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

• Published in: British journal of pharmacology 2001-09, Vol.134 (2), p.257-264
• Main Authors: Bundey, R A, Nahorski, S R
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell receptors; Cell structures and functions; desensitization; Fundamental and applied biological sciences. Psychology; Gq/11‐protein coupling; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); muscarinic acetylcholine receptor; receptor phosphorylation; SH‐SY5Y cell line; α1‐adrenoceptor
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0704229

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. 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. [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). 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. British Journal of Pharmacology (2001) 134, 257–264; doi:10.1038/sj.bjp.0704229