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, for several years, been indicating a connection between caveolae and the actin cytoskeleton. Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-associated 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 filamin-dimerization domain. In in vitro binding assays, recombinant caveolin-1 bound to both nonmuscle and muscle filamin, indicating that the interaction 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, indicating that there was a physical link connecting them. Immunogold double-labeling 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.