Light or tyrosine phosphorylation recruits retinal rod outer segment proteins to lipid rafts

Abstract

Lipid rafts are localized liquid-ordered regions of the plasma membrane that contain high levels of cholesterol and glycosphingolipids, and are resistant to extraction with nonionic detergents. Retinal photoreceptor cells contain detergent-resistant membrane microdomains (DRM), which were isolated here from bovine rod outer segments (ROS) under dark and light conditions. Rhodopsin (R) was present in both DRM and detergent soluble fractions (DSF), and detergent-insoluble ROS rafts were enriched in caveolin 1 (Cav-1) and c-Src. In the dark, arrestin and its 44-kDa truncated form (p44) were present mainly in DSF; however, p44 was translocated to DRM under illumination. Similarly, transducin (T) was mainly present in DSF in the dark, but it was recruited toward the DRM fraction following photolysis. DRM were also prepared in the absence or presence of Mg-ATP, guanosine 5′-3-O-(thio)triphosphate (GTPγS), or both. Although GTPγS released T into DSF in the light, GTPγS-activated T was retained in DRM when Mg2+ and ATP were added. Moreover, T was always tyrosine-phosphorylated under light conditions, which suggested that T phosphorylation prevents its GTPγS-induced release from DRM. In addition, treatment with the tyrosine kinase inhibitor genistein prevented the segregation of T to the rafts. In contrast, no localization difference was seen in the presence of Mg-ATP for Cav-1, c-Src, R and both forms of arrestin. Interestingly, immunoprecipitation assays followed by Western blot analyses under light conditions showed the formation of multimeric complexes containing R, T, c-Src, p44 and Cav-1 in DRM, where T and c-Src were tyrosine-phosphorylated.