Homologous and heterologous uncoupling of muscarinic M(3) and alpha(1B) adrenoceptors to Galpha(q/11) in SH-SY5Y human neuroblastoma cells.
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.
Bundey
R A
RA
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, Leicester, LE1 9HN. rbundey@buckinstitute.org
Nahorski
S R
SR
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D016210:Methacholine Chloride / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D009638:Norepinephrine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011869:Radioligand Assay; D043587:Receptor, Muscarinic M3; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
C547716:ADRA1B protein, human; D000316:Adrenergic alpha-Agonists; D018721:Muscarinic Agonists; D043587:Receptor, Muscarinic M3; D018340:Receptors, Adrenergic, alpha-1; D011976:Receptors, Muscarinic; D013462:Sulfur Radioisotopes; D016210:Methacholine Chloride; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D009638:Norepinephrine