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.
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