Abstract
AimsTo compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype.MethodsUsing CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot).Results and ConclusionsConstitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin
Highlights
There is evidence that for the a1-ARs the constitutive and agonist-driven cellular internalization differs between a1A- and a1B- subtypes
Many lines of evidence suggest that activation of a1-ARs induce phosphorylation of mitogenactivated protein kinases (MAPKs) as p38 and extracellular signalregulated kinase (ERK) [6,7,8]
In order to establish if the internalization pattern determines the signaling pathways and explains differences in the functional role of each subtype, we 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 (ERK1/2 and p38)
Summary
Using CypHer technology and VSV-G epitope tagged a1A- and a1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot)
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