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

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

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