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The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D? Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal.


ABSTRACT: This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D? 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) ?-arrestin 2 (?arr2) 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 ?arr2 recruitment and G?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 D2L-R appears to be the ER retention signal.

SUBMITTER: Kubale V 

PROVIDER: S-EPMC4964525 | BioStudies | 2016-01-01T00:00:00Z

REPOSITORIES: biostudies

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