title: Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons: evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases.
abstract: 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.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom. jmw23@le.ac.uk
authors.forename: Jonathon M
authors.initials: JM
authors.lastname: Willets
authors.forename: Mark S
authors.initials: MS
authors.lastname: Nash
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Nahorski
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D016210:Methacholine Chloride / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D054803:Phospholipase C delta; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D043643:Receptor, Muscarinic M1 / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D010738:Type C Phospholipases / Q000235:genetics; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D007527:Isoenzymes; D008164:Luminescent Proteins; D018721:Muscarinic Agonists; D043643:Receptor, Muscarinic M1; D011993:Recombinant Fusion Proteins; D016210:Methacholine Chloride; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517897:Grk5 protein, rat; D010738:Type C Phospholipases; D054803:Phospholipase C delta; C518026:Plcd1 protein, rat