Endogenous G Protein-coupled Receptor Kinase 6 Regulates M3 Muscarinic Acetylcholine Receptor Phosphorylation and Desensitization in Human SH-SY5Y Neuroblastoma Cells

Jonathon M Willets,R A John Challiss,Stefan R Nahorski

doi:10.1074/jbc.m111217200

Abstract

We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M(3) muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M(3) mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 ((K215R)GRK6). (K215R)GRK6 expression inhibited methacholine-stimulated M(3) mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5'-O-(3-[(35)S]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M(3) mACh receptor-G alpha(q/11) uncoupling was attenuated by 50% in cells expressing (K215R)GRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase (K215R)GRK5 had no effect on M(3) mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [(3)H]inositol phosphate accumulations were more sustained in cells expressing (K215R)GRK6 compared with control and (K215R)GRK5-expressing cells, whereas (K215R)GRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF(4)(-). The ability of (K215R)GRK6 to inhibit agonist-mediated M(3) mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M(3) mACh receptor desensitization in the SH-SY5Y cell line.

Highlights

(7, 8) have been implicated in G protein-coupled receptor desensitization through phosphorylation of serine and threonine residues within the third intracellular loop or C-terminal tail of G protein-coupled receptors
We have recently shown that recombinant G protein-coupled receptor kinase 6 (GRK6) can enhance both M3 muscarinic acetylcholine (mACh) receptor phosphorylation and receptor/ G␣q/11 uncoupling, subsequently increasing desensitization of the endogenously expressed M3 mACh receptor in the SHSY5Y neuroblastoma cell line [13]
These data are suggestive of a role for endogenous GRK6 in the regulation of M3 mACh receptor desensitization

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Creation of Stably Transfected Dominant-negative GRK Cell Lines—SH-SY5Y human neuroblastoma cells were cultured in minimal essential medium containing 5% fetal and 5% new born calf serum, penicillin (100 units/ml), streptomycin (100 ␮g/ml), and fungizone (2.5 ␮g/ml) (Invitrogen). M3 mACh receptor internalization was assessed after treatment with either vehicle or methacholine (100 ␮M) for 0 – 60 min at 37 °C, prior to intensive washing (four times with 1 ml of ice-cold Krebs buffer, pH 7.4). Receptor internalization was determined as the percentage of loss of specific [3H]NMS-binding sites after methacholine treatment when compared with vehicle-treated controls. NaCl. After centrifugation the cells were resuspended in Krebs buffer, pH 7.4, at 37 °C for 15 min prior to the addition of either vehicle or MCh (100 ␮M). In addition to desensitization experiments, M3 mACh receptor activation of G␣q/11 was assessed in nonpretreated cell membranes via generation of methacholine (300 nM to 1 mM) concentration-response curves.

RESULTS

Kinase activity was assessed by addition of dephosphorylated

DISCUSSION