Abstract
Abstract: :
Purpose: We have previously identified caveolin-1 (Cav-1), a putative scaffolding protein, in detergent-resistant membrane fractions isolated from photoreceptor rod outer segments (ROS) and demonstrated, via co-immunoprecipitation, its association with the transducin alpha (Tα) subunit (IOVS, 2001, suppl. 42, S184). In this report, we identify the region of Cav-1 that is required for interaction with Tα. Furthermore, we demonstrate that activation of Tα inhibits its ability to interact with Cav-1. Methods: Full-length Cav-1 (residues 1-178) and various deletion mutants of Cav-1 (residues 1-140, 61-101, 102-134, and 135-178) were expressed as glutathione S-transferase (GST) fusions and immobilized on glutathione-Sepharose beads. Expressed GST-fusion proteins were incubated with solubilized bovine ROS and assessed for their ability to interact with Tα by GST-pull-down followed by immunoblot analysis. In other experiments, dark-adapted bovine ROS were incubated in the presence and absence of the non-hydrolyzable GTP analog GTPγS, exposed to light, solubilized, and subjected to GST pull-down assays using full-length Cav-1. Finally, we constructed and expressed a putative Cav-1 interacting domain in Tα (residues 150-207) in frame fusion to a polyhistidine tag. Results: In GST-fusion pull-down assays, Tα was found to interact with full-length GST-(Cav-1), GST-(1-140), and GST-(61-101), but not with GST-(102-134), GST-(135-178), or GST alone. In addition, incubation of ROS with GTPγS significantly reduced the recovery of Tα by full-length GST-(Cav-1) in pull-down experiments. Finally, in E. coli, we have successfully expressed the putative polyhistidine-tagged Cav-1 binding domain of Tα for cell-free protein-protein interaction studies. Conclusions: Our results demonstrate that residues 61-101 of Cav-1 are sufficient to promote binding with Tα. This region contains a reported "caveolin scaffolding domain" that has been shown to interact with a wide variety of signaling molecules including heterotrimeric G-protein alpha subunits. Furthermore, our results suggest that the interaction of Tα with Cav-1 is dependent upon the activation state of Tα, implying functional consequences of Cav-1/Tα association in photoreceptor physiology.
Keywords: protein structure/function • signal transduction • cytoskeleton