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 signalKubale, Valentina (Avtor) Blagotinšek Cokan, Kaja (Avtor) Larsen, Jane N. (Avtor) Eidne, Karin A (Avtor) Vrecl, Milka (Avtor) D$_2$ dopamine receptorsendoplasmic reticulum (ER) retention motifconfocal microscopysurface expressionbioluminescence resonance energy transfer (BRET$^2$)cAMP signalingmolecular biologyenergy transfer techniquesmetabolismThis 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.20162021-09-13 09:07:06Članek v reviji130293UDK: 576ISSN pri članku: 1422-0067DOI: 10.3390/ijms17071152COBISS_ID: 4174970sl