The conserved arginine cluster in the insert of the third cytoplasmic loop of the long form of the D$_2$ dopamine receptor (D$_{2L}$-R) acts as an intracellular retention signal

Kubale, Valentina (author), Blagotinšek Cokan, Kaja (author), Larsen, Jane N. (author), Eidne, Karin A (author), Vrecl, Milka (author)

This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D$_2$ dopamine receptor (D$_{2L}$-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 D$_{2L}$-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET$^2$) 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 D$_{2L}$-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gα$_i$ -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 D$_{2L}$-R appears to be the ER retention signal.

• Language: English
• Keywords: D$_2$ dopamine receptors, endoplasmic reticulum (ER) retention motif, confocal microscopy, surface expression, bioluminescence resonance energy transfer (BRET$^2$), cAMP signaling, molecular biology, energy transfer techniques, metabolism
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: VF - Veterinary Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2016
• Number of pages: 16 str.
• Numbering: Vol. 17, iss. 7, art. 1152
• PID: 20.500.12556/RUL-130293
• UDC: 576
• ISSN on article: 1422-0067
• DOI: 10.3390/ijms17071152
• COBISS.SI-ID: 4174970
• Publication date in RUL: 13.09.2021

Record is a part of a journal

• Title: International journal of molecular sciences
• Shortened title: Int. j. mol. sci.
• Publisher: MDPI
• ISSN: 1422-0067
• COBISS.SI-ID: 2779162

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
• Licensing start date: 19.07.2016

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: P4-0053
• Name: Endokrini, imunski in encimski odzivi pri zdravih in bolnih živalih

• Funder: Other - Other funder or multiple funders
• Funding programme: COST
• Project number: CM1207
• Acronym: GLISTEN