Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) IbThe activation and association of membrane receptors with the submembranous cytoskeleton is of key importance in regulating cellular functions such as adhesion, motility, transmembrane signaling, receptor distribution, and receptor function. A major component of the submembranous cytoskeleton is actin-binding protein-280 (ABP-280), 1 also known as filamin. In platelets, ABP-280 cross-links actin filaments and anchors the membrane skeleton to the plasma membrane by interacting with the cytoplasmic tail of the von Willebrand factor receptor, the glycoprotein (GP) Ib-IX complex (1-6). GP Ib-IX is a heterotrimeric complex consisting of two disulfide-linked subunits, GP Ib ␣ (M r 140,000) and GP Ib ␤ (M r 25,000), that are noncovalently associated with GP IX (M r 22,000) (7,8). These glycoproteins are each transmembrane proteins (9 -11). The interaction of the GP Ib-IX complex with ABP-280 is mediated by a region in the central portion of the cytoplasmic domain of GP Ib ␣ (5, 6, 12). In other cells of myeloid lineage that do not express GP Ib-IX, ABP-280 binds the high affinity IgG receptor (Fc␥RI) and the ␤2 integrin (13,14).Biochemical and structural analysis of human ABP-280 and cloning of the human endothelial ABP-280 cDNA reveals a protein of 2647 amino acids with three functional domains (15). ABP-280 subunits self-associate head to head (16) using the most carboxyl repetitive element (15, 17). The opposing aminoterminal end of each subunit contains an actin-binding domain (15,(17)(18)(19). The bulk of ABP-280 forms a semiflexible rod domain and is composed of 24 repeats, each about 96 residues long, that are predicted to fold into 8 ␤-sheets. The rod domain of ABP-280 is interrupted twice by short sequence inserts of 20 -40 residues between repeats 15 and 16 and repeats 23 and 24. These regions are postulated to be flexible hinges, and they both contain a calpain cleavage site. Calpain acts initially at the region between repeats 15 and 16 to generate an aminoterminal fragment of 190 kDa and a carboxyl-terminal fragment of 100 kDa (20). The 100-kDa fragment is subsequently cleaved at the site between repeats 23 and 24 to generate 90-and 10-kDa subfragments. Previous reports showed that in platelet lysates in which ABP-280 has been cleaved, GP Ib-IX co-immunoprecipitates with the 100-kDa fragment and to a lesser extent with the 90-kDa fragment (4, 6, 21), suggesting that the binding site for GP Ib ␣ is located in the carboxylterminal portion of ABP-280.In the present studies, using three different approaches, we characterized the region of ABP-280 that interacts with GP Ib ␣ . In the first approach, a melanoma cell line that lacks ABP-280 (22) was stably transfected with the cDNAs coding for the three subunits of the GP Ib-IX complex, then transiently transfected with cDNAs coding for ABP-280 lacking increasing numbers of amino acids from the carboxyl-terminal end; the ability of the trunc...

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 ؋ g) but the membrane skeleton requires 100,000 ؋ g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin ␣ IIb ␤ 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following ␣ IIb ␤ 3 -ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate a ϳ140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.Duchenne muscular dystrophy is one of the most common inherited human diseases. It is caused by a defective gene that codes for a 427-kDa protein, dystrophin (1-5). The deduced amino acid sequence of dystrophin shows that it consists of four domains and suggests that it is a cytoskeletal protein (6). The major rod-shaped domain contains 24 spectrin-like repeats. This domain is flanked on the amino terminus by a domain that has a high degree of homology to the actin-binding domains of spectrin and ␣-actinin, and on the carboxyl terminus by a cysteine-rich domain that shows some homology to a Ca 2ϩ -binding region in ␣-actinin. The most carboxyl-terminal end of dystrophin consists of a short domain that has no homology to any known protein and appears to play a role in linking the molecule to the plasma membrane (7,8). Recent studies using purified protein or recombinant fragments containing the putative actin-binding domain (9 -11) have shown that the protein can bind to actin filaments in vitro, supporting the idea that this molecule functions as a cytoskeletal protein. The finding that dystrophin exists in a submembranous location (8,12,13) and that the carboxyl-terminal end of the molecule associates tightly with a complex of membrane glycoproteins (termed dystroglycan) (14 -17) suggests that dystrophin is a component of a submembranous cytoskeleton.Although there is now considerable inform...