Gq-coupled muscarinic receptor-induced activation of the ERK5 pathway does not require receptor internalization (original) (raw)

2013

Abstract

<p>(A) CHO cells stably overexpressing <i>wild-type</i> muscarinic M3 receptor or internalization-deficient M3 receptor (SASS motif mutant, characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#B10" target="_blank">10</a>]) were transfected with HA-ERK5. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM). HA-ERK5 was immunoprecipitated with an anti-HA agarose-conjugated antibody as detailed in the Materials and Methods section. ERK5 phosphorylation was assessed in the immunoprecipitate using a phosphospecific antibody. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, two-tailed T-test). (B) Quantification of cell surface receptor density was performed through [³H]-NMS binding at 4°C. The two cell types utilised in (A) were serum-starved for 2h and stimulated with carbachol (100µM) for 30 minutes. Data were normalised to unspecific binding (atropine treatment) and binding percentage was expressed as the mean +/- SEM of 3 independent experiments.</p

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