptor to the cell surface. The presence of the receptors at the plasma membrane will be tested by measuring the binding constants and the IP3 production. These assays will be performed using the fluorescence polarization technology in a specific microplate reader. In addition the determination of [Ca2+]I levels will be performed using the Fluo-3 to label the Ca2+ which will be measured using confocal microscopy. A second hypothesis, that the receptor was not transported from the endoplasmic reticulum to the plasma membrane will be evaluated, to explain the intracellular localization of the mutant receptor. A immunocytochemical assay will be used toward the calnexin, a protein responsible for the correct protein maturation. Colocalization of the wild and mutant AT1 receptor with the calnexin, the endoplasmic reticulum chaperone, will be compared, detecting the fluorescence of the secondary antibody anti-rabbit labeled with Texas Red. In another approach cross talk between AII and kinin receptors will be studied in vascular and non-vascular tissues isolated from mice deficient in kinin B1 or B2 receptors. Recent study showed that the kinin B1 and B2 receptors are constitutively expressed in the stomach and in aorta isolated from these transgenic animals. It was already reported that AT1 and B2 receptors can interact each other to form heterodimers and that intra- and intermolecular interactions can be occur between the two sub-types of AII receptors as well as of kinin receptors, leading to form homo and heterodimers. Since mice with deficiency in kinin B1 or B2 receptors are available, it is of our interest to assess possible cross-talk between kinin and AII receptors. Our preliminary studies showed that the efficacy of the responses to AII in aorta but not in stomach of transgenic animals was lower than in WT mice. Therefore the functional expression of the AII receptors, the remanescent kinin receptor and of ECA-1 will be performed in the tissue and the cultured smooth muscle cells isolated from mice aorta. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
RODRIGUES, ELIETE S.; MARTIN, RENAN P.; FELIPE, SANDRA A.; BADER, MICHAEL; OLIVEIRA, SUZANA M.; SHIMUTA, SUMA I. Cross talk between kinin and angiotensin II receptors in mouse abdominal aorta. Biological Chemistry, v. 390, n. 9, p. 907-913, SEP 2009. Web of Science Citations: 7.

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