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

• Published in: Molecular biology of the cell 2000-01, Vol.11 (1), p.325-337
• Main Authors: Stahlhut, M, van Deurs, B
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Actins - metabolism; Amino Acid Sequence; Animals; Antibodies - immunology; Binding Sites; Caveolin 1; Caveolins; Cell Membrane - metabolism; Cloning, Molecular; Contractile Proteins - genetics; Contractile Proteins - immunology; Contractile Proteins - metabolism; Cytoskeleton - metabolism; Dogs; Enzyme Activation; Filamins; Humans; Ligands; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Microfilament Proteins - genetics; Microfilament Proteins - immunology; Microfilament Proteins - metabolism; Microscopy, Immunoelectron; Molecular Sequence Data; Protein Isoforms - genetics; Protein Isoforms - immunology; Protein Isoforms - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; rho GTP-Binding Proteins - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.11.1.325

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.