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 ERIC 1 and 2 (ERK1/2) by the three alpha(1)-adrenoceptor (alpha(1)-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 ERIC phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting beta-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of alpha(1A)- and alpha(1B)-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 alpha(1A)-AR, but not alpha(1B)-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by p-arrestin 2 knockdown or concanavalin A treatment. alpha(1D)-AR displayed constitutive ERIC phosphorylation, which was reduced by incubation with prazosin or the selective am antagonist BMY7378. Following activation by phenylephrine, alpha(1D)-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the alpha(1D)-AR subtype was not observed via CypHer5 technology. The three alpha(1)-AR subtypes present different spatio-temporal patterns of receptor internalization, and only alpha(1A)-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on beta-arrestin 2 mediated internalization. (C) 2013 Elsevier B.V. All rights reserved.