Homologous and heterologous uncoupling of muscarinic M3 and α1B adrenoceptors to Gαq/11in SH-SY5Y human neuroblastoma cells

Abstract

1. The present study employed a [35S]-GTPγS binding protocol in conjunction with immunoprecipitation (IP) of the Gα subunits to investigate the desensitization of Gq/11-coupled receptors at the level of the G-protein activation. Membranes from SH-SY5Y cells expressing the recombinant human α1B-adrenoceptor (α1B-AR) (and endogenously expressing the M3 muscarinic acetylcholine receptor (M3-AChR)) exhibited Gq/11 activation in a concentration-dependent manner in response to noradrenaline or methacholine. 2. Pre-treatment of intact cells with agonist prior to membrane preparation and use in the [35S]-GTPγS IP assay demonstrated that both receptors were homologously desensitized by pre-treatment with agonist since the Gq/11 activation in response to a secondary challenge with agonist was markedly reduced. Stimulation of α1B-AR was effective at heterologously desensitizing the M3-AChR. The PKC inhibitor, Ro-31-8220 (10 μM) was ineffective at preventing the agonist-mediated receptor desensitization. 3. [32P]Pi-labelled cells allowed the detection of increases in receptor phosphorylation. Phorbol 12,13 dibutyrate (PDBu) (1 μM) was effective at producing a Ro-31-8220 (10 μM)-sensitive, detectable increase in α1B-AR but not M3-AChR phosphorylation. Noradrenaline (30 μM) stimulated α1B-AR phosphorylation, which could be partially inhibited by Ro-31-8220 (10 μM). The phosphorylation of M3-AChR was increased by methacholine (100 μM) incubation and this effect appeared to be insensitive to Ro-31-8220 (10 μM). 4. These findings demonstrate that [35S]-GTPγS-Gα-subunit IP can be used to estimate receptor desensitization as a decline in receptor-G-protein coupling. Both the α1B-AR and M3-AChR undergo rapid homologous desensitization that is associated with an increase in receptor phosphorylation. The heterologous desensitization of M3-AChR produced by α1B-AR stimulation is not associated with a detectable increase in M3-AChR phosphorylation, suggesting that receptor phosphorylation is not necessarily a prerequisite for desensitization.