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Janise Deming, Kayleen Lim, Bruce Brown, Joseph Pak, Kathleen Van Craenenbroeck, Cheryl Craft; Interactions between Dopamine Receptor D4 and Visual Arrestins. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2452.
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© ARVO (1962-2015); The Authors (2016-present)
Dopamine (DA) and its G-protein coupled receptors (GPCR) contribute to normal photopic vision, including light adaptation and contrast sensitivity, but the specific cellular pathways by which they contribute are unclear. Similar defective visual phenotypes are observed when either the DA receptor D4 (DRD4) or cone arrestin (ARR4) is not expressed. The purpose of this study was to determine the contribution of ARR4 in the desensitization of DRD4 in vitro and in photopic vision.
Immunohistochemistry (IHC) was performed using specific antibodies on frozen sections of mouse retinas or fixed transfected cells. C57Bl/6J mice were used, along with Drd4-/- and the four ARR knockout mice (J. Chen, USC; R. Lefkowitz, Duke) as negative controls. Co-immunoprecipitation (co-IP) followed by immunoblot analysis was used for protein-protein interactions. DRD4 desensitization was measured using in vitro internalization experiments with tagged plasmid constructs of human DRD4 and four ARRs in transfected HEK 293T cells incubated with or without 10 µM DA.
IHC of control compared to Drd4-/- retinas revealed the co-expression of DRD4 and ARR4 in cone pedicles, with higher DRD4 expression in dark vs. light adapted mice. Both βARR1 and βARR2 were also expressed in photoreceptors. DRD4 interaction with βARR2 is DA independent, but our co-IP data demonstrated a DA dependent interaction between DRD4 and ARR4 but not ARR1. In vitro studies showed DRD4 internalization only when a βARR and a visual ARR were co-transfected with DRD4. Internalization was highest with βARR2 and ARR4, but other combinations revealed limited internalization; however, none was detectable with two βARRs or two visual ARRs.
DRD4 and ARR4 are required for normal photopic vision and are co-expressed with ARR1 and βARRs in cone photoreceptors, particularly in the pre-synaptic terminals. Our studies demonstrate that for complete desensitization of the DRD4, indicated by internalization after DA stimulation, at least one visual ARR must be co-expressed with a βARR. To our knowledge, no GPCR has previously been shown to require the expression of two different ARRs for desensitization. Our in vitro experiments indicate that not only is the interaction of DRD4, a visual arrestin, and a βARR possible in the cone photoreceptors, but their interaction may play an essential role in maintaining normal high acuity vision.
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