Recent studies on the structure-activity of the kinin B1 receptor (B1R) revealed that important residues for its binding to des-Arg9-bradykinin (DBK) are homologous to those in the interaction between angiotensin II and the angiotensin II (AngII) type I receptor (AT1R). Several studies have shown that the ammonium group of Lys199 (512) located in the helix V of AT1R interacts with the carboxyl of the Phe8 AngII, to provide an important positive site to the binding of negative C-terminal portion of AngII (Noda e cols., 1995a; Yamano e cols., 1992). On the other hand, recent studies suggested that Lys128 and Arg212, of the helix III and V respectively, in addition to a sequence (CPYHFFAFL) in the helix VI of the B1R interact with C-terminal residues of DBK (Santos et al., 2008). These data suggest that both receptors, B1R and AT1R presents crucial binding sites at the top of helix V and VI for interaction. Based on these data is our aim: (1) identify important residues to the interaction between DBK and B1R, by binding assays using DBK analogues that will be sintesized and the wild-type receptor; (2) determine the functional expression of wild-type receptor and its mutants, by testing the IP3 production and the determination of [Ca2+]i; (3) verify whether the N-terminal region of DBK (Arg1) interacts with Asp301 (712) of B1R; (4) investigate whether the Asn130 (325) of the B1R, the homologue residue for Asn111 (325) of AT1R, interacts with the DBK Phe5 by the replacement of Asn130 to Gly; (5) evaluate the role of the B1R second disulfide bond (Cys25-Cys294) by replacing the Cys25 for Ser; (6) whether will be confirmed that Arg1 of the peptide is important to interact with Asp301 (712) of the receptor EC-3 loop, we will test the DBK analogue, [Lys1] DBK, to verify the importance of guanide group in this position and (7) if some mutants present low binding index to the peptide DBK, these receptors will be labelled with GFP and fluorescence techynique will be used to find out their preferential distribution. If the most receptors were localized in the intracellular domain, assays will be performed to verify whether the mutants were once expressed on the cell membrane and then they were internalized or the mutants were retained in the endoplasmic reticulum. The hypothesis that constitutive activation was responsible for the internalization of the receptors will be tested treating the cells with specific B1 receptor antagonist which should revert the process. On the other hand if the receptors were not transported from the endoplasmic reticulum because of the deficient maturation of the mutants, it will be assessed using calnexin, a reticulum marker.
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