title: A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells.
abstract: BACKGROUND
  In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for the generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability.
  
  
  METHODS
  We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into a Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking.
  
  
  RESULTS
  KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of the time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H.
  
  
  CONCLUSIONS
  tdT is an alternative to, or even a better tool than, GFPs for fusion to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to the reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity that the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance.
authors.affiliation: Center for Substance Abuse Research and Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, United States.
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Chiu
authors.forename: Chongguang
authors.initials: C
authors.lastname: Chen
authors.forename: Yujun
authors.initials: Y
authors.lastname: Wang
authors.forename: Lee-Yuan
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology; D000818:Animals; D045744:Cell Line, Tumor; D002540:Cerebral Cortex / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D049408:Luminescent Agents / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017473:Receptors, Opioid, kappa / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D018076:DNA, Complementary; D049408:Luminescent Agents; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D017473:Receptors, Opioid, kappa; D000097573:Red Fluorescent Protein
keywords: 17,17′-(dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′-bimorphinan-3,4′,14,14′-tetrol; DYKDDDDK epitope tag; Dynorphin A (1-17); FLAG tag; G protein-coupled receptor; GFP; GFPs; GPCR; H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH; KOPR; Live cell imaging; MAPK; Neurons; RFP; Red fluorescent protein; U50,488H; eGFP; enhanced green fluorescent protein; green fluorescent protein and its spectral variants; kappa opioid receptor; mKOPR; mitogen-activated protein kinase; mouse kappa opioid receptor; norbinaltorphimine; red fluorescent protein; tdT; tdTomato; trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate salt