Abstract
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.
Highlights
Transgenic approaches have helped to define the key roles of M1 muscarinic acetylcholine1 receptors in cholinergic regulation of hippocampal function [5,6,7,8], and gaining a better understanding of the physiological and pathophysiological regulation of this mACh receptor subtype in hippocampal neurons remains a key objective
G protein-coupled receptor kinase 2 (GRK2)-mediated receptor phosphorylation is required for M1 mACh receptor desensitization, we introduced a single point mutation D110A to create both GRK2 and K220RGRK2 mutants, which are incapable of binding G␣q/11 [19]
We have previously shown using the IP3 biosensor eGFPPHPLC␦ that expression of GRK2 in hippocampal neurons leads to an almost complete suppression of M1 mACh receptor-mediated IP3 signaling and that this suppression is independent of kinase activity, because the catalytically inactive K220RGRK2 mutant completely inhibited signaling [18]
Summary
Transgenic approaches have helped to define the key roles of M1 muscarinic acetylcholine (mACh)1 receptors in cholinergic regulation of hippocampal function [5,6,7,8], and gaining a better understanding of the physiological and pathophysiological regulation of this mACh receptor subtype in hippocampal neurons remains a key objective. When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PHPLC␦, 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␣q/11.
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