Abstract
The ability to assess whether individual proteins are involved in the signalling or regulation of G protein-coupled receptor signalling is highly dependent on the pharmacological tools available. In the absence of appropriate pharmacological agents, alternative molecular approaches have been developed to alter either protein function or expression. This has included the use of mutants, for example catalytically inactive (kinase-dead) enzymes, which when overexpressed function as dominant negatives to inhibit endogenous enzyme function, and more latterly small (21–23 bp) interfering RNA dsRNA oligos, whose antisense strand is designed complementary to the target protein mRNA and which can be used to deplete target protein expression. Critically, the success of these approaches depends on the transfection efficiency, and the chosen experimental assay in the cell type studied. Therefore, three transfection techniques and their merits and drawbacks are described. In addition, one method of examining G protein-coupled receptor (GPCR) regulation, combining siRNA-mediated GRK depletion and imaging of fluorescent GPCR signalling reporter biosensors in difficult-to-transfect cells is briefly described.
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References
Fredriksson R., Lagerstrom, M.C., Lundin, L.G. and Schioth, H.B. (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272.
Pierce, K.L., Premont, R.T. and Lefkowitz, R.J. (2002) Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650.
Wilson, S., Bergsma, D.J., Chambers, J.K., Muir, A.I., Fantom, K.G., Ellis, C., Murdock, P.R., Herrity, N.C. and Stadel, J.M. (1998) Orphan G-protein-coupled receptors: the next generation of drug targets? Br. J. Pharmacol. 125, 1387–1392.
Willets, J.M., Challiss, R.A. and Nahorski, S.R. (2003) Non-visual GRKs: are we seeing the whole picture? Trends Pharmacol. Sci. 24, 626–633.
Kong, G., Penn, R. and Benovic, J.L. (1994) A β-adrenergic receptor kinase dominant negative mutant attenuates desensitization of the β2-adrenergic receptor. J. Biol. Chem. 269, 13084–13087.
Simon, V., Robin, M.T., Legrand, C. and Cohen-Tannoudji, J. (2003) Endogenous G protein-coupled receptor kinase 6 triggers homologous beta-adrenergic receptor desensitization in primary uterine smooth muscle cells. Endocrinology 144, 3058–3066.
Debburman, S.K., Kunapuli, P., Benovic, J.L. and Hosey, M.M. (1995) Agonist-dependent phosphorylation of human muscarinic receptors in Spodoptera frugiperda insect cell membranes by G protein-coupled receptor kinases. Mol. Pharmacol. 47, 224–233.
Willets, J.M., Challiss, R.A. and Nahorski, S.R. (2002) Endogenous G protein-coupled receptor kinase 6 Regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells. J. Biol. Chem. 277, 15523–15529.
Willets, J.M., Challiss, R.A., Kelly, E. and Nahorski, S.R. (2001) G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M3 muscarinic acetylcholine receptor in human SH-SY5Y cells. Mol. Pharmacol. 60, 321–330.
Diviani, D., Lattion, A.L., Larbi, N., Kunapuli, P., Pronin, A., Benovic, J.L. and Cotecchia, S. (1996) Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the α1B-adrenergic receptor. J. Biol. Chem. 271, 5049–5058.
Oppermann, M., Freedman, N.J., Alexander, R.W. and Lefkowitz, R.J. (1996) Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C. J. Biol. Chem. 271, 13266–13272.
Schafer, B., Marg, B., Gschwind, A. and Ullrich, A. (2004) Distinct ADAM metalloproteinases regulate G protein-coupled receptor-induced cell proliferation and survival. J. Biol. Chem. 279, 47929–47938.
Willets, J.M., Taylor, A.H., Shaw, H., Konje, J.C. and Challiss, R.A. (2008) Selective regulation of H1 histamine receptor signalling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells. Mol. Endocrinol. 22, 1893–1907.
Morris, G.E., Nelson, C.P., Standen, N.B., Challiss, R.A. and Willets, J.M. (2010) Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2. Cardiovasc. Res. 85, 424–433.
