Abstract

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-PH_PLCδ1). 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.