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

Apr 30, 2004The Journal of neuroscience : the official journal of the Society for Neuroscience

Imaging muscarinic receptor signaling in memory neurons: how receptor kinases regulate it with and without phosphorylation

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Abstract

The introduction of catalytically inactive GRK6 partially reversed the attenuation of receptor responsiveness in hippocampal neurons.

Initial experiments indicated a predominant M1 mACh receptor population linked to IP3 signaling.
Stimulating neurons with methacholine showed a significant decrease in responsiveness after prior exposure to a high concentration of the agonist.
Inhibition of GRK6 activity reduced receptor desensitization, while overexpression of wild-type GRK6 enhanced it.
Manipulating GRK2 activity dramatically inhibited agonist-stimulated IP3 production, suggesting a non-phosphorylation dependent regulation of M1 mACh receptors.
Both phosphorylation-dependent and -independent mechanisms are implicated in the regulation of M1 mACh receptors in hippocampal neurons.

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We used the inositol 1,4,5-trisphosphate (IP3) biosensor, the pleckstrin homology (PH) domain of PLCdelta1 (phospholipase C) tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta)), 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 (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 ((K220R)GRK2) 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 Galpha(q/11). Together, our data suggest a role for both phosphorylation-dependent and -independent regulation of M1 mACh receptors in hippocampal neurons.

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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

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