Cholesterol-dependent association of caveolin-1 with the transducin alpha subunit in bovine photoreceptor rod outer segments: disruption by cyclodextrin and guanosine 5'-O-(3-thiotriphosphate).

Evidence suggests that caveolins, 21-24 kDa cholesterol-binding proteins that generally reside in specialized detergent-resistant membrane microdomains, act as signaling scaffolds. Detergent-resistant membranes isolated from rod outer segments (ROS) have been previously shown to contain the photoreceptor G-protein, transducin. In this report we show, by subcellular fractionation, that caveolin-1 is an authentic component of purified ROS. We demonstrate that caveolin-1 in ROS almost exclusively resides in low-buoyant-density, cholesterol-rich, detergent-resistant membranes that can be disrupted by cholesterol depletion using methyl-beta-cyclodextrin (MCD). Cholesterol depletion was also observed to extract a pool of transducin alpha (Talpha) from ROS membranes. Immunoprecipitation with anti-caveolin-1 revealed the association of Talpha in the absence of Tbetagamma. Treatment of ROS with MCD resulted in a 2-fold decrease in recovery of Talpha in anti-caveolin-1 immunoprecipitates. This interaction was also completely disrupted when ROS were exposed to light in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), a nonhydrolyzable GTP analogue. In addition, caveolin-1/Talpha association in the immune complex was disrupted by a peptide based on the primary sequence of the caveolin-1 scaffolding domain. Finally, we confirm the colocalization of caveolin-1 and Talpha in photoreceptors by immunofluorescence microscopy. These results strongly suggest that the association between Talpha and caveolin-1 occurs in cholesterol-rich, detergent-resistant membranes and is likely to be dependent upon the activation state of Talpha.

Activation of focal adhesion kinase in adenovirus-infected human corneal fibroblasts.

PURPOSE: Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase with protean downstream influences on cell cycle regulation, cytoskeletal dynamics, and cell attachment, is activated by integrin binding and aggregation. Adenoviruses, including those associated with human keratitis, enter permissive cells by an integrin-mediated mechanism. Hence, a possible relationship between adenovirus infection and tyrosine phosphorylation of FAK in human corneal cells was explored. METHODS: Human corneal fibroblasts (HCFs) derived from donor corneas were infected for various periods with adenovirus type 19 (Ad19) or were mock infected with virus-free medium. Parallel experiments included echistatin, which is a snake venom disintegrin and a partial inhibitor of FAK. For immunoblot analysis, Triton-X-soluble and Triton-X-insoluble fractions were analyzed by SDS-PAGE and immunoblotted with phosphospecific antibodies. Expression of the interleukin (IL)-8 gene was analyzed by RT-PCR and ELISA. RESULTS: Ad19 infection of HCFs induced tyrosine phosphorylation of a protein at 125 kDa, which was evident within 15 minutes after infection and was established by immunoprecipitation to be FAK. Immunoblot with antibody to FAK tyrosine-397 confirmed phosphorylation of this key binding site for downstream signaling proteins. Immunoblot analysis further suggested a shift in the intracellular location of phosphorylated FAK from the cytosol (Triton-X-soluble cell lysate fraction) to the cytoskeleton (Triton-X-insoluble cell lysate pellet) on infection. Finally, treatment of HCFs with echistatin reduced virus-induced expression of the neutrophil chemotaxin IL-8, previously implicated in adenoviral pathogenesis. CONCLUSIONS: Ad19 infection of HCFs induces rapid phosphorylation of FAK, and a dramatic change in its intracellular distribution. Activation of FAK may contribute to the inflammatory response to adenovirus infection of the human cornea.

Light-dependent association of Src with photoreceptor rod outer segment membrane proteins in vivo.

In vivo light exposure results in tyrosine phosphorylation of several rod outer segment (ROS) proteins (Ghalayini, A. J., Guo, X. X., Koutz, C. A, and Anderson, R. E. (1998) Exp. Eye Res. 66, 817-821). We now report the presence of Src in ROS and its increased association with bleached ROS membranes. Immunoprecipitation with anti-phosphotyrosine revealed that tyrosine kinase activity recovered from light-adapted ROS membranes was twice that recovered from dark-adapted ROS. Other experiments revealed the presence of both bleached rhodopsin and arrestin in immunoprecipitates of LROS, suggesting the formation of a multimeric complex containing Src, arrestin, and bleached rhodopsin. Additionally, when immobilized Src homology domains 2 and 3 (SH2 and SH3, respectively) were used to study the association of Src with ROS membranes, only bleached opsin and arrestin were found to associate with the SH2 domain of Src. These data strongly suggest that Src through its SH2 domain interacts with bleached rhodopsin and arrestin either directly or indirectly. Similar results were also obtained when dark-adapted and light-adapted retinas were used instead of ROS membranes. Our data strongly suggest that light exposure in vivo activates Src and promotes its association through its SH2 domain with a complex containing bleached rhodopsin and arrestin. A hypothesis for the functional significance of this phenomenon is presented.