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.
Stahlhut
M
M
Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
van Deurs
B
B
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D051242:Caveolin 1; D022461:Caveolins; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003285:Contractile Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation; D064448:Filamins; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D000906:Antibodies; C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D003285:Contractile Proteins; D064448:Filamins; D008024:Ligands; D008565:Membrane Proteins; D008840:Microfilament Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D020741:rho GTP-Binding Proteins