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

Biochim Biophys Acta. 2013 Oct;1833(10):2322-33. doi: 10.1016/j.bbamcr.2013.06.013. Epub 2013 Jun 21.

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 (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.

Keywords: AT(1A)R; Adrenaline α(1) receptors; BSA; Bmax; ConA; Constitutive activity; DMSO; EGFR; ERK1/2; G-protein coupled receptor; G-protein-coupled receptor kinase; GAPDH; GPCR; GRK; HEK; Internalization; KRH; Krebs Ringer Hepes; MAPK; PBST; PCR; PE; PKA; PKC; PTHR; PZ; angiotensin receptor type 1A; bovine serum albumin; concanavalin A; dimethyl sulfoxide; epidermal growth factor receptor; glyceraldehyde-3-phosphate dehydrogenase; human embryonic kidney; maximum number of binding sites; mitogen-activated protein kinase; parathyroid hormone receptor type 1; phenylephrine; phosphate-buffered saline with 0.1% Tween 20; polymerase chain reaction; prazosin; protein kinase A; protein kinase C; α(1) adrenoceptor; α(1)-AR; β(2) adrenoceptor; β(2)-AR.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arrestins / antagonists & inhibitors
  • Arrestins / genetics
  • Arrestins / metabolism
  • Blotting, Western
  • Cells, Cultured
  • Concanavalin A / pharmacology
  • Endocytosis / drug effects
  • Endocytosis / physiology*
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Immunoenzyme Techniques
  • Kidney / cytology
  • Kidney / metabolism
  • Mitogen-Activated Protein Kinase 1 / genetics
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Mitogen-Activated Protein Kinase 3 / genetics
  • Mitogen-Activated Protein Kinase 3 / metabolism*
  • Phosphorylation
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • RNA, Messenger / genetics
  • RNA, Small Interfering / genetics
  • Real-Time Polymerase Chain Reaction
  • Receptors, Adrenergic, alpha-1 / genetics
  • Receptors, Adrenergic, alpha-1 / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • beta-Arrestin 2
  • beta-Arrestins

Substances

  • ARRB2 protein, human
  • Arrestins
  • Enzyme Inhibitors
  • RNA, Messenger
  • RNA, Small Interfering
  • Receptors, Adrenergic, alpha-1
  • beta-Arrestin 2
  • beta-Arrestins
  • Concanavalin A
  • Protein Kinase C
  • MAPK1 protein, human
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3