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F Hayashi, H Liu, K Seno; Stimulus-dependent Recruitment of Transducin and Phosphodiesterase to Raft-like Detergent-resistant Memebranes in Rod Photoreceptor Outer Segment . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2899.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the potential role of raft-like membrane domains in phototransduction on the rod outer segment membranes (ROS). Methods: Bleached or dark-adapted ROS were pretreated with or without GTPγS, and solubilized with an isotonic buffer containing 1 % Triton X-100 (TX100). Insoluble low-density membrane fraction (detergent-resistant membrane: DRM) was prepared by a conventional sucrose density gradient ultracentrifugation. The concentrations of transducin and its effector, phosphodiesterase (PDE), in each fraction from the sucrose density gradient were assessed with SDS-PAGE followed by immunoblotting. Results: DRM was observed at the sucrose density around 1.10 as a buoyant fraction. In dark-adapted ROS, transducin was largely soluble with TX100. Upon illumination, a considerable amount of transducin was recruited to DRM. In contrast, transducin became detergent-soluble again when ROS was exposed to light in the presence of GTPγS. However, a small portion of transducin α subunit (Tα) remained in DRM. On the other hand, inactive PDE in the dark-adapted ROS was TX100-soluble, whereas, the stimulation of ROS by light and GTPγS resulted in massive translocation of active PDE to DRM. We observed a portion of rhodopsin, RGS9, the GAP for GTP-Tα, and its counterpart, Gß5L, in DRM as its steady constituents. ABCR/Rim protein principally was localized in DRM. The DRM was relatively enriched in sphingomyeline, ganglioside GD3, and cholesterol. Conclusion: It is highly likely that light-activated rhodopsin in DRM preferentially recruited transducin to DRM. PDE seemed to be recruited to DRM by binding with GTPγS-Tα, which was fixed to DRM by unknown mechanism. The presence of RGS9/Gß5L in DRM may be convenient to facilitate the deactivation of GTP-Tα after the activation of PDE recruited. These data suggested that the translocation of G protein and its effector between two different domains of disk membrane is crucial for highly effective signaling in photoreceptors. Further, the rim region of the disk membrane containing ABCR/Rim proteins seemed to be involved in the DRM.
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