title: Ligand-induced internalization of the orexin OX(1) and cannabinoid CB(1) receptors assessed via N-terminal SNAP and CLIP-tagging.
abstract: OBJECTIVE
  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(1) receptor and the cannabinoid CB(1) receptor as exemplars.
  
  
  METHODS
  The human OX(1) and CB(1) receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O(6)-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.
  
  
  RESULTS
  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(6)-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
  These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB(1) and OX(1) receptors and their internalization by agonists can be developed based on introduction of variants of O(6)-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors.
authors.affiliation: Molecular Pharmacology Group, Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.forename: John D
authors.initials: JD
authors.lastname: Pediani
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D001583:Benzoxazoles / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D003001:Cloning, Molecular; D003511:Cyclohexanols / Q000378:metabolism / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D009287:Naphthyridines; D009479:Neuropeptides / Q000378:metabolism / Q000494:pharmacology; D064446:Orexin Receptors; D000068797:Orexins; D010671:Phenylurea Compounds / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D011720:Pyrazoles / Q000378:metabolism / Q000494:pharmacology; D011759:Pyrrolidines / Q000378:metabolism / Q000494:pharmacology; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000077285:Rimonabant; D013844:Thiazoles / Q000378:metabolism / Q000494:pharmacology; D014508:Urea / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
chemical_list: C420062:1-(2-methylbenzoxazol-6-yl)-3-(1,5)naphthyridin-4-yl urea; D001583:Benzoxazoles; D003511:Cyclohexanols; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009287:Naphthyridines; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D010671:Phenylurea Compounds; D010880:Piperidines; D011720:Pyrazoles; D011759:Pyrrolidines; D043884:Receptor, Cannabinoid, CB1; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; C542397:SB 408124; C492260:SB674042; D013844:Thiazoles; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D014508:Urea; D019883:Alkyl and Aryl Transferases; D048049:Extracellular Signal-Regulated MAP Kinases; D000077285:Rimonabant