In the last few years has been reported that agonists of G-protein coupled receptors (GPCRs) are capable of promoting a preferential activation profile or biased to a particular intracellular signaling pathway. This phenomenon has received different names in the literature, such as collateral efficacy, pluridimensional efficacy, functional selectivity or biased agonism. In general, this preference of activation has been best studied for canonical signaling (via activation of G protein) and non-canonical signaling (via B-arrestins) that occurs after the formation and internalization of a complex that includes ligand, receptor, B-arrestin (a scaffold protein), and kinases. Various drugs have been discovered as biased agonists to one of these signaling pathways. Despite many studies showing the existence and relevance of biased agonists at different receptor systems, so far there are no studies in the literature evaluating whether a group of biased agonists for the same pathway (e.g. for B-arrestins) may present differences in signaling downstream the recruitment of B-arrestins. Therefore, the aim of this project is to investigate whether activation of the AT1 receptor by AngII and different biased agonists lead to the formation of different signaling complexes. After stimulation and internalization it will be evaluated the interaction of B-arrestins 1 and 2 with complex Raf-1/MEK1/ERK1/2, Ask1/MKK4/JNK3. B-arrestins will be immunoprecipitated and followed by Western blotting with specific antibodies for each protein of the signaling pathways. Furthermore, the activation profile of ERK1/2 and JNK3 will be evaluated by AlphaScreen SureFire (TGR BioSciences / PerkinElmer) and the "biased" activation of the signaling pathway (ERK1/2 or JNK3) for each condition will be calculated by determining the reasons of "coupling efficiencies" tau.
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