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W. Orisme, W. C. Smith; G-Protein Signaling Mediates Arrestin Translocation to Rod Outer Segments. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1671. doi: https://doi.org/.
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Arrestin localizes differentially in response to light in rod photoreceptors. In dark-adapted photoreceptors, arrestin is localized in the inner segments. However, in response to light, arrestin translocates to the rod outer segments. We previously reported arrestin translocation to the rod outer segments could be stimulated by protein kinase C and phospholipase C agonists, which are important molecules in the phosphoinositide pathway. Here, we examine the G-protein utilized in signaling arrestin translocation.
To determine the nature of translocation signaling, transgenic arrestin-GFP (arr-GFP) tadpole eyes were treated with pertussis toxin for 4 hrs and then light adapted for 1 hr. The eyes were fixed immediately in 3.7% formaldehyde and 73% methanol in deionized water, and processed for confocal microscopy. For immunolocalization of Gi, wild-type eyes were dark and light adapted. Sections were immunostained with anti-Gi-3 monoclonal antibody for 14-20 hrs, and visualized with a Cy3-conjugated secondary antibody. In an effort to activate Gi independently of light, we also treated arr-GFP tadpoles with quinpirole (a dopamine receptor agonist) for 5-30 min. The tadpoles were fixed immediately after each time point as above.
Arr-GFP eyes treated with pertussis toxin showed a dramatic slowing of arrestin translocation to the ROS in response to light. Arrestin translocated normally to the ROS in untreated photoreceptors. Gi-3 was detected in the inner segment and along the axoneme by immunocytochemistry. There was no significant difference between Gi-3 immunostaining in dark- or light-adapted photoreceptors. Arrestin distribution in the rod photoreceptors treated with quinpirole changed in the absence of light, moving to the ROS within 5 min of treatment with quinpirole and was sustained for as long as 30 min.
We significantly perturbed arrestin translocation to the ROS when rod photoreceptors were treated with pertussis toxin. This pertussis-sensitivity suggests that the signaling cascade for arrestin translocation may involve a Gi-type G-protein. Immunolocalization of Gi-3 to the axoneme supports this hypothesis. Our ability to co-opt the dopamine receptor to stimulate arrestin translocation independent of rhodopsin adds additional support for signaling of arrestin translocation through a G-protein-regulated pathway.
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