PT  - JOURNAL ARTICLE
AU  - Willets, Jonathon M.
AU  - Mistry, Rajendra
AU  - Nahorski, Stefan R.
AU  - Challiss, R. A. John
TI  - Specificity of G Protein-Coupled Receptor Kinase 6-Mediated Phosphorylation and Regulation of Single-Cell M<sub>3</sub> Muscarinic Acetylcholine Receptor Signaling
AID  - 10.1124/mol.64.5.1059
DP  - 2003 Nov 01
TA  - Molecular Pharmacology
PG  - 1059--1068
VI  - 64
IP  - 5
4099  - http://molpharm.aspetjournals.org/content/64/5/1059.short
4100  - http://molpharm.aspetjournals.org/content/64/5/1059.full
SO  - Mol Pharmacol2003 Nov 01; 64
AB  - Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling. In contrast to the clear attenuating effect of K215RGRK6 expression on M3 mAChR regulation, dominant-negative forms of GRKs (K220RGRK2, K220RGRK3, K215RGRK5) and casein kinase 1α (K46RCK1α) were without effect. In addition, inhibition of a variety of second-messenger-regulated kinases and the tyrosine kinase Src also had no effect upon agonist-stimulated M3 mAChR regulation. To investigate further the desensitization process we have followed changes in inositol 1,4,5-trisphosphate in single SHSY5Y cells using the pleckstrin homology domain of PLCδ1 tagged with green fluorescent protein (eGFP-PHPLCδ1). Stimulation of cells with approximate EC50 concentrations of agonist before and after a desensitizing period of agonist exposure resulted in a marked attenuation of the latter response. Altered GRK6 activity, through overexpression of wild-type GRK6 or K215RGRK6, enhanced or reduced the degree of M3 mAChR desensitization, respectively. Taken together, our data indicate that M3 mAChR desensitization is mediated by GRK6 in human SH-SY5Y cells, and we show that receptor desensitization of phospholipase C signaling can be monitored in 'real-time' in single, living cells.