The three α 1 -adrenoceptor subtypes show different spatio-temporal mechanisms of erk1/2 phosphorylation.

F Montó1, M Perez-Aso1, V Segura1, D Barettino2, MA Noguera1, G Milligan3, P D´Ocon1. 1Universitat de Valencia, Departament de Farmacologia, Spain, 2Instituto de Biomedicina Valencia (CSIC), Biología de la Acción Hormonal, Spain, 3University of Glasgow, Molecular Pharmacology Group, Institute of Neuroscience and Psychology, UK

Background: The α1-adrenoceptors (α1-ARs) stimulate Gq/11 proteins and promote an increase of intracellular IP3 and calcium content as well as PKC activation (1). In addition, α1-ARs promote mitogen-activated protein kinase (MAPK) activation (2). Phosphorylation of α1-ARs by G-protein coupled receptor kinases (GRKs) or second-messenger-dependent protein kinases (PKA and PKC) promotes high affinity binding of cytoplasmatic β-arrestins to the receptor, resulting in its desensitization and endocytosis (3). β-arrestin recruitment is implicated in ERK1/2 activation over time mediated by GPCR, but the intervention of β-arrestin has not been previously examined in the signaling pathways activated by the three α1-AR subtypes. Aims: To study the temporal and spatial patterns of ERK1/2 phosphorilation after stimulation of each human α1-AR stably expressed in HEK293 cells and to determine such signals were modified by β-arrestin 2 transcriptional silencing, by blocking receptor endocytosis with concanavalin A (ConA), and by inhibiting PKC with Ro 31-8425. Methods: HEK293 cells were starved for 4h in serum-free medium. Prior to stimulation with phenylephrine (PHE, 0.1-10 µM), cells were treated for 30 min with ConA (250μg/ml) or with Ro 31-8425 (1μM) for 15 min. In some experiments, the expression of β-arrestin-2 was silenced using HP validated siRNA duplexes targeting human ARRB2 (Hs_ARRB2_10 siRNA). AllStars non-targeting siRNA (Catalog no. 1027284, Qiagen) was used as negative control. Suppression of the target gene was confirmed by western blot. 15μg of cellular extracts were incubated with SDS-sample buffer, separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes for immunoblotting. Results: Activation of the α1A subtype by PHE elicits a fast ERK1/2 activation (peak 1-5 min) followed by a low and progressive reduction of phospho-ERK1/2 which returned to basal levels after 1h. There is a similar initial increase in α1B- and α1D-subtypes which returns to basal level after 10 min. Inhibition of PKC resulted in a dramatic decrease (≈70%) in ERK1/2 activation by α1A and α1B subtypes at early time periods (1-5 min), but little inhibition was observed at longer time points (> 10 min) in the case of α1A -AR. By the contrary, ConA and β-arrestin 2 silencing inhibits the sustained ERK1/2 signal elicited only by the α1A subtype without modification of p-ERK1/2 mediated by α1B and α1D subtypes. Ro 31-8425 did not inhibit the early increased p-ERK level induced by activation of α1D-ARs, but extended the period of ERK1/2 phosphorylation. ConA did not modify the kinetic of p-ERK1/2 signal mediated by α1D-AR. Conclusion: The three α1-AR subtypes present different spatio-temporal patterns of ERK1/2 phosphorylation. Only α1A-AR elicits a late and sustained p-ERK signal which is dependent on β-arrestin and endocytosis. This late ERK1/2 activation may be related to the anti-apoptotic-cardioprotective effect of α1A-AR.

(1) Perez, D. M., DeYoung, M. B., and Graham, R. M. (1993) Mol.Pharmacol. 44, 784-795

(2) Keffel, S., Alexandrov, A., Goepel, M., and Michel, M. C. (2000) Biochem.Biophys.Res.Commun. 272, 906-911

(3) Piascik, M. T. and Perez, D. M. (2001) J.Pharmacol.Exp.Ther. 298, 403-410

ACKNOWLEDGMENTS: Study supported by a grant from Universitat de València (nº 2011-0739).