title: Dopamine receptor D4 internalization requires a beta-arrestin and a visual arrestin.
abstract: OBJECTIVE
  The G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors.
  
  
  METHODS
  To test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4.
  
  
  RESULTS
  Immunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation.
  
  
  CONCLUSIONS
  The results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.
authors.affiliation: Mary D. Allen Laboratory for Vision Research, USC Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, Clinical Science Annex 215, Los Angeles, CA 90033, USA. Electronic address: Janise.deming@gmail.com.
authors.forename: Janise D
authors.initials: JD
authors.lastname: Deming
authors.affiliation: Mary D. Allen Laboratory for Vision Research, USC Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, Clinical Science Annex 215, Los Angeles, CA 90033, USA; Department of Anatomy, School of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea. Electronic address: sja@ewha.ac.kr.
authors.forename: Jung-A
authors.initials: JA
authors.lastname: Shin
authors.affiliation: Mary D. Allen Laboratory for Vision Research, USC Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, Clinical Science Annex 215, Los Angeles, CA 90033, USA. Electronic address: kaylim13@gmail.com.
authors.forename: Kayleen
authors.initials: K
authors.lastname: Lim
authors.affiliation: Mary D. Allen Laboratory for Vision Research, USC Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, Clinical Science Annex 215, Los Angeles, CA 90033, USA; Department of Biomedical Engineering, University of Southern California Viterbi School of Engineering, 1042 Downey Way, Los Angeles, CA 90089-1111, USA. Electronic address: eunjinl@usc.edu.
authors.forename: Eun-Jin
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Laboratory of GPCR Expression and Signal Transduction (L-GEST), Ghent University-UGent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. Electronic address: kathleen.vancraenenbroeck@ugent.be.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Van Craenenbroeck
authors.affiliation: Mary D. Allen Laboratory for Vision Research, USC Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, Clinical Science Annex 215, Los Angeles, CA 90033, USA; Department of Cell & Neurobiology, Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, Clinical Science Center 135H, Los Angeles, CA 90033, USA. Electronic address: CherylMae.Craft@med.usc.edu.
authors.forename: Cheryl Mae
authors.initials: CM
authors.lastname: Craft
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011485:Protein Binding; D021381:Protein Transport; D050638:Receptors, Dopamine D4 / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; C000605255:Arrb1 protein, mouse; C000605258:Arrb2 protein, mouse; D019390:Arrestins; C493600:Drd4 protein, mouse; C000607304:arrestin 4 protein, mouse; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D050638:Receptors, Dopamine D4
keywords: Beta arrestin; Cone arrestin; Dopamine receptor D4; Photoreceptor; Retina; Visual arrestin