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

6533b85bfe1ef96bd12bb52e

RESEARCH PRODUCT

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

Fermí Montó Miguel Pérez-aso Graeme Milligan Pilar D'ocon M.a. Noguera Vanessa Segura Domingo Barettino

subject

MAPK/ERK pathway Arrestins media_common.quotation_subject Blotting Western Kidney Real-Time Polymerase Chain Reaction Immunoenzyme Techniques Constitutive activity Receptors Adrenergic alpha-1 Concanavalin A Humans RNA Messenger PKC Enzyme Inhibitors Phosphorylation RNA Small Interfering Internalization Protein kinase A Molecular Biology Cells Cultured Protein Kinase C beta-Arrestins Protein kinase C media_common Mitogen-Activated Protein Kinase 1 G protein-coupled receptor kinase Mitogen-Activated Protein Kinase 3 ERK1/2 biology Reverse Transcriptase Polymerase Chain Reaction Kinase Chemistry Cell Biology beta-Arrestin 2 Molecular biology Adrenaline α1 receptors Endocytosis Mitogen-activated protein kinase biology.protein Phosphorylation Internalization Signal Transduction

description

AbstractWe analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-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 α1A- and α1B-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 α1A-AR, but not α1B-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. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.

year	journal	country	edition	language
2013-10-01	Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

https://doi.org/10.1016/j.bbamcr.2013.06.013