Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors.
Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors.
复制标题
涉及蛋白质磷酸化的多种机制与毒蕈碱受体的脱敏有关。
DOI：
10.1016/0024-3205(95)00033-3
复制
发表时间：
1995
期刊：
影响因子：
6.1
通讯作者：
Richardson,RM
中科院分区：
文献类型：
--
作者：
Hosey,MM;Benovic,JL;DebBurman,SK;Richardson,RM
激动剂在几种细胞类型中诱导m2毒蕈碱受体（mAChR）的磷酸化。这种磷酸化与脱敏有关。已经使用几种体外方法研究了mAChR磷酸化的潜在机制。蛋白激酶C磷酸化的纯化和重建的m2 mAChR的化学计量的约5摩尔P/摩尔受体，这种磷酸化导致的受体激活G蛋白的能力降低。虽然PKC的磷酸化不受激动剂与mAChR结合的调节，但异源三聚体G蛋白能够完全阻断PKC介导的作用。如果在体内发生显著的受体/G蛋白偶联，则需要激动剂来促进G蛋白从受体解离并揭示PKC的磷酸化位点。G蛋白偶联受体激酶（GRK）家族的成员也磷酸化的纯化和重建的m2 mAChR。与PKC相反，GRKs严格以激动剂依赖性方式磷酸化m2 mAChR。GRK介导的磷酸化干扰受体/G蛋白偶联。此外，磷酸化允许arrestin与m2 mAChR结合，这应该进一步有助于脱敏。使用一种新的策略，不需要纯化和重建受体的GRK研究，m3 mAChR被揭示为基板的GRKs。对于m2和m3受体亚型，最有效的激酶是GRK 2和3。低浓度的G蛋白和膜磷脂刺激这些酶的受体磷酸化。因此，涉及蛋白磷酸化的多种机制似乎有助于mAChR脱敏的整个过程。
Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.