title: The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D₂ Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal.
abstract: This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D₂ dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET²) β-arrestin 2 (βarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal.
authors.affiliation: Institute of Anatomy, Histology &amp; Embryology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, Slovenia. valentina.kubale@vf.uni-lj.si.
authors.forename: Valentina
authors.initials: V
authors.lastname: Kubale
authors.affiliation: Institute of Anatomy, Histology &amp; Embryology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, Slovenia. kaja1.blagotinsek@gmail.com.
authors.forename: Kaja
authors.initials: K
authors.lastname: Blagotinšek
authors.affiliation: Department of Incretin &amp; Islet Biology, Novo Nordisk A/S, DK-2760 M&#229;l&#248;v, Denmark. jnql@novonordisk.com.
authors.forename: Jane
authors.initials: J
authors.lastname: Nøhr
authors.affiliation: Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, The University of Western Australia, WA 6009 Perth, Australia. keidne@gmail.com.
authors.forename: Karin A
authors.initials: KA
authors.lastname: Eidne
authors.affiliation: Institute of Anatomy, Histology &amp; Embryology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, Slovenia. milka.vrecl@vf.uni-lj.si.
authors.forename: Milka
authors.initials: M
authors.lastname: Vrecl
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D020033:Protein Isoforms; D011869:Radioligand Assay; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: D020033:Protein Isoforms; D017448:Receptors, Dopamine D2; C113776:dopamine D2L receptor; D001120:Arginine; D004298:Dopamine
keywords: D2 dopamine receptors; bioluminescence resonance energy transfer (BRET2); cAMP signaling; confocal microscopy; endoplasmic reticulum (ER) retention motif; surface expression