Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons : evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases

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.