20.500.12556/RUL-130293 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 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. 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 true false true Angleški jezik Ni določen Članek v reviji 2021-09-13 09:07:06 2021-09-13 09:07:07 2022-09-09 04:15:44 0000-00-00 00:00:00 2016 0 0 16 str. iss. 7, art. 1152 Vol. 17 2016 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 576 1422-0067 10.3390/ijms17071152 4174970 RAZ_Kubale_Valentina_2016.pdf RAZ_Kubale_Valentina_2016.pdf 1 1FC07B439D9AF0339B03BF5902304E31 fdd98b85a9e300bd4636a063a676fb59ea10234747bfd2bb8c69268e4104e2b2 4ebceb8f-1461-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=147363 https://www.mdpi.com/1422-0067/17/7/1152 0 19fa2135-1461-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=147362 Veterinarska fakulteta 0 0 0