Homologous and heterologous uncoupling of muscarinic M3 and α1B adrenoceptors to Gαq/11 in SH‐SY5Y human neuroblastoma cells
Homologous and heterologous uncoupling of muscarinic M3 and α1B adrenoceptors to Gαq/11 in SH‐SY5Y human neuroblastoma cells
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SH-SY5Y 人神经母细胞瘤细胞中毒蕈碱 M3 和 α1B 肾上腺素受体与 Gαq/11 的同源和异源解偶联
DOI：
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
S. Nahorski
中科院分区：
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作者：
R. Bundey;S. Nahorski
本研究采用[35S] - gtp - γ - s结合方案，结合Gα亚基的免疫沉淀（IP）来研究Gq/11偶联受体在G蛋白激活水平上的脱敏性。来自SH‐SY5Y细胞的膜表达重组人α1B‐肾上腺素能受体（α1B‐AR）（内源性表达M3毒蕈碱乙酰胆碱受体（M3‐AChR）），在对去甲肾上腺素或甲胆碱的反应中表现出浓度依赖性的Gq/11激活。在膜制备和[35S] - gtp - γ - s IP实验中，用激动剂预处理完整细胞表明，由于Gq/11在激动剂二次刺激下的激活显著降低，这两种受体都通过激动剂预处理而同源脱敏。α1B‐AR刺激对M3‐AChR的异源脱敏是有效的。PKC抑制剂Ro‐31‐8220 （10 μM）在阻止激动剂介导的受体脱敏方面无效。[32P]Pi‐标记的细胞可以检测到受体磷酸化的增加。Phorbol 12,13 dibutyrate (PDBu) （1 μM）可以有效地产生Ro‐31‐8220 （10 μM）敏感的α1B‐AR磷酸化，但不能检测到M3‐AChR磷酸化。去甲肾上腺素（30 μM）刺激α1B‐AR磷酸化，Ro‐31‐8220 （10 μM）可部分抑制α1B‐AR磷酸化。甲基胆碱（100 μM）孵育可增加M3‐AChR的磷酸化，而这种效应对Ro‐31‐8220 （10 μM）不敏感。这些发现表明，[35S]‐GTPγS‐Gα‐亚基IP可以用来估计受体脱敏，作为受体- G -蛋白偶联的下降。α1B‐AR和M3‐AChR都经历了快速的同源脱敏，这与受体磷酸化的增加有关。α1B‐AR刺激产生的M3‐AChR的异源脱敏与M3‐AChR磷酸化的可检测到的增加无关，这表明受体磷酸化不一定是脱敏的先决条件。
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.