Willets, J.M., Brighton, P.J., Mistry, R., Morris, G.E., Konje, J.C. and Challiss, R.A. (2009) Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle. Mol. Endocrinol. 23, 1272–1280.
Labasque, M., Reiter, E., Becamel, C., Bockaert, J. and Marin, P. (2008) Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signalling. Mol. Biol. Cell 19, 4640–4650.
Hirotani, S., Otsu, K., Nishida, K., Higuchi, Y., Morita, T., Nakayama, H., Yamaguchi, O., Mano, T., Matsumura, Y., Ueno, H., Tada, M. and Hori, M. (2002) Involvement of nuclear factor-κB and apoptosis signal-regulating kinase 1 in G-protein-coupled receptor agonist-induced cardiomyocyte hypertrophy. Circulation 105, 509–515.
Penela, P., Murga, C., Ribas, C., Tutor, A.S., Peregrin, S. and Mayor, F., Jr. (2006) Mechanisms of regulation of G protein-coupled receptor kinases (GRKs) and cardiovascular disease. Cardiovasc. Res. 69, 46–56.
Gesty-Palmer, D. and Luttrell, L.M. (2008) Heptahelical terpsichory. Who calls the tune? J. Recept. Signal Transduct. Res. 28, 39–58.
Martini, J.S., Raake, P., Vinge, L.E., DeGeorge, B.R., Jr., Chuprun, J.K., Harris, D.M., Gao, E., Eckhart, A.D., Pitcher, J.A. and Koch, W.J. (2008) Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes. Proc. Natl. Acad. Sci. U S A 105, 12457–12462.
Rao, M. and Sockanathan, S. (2005) Molecular mechanisms of RNAi: implications for development and disease. Birth Defects Res. C. Embryo Today 75, 28–42.
Willets, J.M., Nash, M.S., Challiss, R.A. and Nahorski, S.R. (2004) Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons: evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases. J. Neurosci. 24, 4157–4162.
Carman, C.V., Parent, J.L., Day, P.W., Pronin, A.N., Sternweis, P.M., Wedegaertner, P.B., Gilman, A.G., Benovic, J.L. and Kozasa, T. (1999) Selective regulation of Gαq/11 by an RGS domain in the G protein-coupled receptor kinase, GRK2. J. Biol. Chem. 274, 34483–34492.
Sterne-Marr, R., Tesmer, J.J., Day, P.W., Stracquatanio, R.P., Cilente, J.A., O’Connor, K.E., Pronin, A.N., Benovic, J.L. and Wedegaertner, P.B. (2003) G protein-coupled receptor kinase 2/Gαq/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding G alpha subunits. J. Biol. Chem. 278, 6050–6058.
Wang, X., Dong, L., Xie, J., Tong, T. and Zhan, Q. (2009) Stable knockdown of Aurora-A by vector-based RNA interference in human oesophageal squamous cell carcinoma cell line inhibits tumor cell proliferation, invasion and enhances apoptosis. Cancer Biol. Ther. 8, 1852–1859.
Willets, J.M., Mistry, R., Nahorski, S.R. and Challiss, R.A. (2003) Specificity of G protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell M3 muscarinic acetylcholine receptor signalling. Mol. Pharmacol. 64, 1059–1068.
Kim, J., Ahn, S., Ren, X.R., Whalen, E.J., Reiter, E., Wei, H. and Lefkowitz, R.J. (2005) Functional antagonism of different G protein-coupled receptor kinases for β-arrestin-mediated angiotensin II receptor signalling. Proc. Natl. Acad. Sci. U S A 102, 1442–1447.
Ren, X.R., Reiter, E., Ahn, S., Kim, J., Chen, W. and Lefkowitz, R.J. (2005) Different G protein-coupled receptor kinases govern G protein and β-arrestin-mediated signaling of V2 vasopressin receptor. Proc. Natl. Acad. Sci. U S A, 102, 1448–1453.
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Willets, J.M. (2011). Approaches to Study GPCR Regulation in Native Systems. In: Willars, G., Challiss, R. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology, vol 746. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-126-0_6
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DOI: https://doi.org/10.1007/978-1-61779-126-0_6
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