Cheryl Mae Craft, Ph.D.

Previous studies discovered cone phototransduction shutoff occurs normally for Arr1-/- and Arr4-/-; however, it is defective when both visual arrestins are simultaneously not expressed (Arr1-/-Arr4-/-). We investigated the roles of visual arrestins in an all-cone retina (Nrl-/-) since each arrestin has differential effects on visual function, including ARR1 for normal light adaptation, and ARR4 for normal contrast sensitivity and visual acuity.We examined Nrl-/-, Nrl-/-Arr1-/-, Nrl-/-Arr4-/-… Show more

Previous studies discovered cone phototransduction shutoff occurs normally for Arr1-/- and Arr4-/-; however, it is defective when both visual arrestins are simultaneously not expressed (Arr1-/-Arr4-/-). We investigated the roles of visual arrestins in an all-cone retina (Nrl-/-) since each arrestin has differential effects on visual function, including ARR1 for normal light adaptation, and ARR4 for normal contrast sensitivity and visual acuity.We examined Nrl-/-, Nrl-/-Arr1-/-, Nrl-/-Arr4-/-, and Nrl-/-Arr1-/-Arr4-/- mice with photopic electroretinography (ERG) to assess light adaptation and retinal responses, immunoblot and immunohistochemical localization analysis to measure retinal expression levels of M- and S-opsin, and optokinetic tracking (OKT) to measure the visual acuity and contrast sensitivity.Study results indicated that Nrl-/- and Nrl-/-Arr4-/- mice light adapted normally, while Nrl-/-Arr1-/- and Nrl-/-Arr1-/-Arr4-/- mice did not. Photopic ERG a-wave, b-wave, and flicker amplitudes followed a general pattern in which Nrl-/-Arr4-/- amplitudes were higher than the amplitudes of Nrl-/-, while the amplitudes of Nrl-/-Arr1-/- and Nrl-/-Arr1-/-Arr4-/- were lower. All three visual arrestin knockouts had faster implicit times than Nrl-/- mice. M-opsin expression is lower when ARR1 is not expressed, while S-opsin expression is lower when ARR4 is not expressed. Although M-opsin expression is mislocalized throughout the photoreceptor cells, S-opsin is confined to the outer segments in all genotypes. Contrast sensitivity is decreased when ARR4 is not expressed, while visual acuity was normal except in Nrl-/-Arr1-/-Arr4-/-.Based on the opposite visual phenotypes in an all-cone retina in the Nrl-/-Arr1-/- and Nrl-/-Arr4-/- mice, we conclude that ARR1 and ARR4 perform unique modulatory roles in cone photoreceptors. Show less

Well-established laboratory mouse lines are important in creating genetically engineered knockout mouse models; however, these routinely used inbred strains are prone to spontaneous and deleterious mutations. One of these strains, the commonly used C57BL/6N (B6N), was discovered to carry a point mutation in the Crumbs homolog 1 (Crb1rd8 ) gene, which codes for a developmental protein involved in tight junction formation at the outer limiting membrane (OLM). This mutation disrupts photoreceptor… Show more

Well-established laboratory mouse lines are important in creating genetically engineered knockout mouse models; however, these routinely used inbred strains are prone to spontaneous and deleterious mutations. One of these strains, the commonly used C57BL/6N (B6N), was discovered to carry a point mutation in the Crumbs homolog 1 (Crb1rd8 ) gene, which codes for a developmental protein involved in tight junction formation at the outer limiting membrane (OLM). This mutation disrupts photoreceptor polarity and leads to retinal degeneration. It was hypothesized that the G-protein receptor kinase 1 knockouts (Grk1-/- ), which were based on the B6N strain, would exhibit abnormal morphological phenotypes in their offspring not related to GRK1's major phosphorylation function. The hypothesis was tested by examining Grk1-/- with or without the Crb1rd8 mutation. This study demonstrates that the B6N background can influence the phenotype of a genetic mouse knockout and introduces potential visual functional consequences of the Crb1 mutation.


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ABSTRACT: The G-protein coupled receptor (GPCR) Dopamine receptor D4 (Drd4) plays an important role in cAMP regulation and gap junctional coupling in the photoreceptors, where expression of Drd4 mRNA is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when either of the beta-arrestins is co-transfected. We hypothesized that the visual arrestins, Arr1 and Arr4, play a modulatory… Show more

ABSTRACT: The G-protein coupled receptor (GPCR) Dopamine receptor D4 (Drd4) plays an important role in cAMP regulation and gap junctional coupling in the photoreceptors, where expression of Drd4 mRNA is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when either of the beta-arrestins is co-transfected. We hypothesized that the visual arrestins, Arr1 and Arr4, play a modulatory role in desensitization of Drd4 in the photoreceptors. IHC studies confirmed the presence of both beta-arrestins in the mouse photoreceptors, as well as co-localization of Drd4 and Arr4 in mouse cone photoreceptor inner segments. Co-IP experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no 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 pair of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either both visual arrestins or both beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to dopamine stimulation.
Conclusions: The results have functional implications and we hypothesize that the desensitization and internalization 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 GPCR, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other GPCRs may require more than one arrestin for desensitization and/or internalization. Show less