Others undergo downregulation by being targeted to lysosomes where they are degraded. Some GPCRs remain intact and continue to signal, or initiate new signaling Folinic acid calcium salt pentahydrate pathways independent of arrestins. The best studied example of an arrestin-activated signaling pathway is the ERK cascade. Recent studies have shown that adrenoceptors can initiate ERK signaling by both G-protein- and b-arrestin-dependent processes. b-arrestins function as membrane-tethered scaffolds capable of recruiting elements of the MAPK pathways to membranes of endosomes, thus facilitating ERK activation. Furthermore, by anchoring activated ERK to endosomes, b-arrestins might prevent ERK translocation to the nucleus, thus favoring cytoplasmic ERK signaling. Interestingly, the time course and molecular consequences of activating ERK signaling through G-protein mediated pathways versus b-arrestin mediated pathways are considerably different. A few studies have investigated the internalization properties of the a1-AR subtypes with divergent conclusions. Following agonist stimulation, a1B-AR is rapidly desensitized by GRKs and its clathrin-mediated internalization involves b-arrestin interaction. This subtype undergoes constitutive internalization according to Stanasila et al. but not according to Morris et al.. The most intriguing results have been observed with the a1AAR. This subtype has been observed in lipid rafts under basal conditions and, according to some authors, undergoes constitutive and phenylephrine -mediated internalization via clathrin-coated vesicles. In contrast, other authors did not find constitutive internalization for this subtype possibly due to differences in the expression system, the receptor constructs or the methods used to measure endocytosis. The aim of the present work was to explore the constitutive and agonist-dependent endosomal trafficking of a1A- and a1B-ARs, using Yunaconitine CypHer5 technology and VSV-G epitope tagged receptors stably and transiently expressed in HEK 293 cells. In order to establish if the internalization pattern determines the signaling pathways and explains differences in the functional role of each subtype, we also analyzed the temporal relationship between internalization and the increase in intracellular calcium, a signal directly related to the interaction of ARs with the G-protein in the membrane, as well as intracellular signals not necessarily dependent on G proteins, such as activation of MAPKs. We analyzed the internalization kinetics of the a1-ARs, as well as the specific subcellular distribution of each subtype related to its internalization kinetics. For this purpose, the VSV-G tag was inserted into the amino terminal sequence 
of the a1A- anda1B-ARs that had been stably or transiently transfected in HEK293 cells. This tag is highly detectable with an anti VSV-G antibody labeled with the CypHer 5 fluorochrome which is able to monitor the trafficking of the receptors from the cell surface into acidic endosomal pathways in live cells. This dye is pH-sensitive and fluorescent only in acidic environments, but is non fluorescent at a neutral pH. Compared to most common methods such as green fluorescent proteins tags, this approach has the advantage of avoiding the fluorescent signal of membrane receptors. Given this particular property, the intracellular fluorescence corresponding to surface VSV-G tagged-receptor internalization could be assessed by real-time live cell imaging. We present evidence for two major conclusions. The first is that, a1A- and a1B-ARs, one of them located close to the inner face of the cell membrane whereas the other is deeper distributed in the cytosol.