title: Molecular characterization of dopamine D2 receptor isoforms tagged with green fluorescent protein.
abstract: In this study, a cleavable signal peptide fused to the enhanced green fluorescent protein (EGFP) was tagged to the extracellular N-terminus of the human dopamine D2 receptor short and long isoforms (D2S and D2L). Ligand-binding properties of EGFP-tagged receptors were essentially unchanged in comparison to their respective wild-type receptors. The dopamine-mediated activation of both EGFP-D2 isoforms generated a robust inhibition of adenylyl cyclase type 5 in intact cells. In addition, the receptor density of EGFP-D2S and EGFP-D2L in transfected human embryonic kidney 293 (HEK293) cells was not altered when compared to cells transfected with the untagged D2S and D2L. However, the receptor densities of untagged and EGFP-tagged D2L were significantly lower in comparison to those measured with D2S constructs. Moreover, the receptor density of EGFP-D2S and EGFP-D2L was differentially upregulated in cells treated with antipsychotic drugs. As assessed by confocal microscopy, both EGFP-D2 isoforms were present on the cell surface. Notably, in contrast to the predominant plasma membrane localization of EGFP- D2S, EGFP-D2L was visualized both on the plasma membrane and intracellularly before dopamine exposure. Importantly, EGFP-D2S and EGFP-D2L are robustly internalized after dopamine treatment. Overall, our data suggest a differential intracellular sorting of D2S and D2L.
authors.affiliation: Ottawa Health Research Institute, Moses and Rose Loeb Research Centre (Neuroscience Program) 725 Parkdale Ave., Ottawa, ON, K1Y 4E9, Canada.
authors.forename: Keyvan
authors.initials: K
authors.lastname: Sedaghat
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Nantel
authors.forename: Simon
authors.initials: S
authors.lastname: Ginsberg
authors.forename: Véronique
authors.initials: V
authors.lastname: Lalonde
authors.forename: Mario
authors.initials: M
authors.lastname: Tiberi
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D020033:Protein Isoforms / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins