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Title: Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons : evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases
Authors: Willets, Jonathon M.
Nash, Mark S.
Challiss, R. A.
Nahorski, Stefan R.
First Published: 28-Apr-2004
Publisher: Society for Neuroscience
Citation: The Journal of Neuroscience, 2004, 24 (17), pp. 4157-4162
Abstract: We used the inositol 1,4,5-trisphosphate (IP[subscript 3]) biosensor, the pleckstrin homology (PH) domain of PLCδ1 (phospholipase C) tagged with enhanced green fluorescent protein (eGFP-PH[subscript PLCδ]), to examine muscarinic acetylcholine (mACh) receptor regulation of phospholipase C/IP[subscript 3] signaling in intact single hippocampal neurons in "real time." Initial experiments produced a pharmacological profile consistent with the presence of a predominant M[subscript 1] mACh receptor population coupled to the IP[subscript 3] response. To investigate M[subscript 1] mACh receptor regulation, neurons were stimulated with approximate EC[subscript 50] 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 [superscript K215R]GRK6, 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 ([superscript K220R]GRK2) almost completely inhibited agonist-stimulated IP[subscript 3] 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 Gα[subscript q/11]. Together, our data suggest a role for both phosphorylation-dependent and -independent regulation of M[subscript 1] mACh receptors in hippocampal neurons.
DOI Link: 10.1523/JNEUROSCI.5506-03.2004
ISSN: 0270-6474
eISSN: 1529-2401
Version: Publisher Version
Status: Peer-reviewed
Type: Journal Article
Rights: Copyright © 2004, Society for Neuroscience. Deposited with reference to the publisher’s archiving policy available on the SHERPA/RoMEO website.
Appears in Collections:Published Articles, Dept. of Cell Physiology and Pharmacology

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