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
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...
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doaj-177b4a15f84d43db9cfbbeec06b80d8b2020-11-25T01:13:46ZengMDPI AGInternational Journal of Molecular Sciences1422-00672016-07-01177115210.3390/ijms17071152ijms17071152The 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 SignalValentina Kubale0Kaja Blagotinšek1Jane Nøhr2Karin A. Eidne3Milka Vrecl4Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, SloveniaInstitute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, SloveniaDepartment of Incretin & Islet Biology, Novo Nordisk A/S, DK-2760 Måløv, DenmarkLaboratory 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, AustraliaInstitute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana, SloveniaThis 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.http://www.mdpi.com/1422-0067/17/7/1152D2 dopamine receptorsendoplasmic reticulum (ER) retention motifconfocal microscopysurface expressionbioluminescence resonance energy transfer (BRET2)cAMP signaling |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Valentina Kubale Kaja Blagotinšek Jane Nøhr Karin A. Eidne Milka Vrecl |
spellingShingle |
Valentina Kubale Kaja Blagotinšek Jane Nøhr Karin A. Eidne Milka Vrecl 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 International Journal of Molecular Sciences D2 dopamine receptors endoplasmic reticulum (ER) retention motif confocal microscopy surface expression bioluminescence resonance energy transfer (BRET2) cAMP signaling |
author_facet |
Valentina Kubale Kaja Blagotinšek Jane Nøhr Karin A. Eidne Milka Vrecl |
author_sort |
Valentina Kubale |
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 |
title_short |
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 |
title_full |
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 |
title_fullStr |
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 |
title_full_unstemmed |
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 |
title_sort |
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 |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2016-07-01 |
description |
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. |
topic |
D2 dopamine receptors endoplasmic reticulum (ER) retention motif confocal microscopy surface expression bioluminescence resonance energy transfer (BRET2) cAMP signaling |
url |
http://www.mdpi.com/1422-0067/17/7/1152 |
work_keys_str_mv |
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