Differences in the signalling pathways of α 1A - and α 1B -adrenoceptors are related to different endosomal targeting

C Arce, V Segura, M Perez-Aso, F Montó, E Carceller, MA Noguera, MD Ivorra, P D´Ocon. Universidad de Valencia, Departamento de Farmacologia, Spain

AIMS: The three α1 adrenoceptor (ARs) subtypes (α1A, α1B and α1D) posses different characteristics related to its cellular location, trafficking, constitutive activity and signalling pathways (1). The aims of the present work were to explore the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-ARs and to establish if the internalization pattern conditions the signaling pathways of each subtype.

METHODS: VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, were obtained by electroporation (300V, 50ms, 2mm gap) or using LipofectamineTM, respectively. After incubation with CypHer5 linked anti-VSV antibody (5 μg/ml in Krebs-Ringer-Hepes buffer, KHB, at 4ºC for 1h), we analyzed by confocal microscopy the constitutive and agonist-induced (phenylephrine: PHE, 100 μM) internalization of each subtype, and the temporal relationship (1-15 min) between agonist induced internalization and the increase in intracellular calcium (determined by confocal microscopy after incubation with FLUO-3, 5μM, 2h at 4ºC) or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). The experiments were performed in KHB at 37ºC aerated with a mixture of 95% O2/5% CO2. To determine the influence of the receptor endocytosis and the membrane integrity in the signalling pathways, some experiments were performed after incubation (30 min) with the inhibitor of endocytosis concanavalin A (250 μg/ml) or the cholesterol-depleting agent methyl-β-ciclodextrin (mβcd, 10mM).

RESULTS AND CONCLUSIONS: Confocal experiments demonstrated constitutive as well as PHE-induced trafficking of α1A- and α1B-ARs which maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that condition their signaling pathways by PHE activation: α1A-ARs when located in the plasma membrane, signal through calcium, p38 and ERK1/2 pathways but, when translocate to profound endosomes, continue signaling through ERK1/2 by a mechanism sensitive to β-arrestin-2 (2) and concanavalin A, but insensitive to mβcd treatment. PHE-activated α1B-ARs signals through calcium, ERK1/2 and p38 only when located in the membrane, but the signals disappear after endocytosis or by disruption of the membrane lipid rafts by mβcd.

ACKNOWLEDGMENTS: Study supported by a grant from Universitat de València (nº 2011-0739). Graeme Milligan from the University of Glasgow provided VSV-tagged α1A- and α1B-ARs.

(1) Koshimizu TA, et al. (2003). Recent advances in alpha1-adrenoceptor pharmacology. Pharmacol Ther 98: 235-244.

(2) Perez-Aso M, et al. The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation. Mol Pharmacol (in review).