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S.K. Nair, S.M. Buckheister, V.V. Gurevich, V.Z. Slepak; Mechanisms and Effects of Signal-Dependent Translocation of the G protein, RGS, and Arrestin to Lipid Rafts in Rod Outer Segment . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3179.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cellular membranes contain "lipid rafts", microdomains different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. We investigated if lipid rafts have a role in G protein signaling using vertebrate phototransduction as a model. Methods: Lipid rafts from rod outer segments were isolated using the property of lipid rafts being resistant to detergents and to float in the interphase of 5-30 % sucrose in a discontinuous sucrose gradient upon centrifugation. Results: Our finding show that photoreceptor membranes contain detergent-resistant fraction that floats on density gradients and can be dissolved by cholesterol-depleting agent methyl-cyclodextrin, a distinctive feature of cholesterol-rich rafts. Receptor (rhodopsin) and effector (cGMP phosphodiesterase) are present in both raft and fluid portions of the membrane; guanylate cyclase is found exclusively in the raft along with the raft marker caveolin-1. Distribution of these proteins does not change in light or dark. In contrast, the G protein (transducin, Gt) and the RGS9/Gß5L dimer undergo dramatic translocation to the raft upon illumination. GTPγS or pertussis toxin prevent this translocation, whereas AMF causes both Gt and RGS9/Gß5L to move to the raft in the dark. This shows that while receptor-mediated activation of the G protein is necessary for translocation, it is the Gαt-RGS complex, not simply active Gt, has the highest affinity to rafts. In the raft, GTPγS binds to transducin at an approximately 50-fold slower rate, indicating that this translocation results in a reduced receptor-G protein coupling. Similarly to the G protein and RGS, arrestin p44 also translocates to the rafts. Translocation of p44 is G protein-independent, but requires active rhodopsin; we also identified an additional anchor protein necessary for arrestin recruitment to the raft. Interestingly, the longer splice version of arrestin, p48, always remains in the fluid portion of the membrane; structure-function analysis of p44 and p48 provided us with a significant insight into this mechanism. Conclusions: In summary, these studies show that an external signal can rearrange localization of components of a G protein pathway in specific domains of the cell membrane, and change the signaling properties of the pathway.
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