Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling - PubMed

Affiliations

• PMID: 14573754
• DOI: 10.1124/mol.64.5.1059

Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling

Jonathon M Willets et al. Mol Pharmacol. 2003 Nov.

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 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.

Similar articles

• Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons: evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases.

  Willets JM, Nash MS, Challiss RA, Nahorski SR. Willets JM, et al. J Neurosci. 2004 Apr 28;24(17):4157-62. doi: 10.1523/JNEUROSCI.5506-03.2004. J Neurosci. 2004. PMID: 15115810 Free PMC article.

• Cross talk between m3-muscarinic and beta(2)-adrenergic receptors at the level of receptor phosphorylation and desensitization.

  Budd DC, Challiss RA, Young KW, Tobin AB. Budd DC, et al. Mol Pharmacol. 1999 Oct;56(4):813-23. Mol Pharmacol. 1999. PMID: 10496966

• [Mechanisms of regulation and function of G-protein coupled receptor kinases].

  Sobierajska K, Fabczak H, Fabczak S. Sobierajska K, et al. Postepy Biochem. 2005;51(4):421-9. Postepy Biochem. 2005. PMID: 16676577 Review. Polish.

• Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors.

  Hosey MM, Benovic JL, DebBurman SK, Richardson RM. Hosey MM, et al. Life Sci. 1995;56(11-12):951-5. doi: 10.1016/0024-3205(95)00033-3. Life Sci. 1995. PMID: 10188798 Review.

Cited by

• Alterations of Expression of the Serotonin 5-HT4 Receptor in Brain Disorders.

  Rebholz H, Friedman E, Castello J. Rebholz H, et al. Int J Mol Sci. 2018 Nov 13;19(11):3581. doi: 10.3390/ijms19113581. Int J Mol Sci. 2018. PMID: 30428567 Free PMC article. Review.

• Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle.

  Morris GE, Nelson CP, Brighton PJ, Standen NB, Challiss RA, Willets JM. Morris GE, et al. Am J Physiol Cell Physiol. 2012 Mar 1;302(5):C723-34. doi: 10.1152/ajpcell.00202.2011. Epub 2011 Dec 7. Am J Physiol Cell Physiol. 2012. PMID: 22159081 Free PMC article.

• Beneficial metabolic effects caused by persistent activation of beta-cell M3 muscarinic acetylcholine receptors in transgenic mice.

  Gautam D, Ruiz de Azua I, Li JH, Guettier JM, Heard T, Cui Y, Lu H, Jou W, Gavrilova O, Zawalich WS, Wess J. Gautam D, et al. Endocrinology. 2010 Nov;151(11):5185-94. doi: 10.1210/en.2010-0519. Epub 2010 Sep 15. Endocrinology. 2010. PMID: 20843999 Free PMC article.

• Targeting G protein-coupled receptor kinases (GRKs) in Heart Failure.

  Brinks H, Koch WJ. Brinks H, et al. Drug Discov Today Dis Mech. 2010 Summer;7(2):e129-e134. doi: 10.1016/j.ddmec.2010.07.007. Drug Discov Today Dis Mech. 2010. PMID: 21218155 Free PMC article.

• Crosstalk between beta-2-adrenoceptor and muscarinic acetylcholine receptors in the airway.

  Pera T, Penn RB. Pera T, et al. Curr Opin Pharmacol. 2014 Jun;16:72-81. doi: 10.1016/j.coph.2014.03.005. Epub 2014 Apr 17. Curr Opin Pharmacol. 2014. PMID: 24747364 Free PMC article. Review.

Publication types

MeSH terms

Substances

LinkOut - more resources

• Full Text Sources

  • Elsevier Science

• Miscellaneous

  • NCI CPTAC Assay Portal