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

Perez-Aso, M., Segura, V., Montó, F., Barettino, D., Noguera, M.A., Milligan, G. and D'Ocon, P. (2013) The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation. Biochimica et Biophysica Acta: Molecular Cell Research, 1833(10), pp. 2322-2333. (doi: 10.1016/j.bbamcr.2013.06.013)

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Abstract

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Milligan, Professor Graeme
Authors: Perez-Aso, M., Segura, V., Montó, F., Barettino, D., Noguera, M.A., Milligan, G., and D'Ocon, P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Biochimica et Biophysica Acta: Molecular Cell Research
ISSN:0167-4889

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