Identification of filamin as a novel ligand for caveolin-1: Evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton

Reports on the ultrastructure of cells as well as biochemical data have, fo r several years, been indicating a connection between caveolae and the acti n cytoskeleton. Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-ass ociated protein caveolin-1. Binding of caveolin-1 to filamin involved the N -terminal region of caveolin-1 and the C terminus of filamin close to the f ilamin-dimerization domain. In in vitro binding assays, recombinant caveoli n-1 bound to both nonmuscle and muscle filamin, indicating that the interac tion might not be cell type specific. With the use of confocal microscopy, colocalization of caveolin-1 and filamin was observed in elongated patches at the plasma membrane. Remarkably, when stress fiber formation was induced with Rho-stimulating Escherichia coli cytotoxic necrotizing factor 1, the caveolin-1-positive structures became coaligned with stress fibers, indicat ing that there was a physical link connecting them. Immunogold double-label ing electron microscopy confirmed that caveolin-1-labeled racemose caveolae clusters were positive for filamin. The actin network, therefore, seems to be directly involved in the spatial organization of caveolin-1-associated membrane domains.