Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin α and β, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin α contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin α and β localized to focal adhesions in NIH 3T3 fibroblasts, and to cell–cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin α also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin β showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell– cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

Adaptor proteins, composed of two or more protein-protein interacting modules without enzymatic activity, regulate various cellular functions. Vinexin, CAP/ponsin, and ArgBP2 constitute a novel adaptor protein family. They have a novel conserved region homologous to the active peptide sorbin, as well as three SH3 (src homology 3) domains. A number of proteins binding to this adaptor family have been identified. There is accumulating evidence that this protein family regulates cell adhesion, cytoskeletal organization, and growth factor signaling. This review will summarize the structure and the function of proteins in this family.

Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin β by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the halftype ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral