Abstract
Epitope tagging of a receptor involves introducing a defined amino acid sequence, to which an antibody has already been produced, into the primary amino acid sequence of the receptor. The new sequence can be as short as 10–15 amino acids and the method allows the receptor to be monitored without having to raise an antibody specific to it, and so permits its biochemical characterization and immunolocalization within cells. Other related techniques involve the introduction of functional domains of proteins such as green fluorescence protein or one of its derivatives into receptors to allow their direct visualization. There are a wide range of amino acid tag sequences, and fluorescent proteins, available for use as tags, and choice of tag will depend on several factors including the availability of antisera and cost. The position for the introduction of the tag into the native receptor will depend on precisely what the requirements of the experiments are but it must always be such that the receptor is normally processed, trafficked, and remains functional after tagging. These considerations are discussed fully in this chapter, which also describes examples of strategies for introducing tags, and some general methods for use in the characterization of the tagged proteins
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Stowell, J. N. and Craig, A. M., (1999) Axon/dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains. Neuron 22, 525–536.
Standley, S., Roche, K. W., McCallum, J., Sans, N., and Wenthold, R. J. (2000) PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron 28, 887–898.
Chan, W. Y., Soloviev, M. M., Ciruela, F., and McIlhinney, R. A. J. (2001) Molecular determinants of metabotropic glutamate receptor 1B trafficking. Mol. Cell Neurosci. 17, 577–588.
Szollosi, J., Nagy, P., Sebestyen, Z., et al. (2002) Applications of fluorescence resonance energy transfer for mapping biological membranes. J. Biotechnol. 82, 251–266.
Xu, Y., Johnson, C. H., and Piston, D. (2002) Bioluminescence resonance energy transfer assays for protein-protein interactions in living cells. Methods Mol. Biol. 183, 121–133.
Romano, C., Yang, W. L., and Omalley, K. L. (1996) Metabotropic glutamate receptor 5 is a disulfide-linked dimer. J. Biol. Chem. 271, 28612–28616.
Rios, C. D., Jordan, B. A., Gomes, I., and Devi, L. A. (2001) G-protein coupled receptor dimerization: modulation of receptor function. Pharmacol. Ther. 92, 71–78.
Gomes, I., Jordan, B. A., Gupta, A., et al. (2001) G protein coupled receptor dimerization: implications in modulating receptor function. J. Mol. Med. 79, 226–242.
Bennett, J. A. and Dingledine, R. (1995) Topology profile for a glutamate receptor: three transmembrane domains and a channel-lining reentrant membrane loop. Neuron 14, 373–384.
Anand, R. (2000) Probing the topology of the glutamate receptor GluR1 subunit using epitope-tag insertions. Biochem. Biophys. Res. Commun. 276, 157–161.
Murkerji, J., Haghighi, A., and Seguela, P. (1996) Immunological characterization and transmembrane topology of 5-hydroxytryptamine 3 receptors by functional epitope tagging. J. Neurochem. 66, 1027–1032.
Xie, L. Y. and Abou-Samra, A. B. (1998) Epitope tag mapping of the extracellular and cytoplasmic domains of the rat parathyroid hormone PTH-related peptide receptor. Endocrinology 139, 4563–4567.
Han, G. M. and Hampson, D. R. (1999) Ligand binding to the amino-terminal domain of the mGluR4 subtype of metabotropic glutamate receptor. J. Biol. Chem. 274, 10008–10013.
Selkirk, J. V., Challis, R. A. J., and McIlhinney, R. A. J. (2002) Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line. J. Neurochem. 80, 346–353.
Niethammer, M., Kim, E., and Sheng, M. (1996) Interaction between the C terminus of the NMDA receptor subunits and multiple members of the PSD-95 family of membrane associated guanylate kinases. J. Neurosci. 16, 2157–2163.
Brakeman, P. R., Lanahan, A. A., O’Brien, R., et al. (1997) Homer: a protein that selectively binds metabotropic glutamate receptors. Nature 386, 284–288.
Kitano, J., Kimura, K., Yamazaki, Y., et al. (2002) Tamalin, a PDZ domaincontaining protein, links a protein complex formation of group 1 metabotropic glutamate receptors and the guanine nucleotide exchange factor cytohesins. J. Neurosci. 22, 1280–1289.
Sheng, M. and Wyszynski, M. (1997) Ion channel targeting in neurons. Bioessays 19, 847–853.
Sheng, M. and Sala, C. (2001) PDZ domains and the organization of supramolecular complexes. Annu. Rev. Neurosci. 24, 1–29.
Brose, N., Gasic, G., Vetter, D., Sullivan, J. M., and Heinemann, S. F. (1993) Protein chemical characterization and immunocytochemical localization of the NMDA receptor subunit NMDA R1. J. Biol. Chem. 268, 22663–22671.
McIlhinney, R. A. J., Molnar, E., Atack, J. R., and Whiting, P. J. (1996) Cell surface expression of the human N-methyl-d-aspartate receptor subunit 1a (NMDAR1a) requires the co-expression of other NR2 subunits in transfected cells. Neuroscience 70, 989–997.
Ciruela, F., Soloviev, M. M., and McIlhinney, R. A. J. (1999) Co-expression of metabotropic glutamate receptor type 1 alpha with Homer-1a/Vesl-1S increases the cell surface expression of the receptor. Biochem. J. 341, 795–803.
Chan, W. Y. (2002) The targeting of metabotropic glutamate receptors, D. Phil Thesis Physiological Sciences. University of Oxford, Oxford.
Ciruela, F., Soloviev, M. M., Chan, W. Y., and McIlhinney, R. A. J. (2000) Homer-1c/Vesl-1L modulates the cell surface targeting of metabotropic glutamate receptor type 1 alpha: evidence for an anchoring function. Mol. Cell Neurosci. 15, 36–50.
Ango, F., Pin, J. P., Tu, J. C., et al. (2000) Dendritic and axonal targeting of type 5 metabotropic glutamate receptor is regulated by Homer 1 proteins and neuronal excitation. J. Neurosci. 20, 8710–8716.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
McIlhinney, R.A.J. (2004). Generation and Use of Epitope-Tagged Receptors. In: Willars, G.B., Challiss, R.A.J. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology, vol 259. Humana Press. https://doi.org/10.1385/1-59259-754-8:081
Download citation
DOI: https://doi.org/10.1385/1-59259-754-8:081
Publisher Name: Humana Press
Print ISBN: 978-1-58829-329-9
Online ISBN: 978-1-59259-754-3
eBook Packages: Springer Protocols