Transformation of mammary epithelial cells into invasive carcinoma results in alterations in their integrin-mediated responses to the extracellular matrix, including a loss of normal epithelial polarization and differentiation, and a switch to a more motile, invasive phenotype. Changes in the actin cytoskeleton associated with this switch suggest that the small GTPases Cdc42 and Rac, which regulate actin organization, might modulate motility and invasion. However, the role of Cdc42 and Rac1 in epithelial cells, especially with respect to integrin-mediated events, has not been well characterized. Here we show that activation of Cdc42 and Rac1 disrupts the normal polarization of mammary epithelial cells in a collagenous matrix, and promotes motility and invasion. This motility does not require the activation of PAK, JNK, p70 S6 kinase, or Rho, but instead requires phosphatidylinositol-3-OH kinase (PI(3)K). Further, direct PI(3)K activation is sufficient to disrupt epithelial polarization and induce cell motility and invasion. PI(3)K inhibition also disrupts actin structures, suggesting that activation of PI(3)K by Cdc42 and Rac1 alters actin organization, leading to increased motility and invasiveness.

Sensorineural hearing loss is genetically heterogeneous. Here we report that mutations in CIB2, encoding a Ca2+- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). There is one mutation of CIB2 that is a prevalent cause of DFNB48 deafness in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In rodents, CIB2 is localized in the mechanosensory stereocilia of inner ear hair cells and in retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of Ca2+ binding, CIB2 significantly decreased the ATP-induced Ca2+ responses in heterologous cells, while DFNB48 mutations altered CIB2 effects on Ca2+ responses. Furthermore, in zebrafish and Drosophila, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We show that CIB2 is a new member of the vertebrate Usher interactome.

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The mechanism by which platelets regulate the function of integrin ␣ IIb ␤ 3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, we screened a human fetal liver cDNA library in the yeast two-hybrid system, using the ␣ IIb cytoplasmic domain as "bait," and isolated a novel 855-base pair clone. The open reading frame encodes a novel 191-amino acid polypeptide (termed CIB for calciumand integrin-binding protein) that appears to be specific for the cytoplasmic domain of ␣ IIb , since it does not interact with the ␣ v , ␣ 2 , ␣ 5 , ␤ 1 , or ␤ 3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has sequence homology to two known Ca 2؉ -binding regulatory proteins, calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs corresponding to the two C-terminal Ca 2؉ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45 Ca 2؉ in blot overlay assays. Using reverse transcriptase-polymerase chain reaction and Western blot analysis, we detected CIB mRNA and protein (ϳ25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured ␣ IIb ␤ 3 indicates a specific interaction between CIB and intact ␣ IIb ␤ 3 . These results suggest that CIB is a candidate regulatory molecule for integrin ␣ IIb ␤ 3 .Integrin-mediated adherence of cells to extracellular matrix is important for a variety of physiological processes, such as hemostasis, angiogenesis, and cell differentiation (1). Integrin ␣ IIb ␤ 3 , the platelet fibrinogen receptor, is specific to the megakaryocytic lineage. In unstimulated platelets the majority of ␣ IIb ␤ 3 is in an inactive conformation or low affinity state and is unable to bind soluble ligands. Platelet agonists through their respective receptors transduce a cascade of intracellular signals ultimately converting ␣ IIb ␤ 3 to an active, high affinity state, capable of binding soluble ligands. This activation process is termed inside-out signaling (2).The role of the ␣ IIb and ␤ 3 cytoplasmic domains in inside-out signaling has been indicated using mutants of ␣ IIb ␤ 3 transfected into Chinese hamster ovary cells (3). When wild type ␣ IIb ␤ 3 is expressed in Chinese hamster ovary cells, it is unable to bind soluble fibrinogen. However, when an ␣ IIb ␤ 3 construct lacking the ␣ IIb cytoplasmic domain is expressed, the integrin becomes constitutively active and binds soluble fibrinogen with high affinity (4). Moreover, deletion of the conserved GFFKR motif of the ␣ IIb cytoplasmic domain makes the integrin constitutively active. These data suggest that the ␣ IIb cytoplasmic domain helps to maintain a default low affinity state of ␣ IIb ␤ 3 . Interestingly, a naturally occurring point mutation, Ser-752 3 Pro in the ␤ 3 cytoplasmic domain, disrupts inside-out signaling (5), indica...

The GPIIb/IIIa complex functions as the aggregation site on the platelet membrane surface. This complex has been purified, characterized biochemically and morphologically, and reconstituted into phospholipid vesicles. Fibrinogen and fibronectin bind to reconstituted GPIIb/IIIa with many of the properties that characterize their binding to intact platelets. The GPIIb/IIIa complex appears to be a member of a widely distributed family of cell-surface glycoproteins that mediate cellular interactions. The terms cytoadhesins30 and integrins39 have been suggested for the members of this family of two-subunit molecules. The aminotermini of the alpha subunits of these molecules have been sequenced and appear to be homologous. Three beta subunits have been identified for this family of receptors, indicating that many alpha subunits have a common beta subunit. The three beta subunits have been sequenced, and there is about a 40 to 50% identity among their amino acid sequences. It thus appears that the receptors mediating cellular interactions have evolved from a common ancestral gene.