Differences in the signaling pathways of α1A- and α1B-adrenoceptors are related to different endosomal targeting

Aims: To 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. Methods: Using 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 Conclusions: Constitutive 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

The three α₁-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α<sub>1</sub>-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α<sub>1A</sub>- and α<sub>1B</sub>-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α<sub>1A</sub>-AR, but not α<sub>1B</sub>-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α<sub>1D</sub>-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α<sub>1D</sub> antagonist BMY7378. Following activation by phenylephrine, α<sub>1D</sub>-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α<sub>1D</sub>-AR subtype was not observed via CypHer5 technology. The three α<sub>1</sub>-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α<sub>1A</sub>-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization