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Int. J. Mol. Sci. 2016, 17(7), 1152; doi:10.3390/ijms17071152

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

1
Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, Slovenia
2
Department of Incretin & Islet Biology, Novo Nordisk A/S, DK-2760 Måløv, Denmark
3
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
These authors contributed equally to this work.
Present address: Institute of Biochemistry, Center for Functional Genomics and Bio-Chips, Medical Faculty, University of Ljubljana, SI-1000 Ljubljana, Slovenia
*
Author to whom correspondence should be addressed.
Academic Editor: Kathleen Van Craenenbroeck
Received: 17 March 2016 / Revised: 5 July 2016 / Accepted: 9 July 2016 / Published: 19 July 2016
(This article belongs to the Collection G Protein-Coupled Receptor Signaling and Regulation)
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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. View Full-Text
Keywords: D2 dopamine receptors; endoplasmic reticulum (ER) retention motif; confocal microscopy; surface expression; bioluminescence resonance energy transfer (BRET2); cAMP signaling D2 dopamine receptors; endoplasmic reticulum (ER) retention motif; confocal microscopy; surface expression; bioluminescence resonance energy transfer (BRET2); cAMP signaling
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Kubale, V.; Blagotinšek, K.; Nøhr, J.; Eidne, K.A.; Vrecl, M. 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. Int. J. Mol. Sci. 2016, 17, 1152.

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