Ligand-induced internalisation of the orexin OX1 and cannabinoid CB1 receptors assessed via N-terminal SNAP and CLIP-tagging (original) (raw)
Ward, Richard J., Pediani, John D. 
ORCID: https://orcid.org/0000-0001-6615-537X and Milligan, Graeme ORCID: https://orcid.org/0000-0002-6946-3519(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
BACKGROUND AND PURPOSE 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 OX1 receptor and the cannabinoid CB1 receptor as exemplars.
EXPERIMENTAL APPROACH The human OX1 and CB1 receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O6-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. KEY RESULTSCell surface forms of each receptor construct were detected by both antibody recognition of the epitope tags and covalent binding of fluorophores to the O6-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. CONCLUSIONS AND IMPLICATIONS These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB1 and OX1 receptors and their internalization by agonists can be developed based on introduction of variants of O6-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors.| Item Type: | Articles |
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| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Milligan, Professor Graeme and Pediani, Dr John and Ward, Dr Richard |
| 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 |
| Related URLs: | PubMed |
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