Abstract
Abstract: :
Purpose: We have found that RPE65 contains a conserved caveolin–binding motif and can bind to caveolin–1. Since caveolin–binding proteins interact with either the aa82–101 scaffolding domain or the C–terminal aa134–178 domain of caveolin–1, or both, we wished to determine which of the protein interaction domains of caveolin–1 associate with RPE65. Methods: An expression construct encoding GST–caveolin 1 (GST–Cav1) was subjected to site directed mutagenesis and/or domain removal to generate a panel of GST–Cav1 deletion fusion proteins. All constructs were sequenced to validate identity. Mutated fusion proteins were expressed and purified by binding onto glutathione sepharose. GST pulldown assay was used to assess interaction of the various GST–Cav1 fusion proteins with RPE65. Results: The GST–Cav1deletion constructions GST–Cav1/del82–101, GST–Cav1/delCTerm, GST–Cav1/del82–101/CTerm, GST–Cav1/del62–101/CTerm, GST–Cav1/del82–178 were generated. In addition, GST–Cav1 was employed as a positive control, while unfused GST was used as a control to establish the background binding of RPE65 to GST–sepharose. When these fusion proteins were used as bait in GST pulldown assays with RPE microsomal extract as prey, it was observed that RPE65 bound to the GST–Cav1 deletion lacking either the aa82–101 scaffolding domain or the C–terminal 134–182 just as well as full–length GST–Cav1. Surprisingly, when both these domains were deleted (construct GST–Cav1/del82–101/Cterm) there was still significant binding of RPE65. Even when the deletion of the scaffolding domain was extended to aa62–101 binding was maintained. All four of these constructs have in common the aa102–133 caveolin–1 transmembrane domain, implicating this region in RPE65 binding. When the scaffolding domain, the transmembrane domain and the C–terminal domain were all deleted (construct GST–Cav1/del82–178), binding of RPE65 was reduced to the background level. Conclusions: Both the best–characterized protein interaction domains of caveolin–1 (the scaffolding and the C–terminal domains) are implicated in the binding of RPE65. In addition, the transmembrane domain also contributes to RPE65 binding. However, it is not clear whether this is a specific interaction of the RPE65 caveolin–binding motif or if it is a generalized hydrophobic interaction.
Keywords: protein structure/function • retinal pigment epithelium • proteins encoded by disease genes