Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling.
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 1alpha (K46RCK1alpha) 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 PLCdelta1 tagged with green fluorescent protein (eGFP-PHPLCdelta1). 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.
Willets
Jonathon M
JM
Department of Cell Physiology and Pharmacology, University of Leicester, Maurice Shock Medical Sciences Building, University Road, Leicester, LE1 9HN, UK. jmw23@le.ac.uk.
Mistry
Rajendra
R
Nahorski
Stefan R
SR
Challiss
R A John
RA
D001285:Atropine / Q000494:pharmacology; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis; D004347:Drug Interactions; D054768:G-Protein-Coupled Receptor Kinases; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity; D051552:beta-Adrenergic Receptor Kinases
D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043587:Receptor, Muscarinic M3; D001285:Atropine; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6