Willets Jonathon M, Nahorski Stefan R, Challiss R A John
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom.
J Biol Chem. 2005 May 13;280(19):18950-8. doi: 10.1074/jbc.M412682200. Epub 2005 Mar 2.
When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PH(PLC delta), G protein-coupled receptor kinase 2 (GRK2) can suppress M1 muscarinic acetylcholine (mACh) receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism, most likely involving the direct binding of the RGS homology domain of GRK2 to G alpha(q/11). To define the importance of this mechanism in comparison with classical, phosphorylation-dependent receptor regulation by GRKs, we have examined M1 mACh receptor signaling in hippocampal neurons following depletion of GRK2 and also in the presence of non-G alpha(q/11)-binding GRK2 mutants. Depletion of neuronal GRK2 using an antisense strategy almost completely inhibited M1 mACh receptor desensitization without enhancing acute agonist-stimulated phospholipase C activity. By stimulating neurons with a submaximal agonist concentration before (R1) and after (R2) a period of exposure to a maximal agonist concentration, an index (R2/R1) of agonist-induced desensitization of signaling could be obtained. Co-transfection of neurons with either a non-G alpha(q/11)-binding (D110A) GRK2 mutant or the catalytically inactive (D110A,K220R)GRK2 did not suppress acute M1 mACh receptor-stimulated inositol 1,4,5-trisphosphate production. However, using the desensitization (R2/R1) protocol, it could be shown that expression of (D110A)GRK2 enhanced, whereas (D110A,K220R)GRK2 inhibited, agonist-induced M1 mACh receptor desensitization. In Chinese hamster ovary cells, the loss of G alpha(q/11) binding did not affect the ability of the (D110A)GRK2 mutant to phosphorylate M1 mACh receptors, whereas expression of (D110A,K220R)GRK2 had no effect on receptor phosphorylation. These data indicate that in hippocampal neurons endogenous GRK2 is a key regulator of M1 mACh receptor signaling and that the regulatory process involves both phosphorylation-dependent and -independent mechanisms.
当与肌醇1,4,5 - 三磷酸生物传感器eGFP - PH(PLCδ)共表达时，G蛋白偶联受体激酶2（GRK2）可通过一种不依赖磷酸化的机制抑制海马神经元中M1毒蕈碱型乙酰胆碱（mACh）受体介导的磷脂酶C信号传导，这很可能涉及GRK2的RGS同源结构域与Gα(q/11)的直接结合。为了确定与GRKs经典的、依赖磷酸化的受体调节相比，这种机制的重要性，我们检测了GRK2缺失后以及存在非Gα(q/11)结合的GRK2突变体时海马神经元中M1 mACh受体的信号传导。采用反义策略耗尽神经元中的GRK2几乎完全抑制了M1 mACh受体脱敏，而没有增强急性激动剂刺激的磷脂酶C活性。通过在最大激动剂浓度暴露一段时间之前（R1）和之后（R2）用次最大激动剂浓度刺激神经元，可以获得激动剂诱导的信号脱敏指数（R2/R1）。用非Gα(q/11)结合（D110A）GRK2突变体或催化失活的（D110A，K220R）GRK2共转染神经元，均未抑制急性M1 mACh受体刺激的肌醇1,4,5 - 三磷酸生成。然而，使用脱敏（R2/R1）方案可以表明，（D110A）GRK2的表达增强了激动剂诱导的M1 mACh受体脱敏，而（D110A，K220R）GRK2则抑制了这种脱敏。在中国仓鼠卵巢细胞中，Gα(q/11)结合的丧失并不影响（D110A）GRK2突变体磷酸化M1 mACh受体的能力，而（D110A，K220R）GRK2的表达对受体磷酸化没有影响。这些数据表明，在海马神经元中内源性GRK2是M1 mACh受体信号传导的关键调节因子，并且调节过程涉及依赖磷酸化和不依赖磷酸化的机制。
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