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.

The ATP-sensitive potassium (K ATP ) channels in pancreatic ␤ cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of K ATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of K ATP -channel regulation remains unclear. The K ATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that -channel openers. Sulfonylureas, commonly used in the treatment of non-insulin-dependent diabetes mellitus, stimulate insulin secretion by closing the K ATP channels, whereas K ϩ -channel openers inhibit insulin secretion by opening the K ATP channels (7).The pancreatic ␤ cell K ATP channel is a complex of four SUR1 subunits of the ATP-binding cassette (ABC) superfamily with two nucleotide-binding folds (NBF1 and NBF2) and four Kir6.2 subunits of the inwardly rectifying K ϩ -channel family (8, 9). SUR1 is thought to mediate the stimulatory effect of MgADP and is the primary target for pharmacological agents, such as the sulfonylurea glibenclamide and the K ϩ -channel opener diazoxide. The primary site of ATP inhibition of K ATP -channel activity seems to be in Kir6.2 (10, 11). However, the regulation of the ␤ cell K ATP channels by adenine nucleotides and pharmacological agents is complex. In addition to the inhibitory effect, MgATP enhances ␤ cell K ATPchannel activity (12, 13). ADP also has both stimulatory and inhibitory effects (14,15). Although the interaction of sulfonylureas with SUR1 abolishes the stimulatory effect of MgADP on K ATP channels (10), the sensitivity of the K ATP channel to sulfonylureas is increased in the presence of .Previously, we found that SUR1 strongly binds 8-azido-ATP at NBF1, whereas MgADP is bound at NBF2, and that preincubation of SUR1 with MgADP efficiently inhibits 8-azido-ATP binding to SUR1 (ref. 19; Fig. 1A, preincubation procedure). Because this inhibitory effect of MgADP was reduced by mutations in NBF2, we thought the MgADP bound at NBF2 might facilitate MgADP binding at NBF1 and thereby prevent 8-azido-ATP binding to NBF1. The strong 8-azido-ATP binding to SUR1 made it possible to investigate the biochemical basis of such cooperative interaction. Here, we provide direct biochemical evidence, obtained by postincubation procedure, of the cooperative interaction in nucleotide binding of the two NBFs of SUR1. In addition, we show that the sulfonylurea glibenclamide modulates this cooperative interaction in nucleotide binding of the two NBFs of SUR1. MATERIALS AND METHODS Materials

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