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The Journal of Neuroscience, April 28, 2004, 24(17):4157-4162; doi:10.1523/JNEUROSCI.5506-03.2004

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Imaging of Muscarinic Acetylcholine Receptor Signaling in Hippocampal Neurons: Evidence for Phosphorylation-Dependent and -Independent Regulation by G-Protein-Coupled Receptor Kinases

Jonathon M. Willets, Mark S. Nash, R. A. John Challiss, and Stefan R. Nahorski

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom

We used the inositol 1,4,5-trisphosphate (IP₃) biosensor, the pleckstrin homology (PH) domain of PLC1 (phospholipase C) tagged with enhanced green fluorescent protein (eGFP-PH_PLC), to examine muscarinic acetylcholine (mACh) receptor regulation of phospholipase C/IP₃ signaling in intact single hippocampal neurons in "real time." Initial experiments produced a pharmacological profile consistent with the presence of a predominant M₁ mACh receptor population coupled to the IP₃ response. To investigate M₁ mACh receptor regulation, neurons were stimulated with approximate EC₅₀ concentrations of the mACh receptor agonist methacholine before (R1) and after (R2) a short (60 sec) exposure to a high concentration of agonist. This resulted in a marked attenuation in the R2 relative to R1 response. Inhibition of endogenous GRK6 (G-protein-coupled receptor kinase) activity, by the introduction of catalytically inactive ^K215RGRK6, partially reversed the attenuation of agonist-induced responsiveness, whereas overexpression of wild-type GRK6 increased receptor desensitization. Manipulation of endogenous GRK2 activity through introduction of either wild-type or catalytically inactive GRK2 (^K220RGRK2) almost completely inhibited agonist-stimulated IP₃ production, implying a phosphorylation-independent regulation of M₁ mACh receptor signaling, most probably mediated by a GRK2 N-terminal RGS-like (regulator of G-protein signaling) domain interaction with GTP-bound G_q/11. Together, our data suggest a role for both phosphorylation-dependent and -independent regulation of M₁ mACh receptors in hippocampal neurons.

Key words: G-protein-coupled receptor kinase; GRK; GRK2; GRK6; M₁ muscarinic acetylcholine receptor; hippocampal neurons; inositol 1,4,5-trisphosphate; IP₃ biosensor; confocal imaging

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Received Dec 15, 2003; revised March 10, 2004; accepted March 10, 2004.

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