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 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.