Ward, R. J., Pediani, J. D. and Milligan, G. (2011) Ligand-induced internalisation of the orexin OX1 and cannabinoid CB1 receptors assessed via N-terminal SNAP and CLIP-tagging. British Journal of Pharmacology, 162(6), pp. 1439-1452. (doi: 10.1111/j.1476-5381.2010.01156.x)
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Publisher's URL: http://dx.doi.org/10.1111/j.1476-5381.2010.01156.x
Abstract
<b>BACKGROUND AND PURPOSE</b> Many G protein-coupled receptors internalize following agonist binding. The studies were designed to identify novel means to effectively quantify this process using the orexin OX<sub>1</sub> receptor and the cannabinoid CB1 receptor as exemplars.<p></p> <b>EXPERIMENTAL APPROACH</b> The human OX1 and CB1 receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O<sub>6</sub>-alkylguanine-DNA-alkyltransferase within their extracellular, N-terminal domain. Cells able to regulate expression of differing amounts of these constructs upon addition of an antibiotic were developed and analysed.<p></p> <b>KEY RESULTS</b>Cell surface forms of each receptor construct were detected by both antibody recognition of the epitope tags and covalent binding of fluorophores to the O<sub>6</sub>-alkylguanine-DNA-alkyltransferase variants. Receptor internalization in response to agonists but not antagonists could be monitored by each approach but sensitivity was up to six- to 10-fold greater than other approaches when employing a novel, time-resolved fluorescence probe for the SNAP tag. Sensitivity was not enhanced, however, for the CLIP tag, possibly due to higher levels of nonspecific binding.<p></p> <b>CONCLUSIONS AND IMPLICATIONS</b> These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB<sub>1</sub> and OX<sub>1</sub> receptors and their internalization by agonists can be developed based on introduction of variants of O<sub>6</sub>-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors.<p></p>
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Pediani, Dr John and Ward, Dr Richard and Milligan, Professor Graeme |
| Authors: | Ward, R. J., Pediani, J. D., and Milligan, G. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience |
| Journal Name: | British Journal of Pharmacology |
| Publisher: | John Wiley & Sons Ltd. |
| ISSN: | 0007-1188 |
| ISSN (Online): | 1476-5381 |
| Published Online: | 22 February 2011 |
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