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Molecular Pharmacology

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Research ArticleArticle

Specificity of G Protein-Coupled Receptor Kinase 6-Mediated Phosphorylation and Regulation of Single-Cell M3 Muscarinic Acetylcholine Receptor Signaling

Jonathon M. Willets, Rajendra Mistry, Stefan R. Nahorski and R. A. John Challiss
Molecular Pharmacology November 2003, 64 (5) 1059-1068; DOI: https://doi.org/10.1124/mol.64.5.1059
Jonathon M. Willets
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Rajendra Mistry
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Stefan R. Nahorski
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R. A. John Challiss
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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-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.

  • Received April 15, 2003.
  • Accepted July 28, 2003.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 64 (5)
Molecular Pharmacology
Vol. 64, Issue 5
1 Nov 2003
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Research ArticleArticle

Specificity of G Protein-Coupled Receptor Kinase 6-Mediated Phosphorylation and Regulation of Single-Cell M3 Muscarinic Acetylcholine Receptor Signaling

Jonathon M. Willets, Rajendra Mistry, Stefan R. Nahorski and R. A. John Challiss
Molecular Pharmacology November 1, 2003, 64 (5) 1059-1068; DOI: https://doi.org/10.1124/mol.64.5.1059

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Research ArticleArticle

Specificity of G Protein-Coupled Receptor Kinase 6-Mediated Phosphorylation and Regulation of Single-Cell M3 Muscarinic Acetylcholine Receptor Signaling

Jonathon M. Willets, Rajendra Mistry, Stefan R. Nahorski and R. A. John Challiss
Molecular Pharmacology November 1, 2003, 64 (5) 1059-1068; DOI: https://doi.org/10.1124/mol.64.5.1059
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