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 (avtor), Blagotinšek Cokan, Kaja (avtor), Larsen, Jane N. (avtor), Eidne, Karin A (avtor), Vrecl, Milka (avtor)

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.

• Jezik: Angleški jezik
• Ključne besede: 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
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: VF - Veterinarska fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2016
• Št. strani: 16 str.
• Številčenje: Vol. 17, iss. 7, art. 1152
• PID: 20.500.12556/RUL-130293
• UDK: 576
• ISSN pri članku: 1422-0067
• DOI: 10.3390/ijms17071152
• COBISS.SI-ID: 4174970
• Datum objave v RUL: 13.09.2021

Gradivo je del revije

• Naslov: International journal of molecular sciences
• Skrajšan naslov: Int. j. mol. sci.
• Založnik: MDPI
• ISSN: 1422-0067
• COBISS.SI-ID: 2779162

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.
• Začetek licenciranja: 19.07.2016

Projekti

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P4-0053
• Naslov: Endokrini, imunski in encimski odzivi pri zdravih in bolnih živalih

• Financer: Drugi - Drug financer ali več financerjev
• Program financ.: COST
• Številka projekta: CM1207
• Akronim: GLISTEN