Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin ␣v␤3 (K D about 1 M). Antagonists to ␣v␤3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. ␣v␤3 was the predominant, if not the only, integrin that bound to FGF1, because FGF1 bound only weakly to several ␤1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of ␤3 plays a role in FGF1 binding. We found that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1 and FRS2␣ and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful tool to identify the role of integrins in FGF signaling. Fibroblast growth factors (FGFs)2 constitute a family of heparin-binding polypeptides involved in the regulation of biological responses such as growth, differentiation, and angiogenesis (1-4). The FGF family currently consists of 22 members with FGF1 (acidic FGF) and FGF2 (basic FGF) the most extensively studied. The biological effects of FGFs are mediated by four structurally related receptor tyrosine kinases designated FGFR1-4. The binding of FGF to its receptor results in receptor dimerization and subsequent autophosphorylation of specific tyrosine residues within the cytoplasmic domain (1-4). Activation of the receptor allows proteins containing Src homology 2 or phosphotyrosine binding domains to bind to sequence recognition motifs in the FGFR, resulting in phosphorylation and activation of these proteins (5). This leads to the activation of intracellular signaling cascades. The main signaling cascade activated through the stimulation of FGFR is the Ras/MAPK pathway.FGF signaling enhances multiple biological processes that promote tumor progression (6). Therapies targeting FGF receptors and/or FGF signaling not only affect the growth of the tumor cells but also modulate tumor angiogenesis (7). FGF1 and FGF2 are responsible for resistance to chemotherapeutic agents in cancer (8 -11) and are also pro-inflammatory growth factors that play a role in pathological angiogenesis in chronic inflammatory diseases (12). Thus FGF signaling is a potential therapeutic target for cancer and pathological angiogenesis in chronic inflammatory diseases.It has been proposed that cross-talk between i...

Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH 4 Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses. IMPORTANCEBovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin-and cholesteroldependent, clathrin-mediated endocytosis that requires Rab5 and Rab7....

Plasmin is a major extracellular protease that elicits intracellular signals to mediate platelet aggregation, chemotaxis of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types in a G protein-dependent manner. Angiostatin, a fragment of plasmin(ogen), is a ligand and an antagonist for integrin ␣ 9 ␤ 1 . Here we report that plasmin specifically interacts with ␣ 9 ␤ 1 and that plasmin induces migration of cells expressing recombinant ␣ 9 ␤ 1 (␣ 9 -Chinese hamster ovary (CHO) cells). Migration was dependent on an interaction of the kringle domains of plasmin with ␣ 9 ␤ 1 as well as the catalytic activity of plasmin. Angiostatin, representing the kringle domains of plasmin, alone did not induce the migration of ␣ 9 -CHO cells, but simultaneous activation of the G protein-coupled protease-activated receptor (PAR)-1 with an agonist peptide induced the migration on angiostatin, whereas PAR-2 or PAR-4 agonist peptides were without effect. Furthermore, a small chemical inhibitor of PAR-1 (RWJ 58259) and a palmitoylated PAR-1-blocking peptide inhibited plasmin-induced migration of ␣ 9 -CHO cells. These results suggest that plasmin induces migration by kringle-mediated binding to ␣ 9 ␤ 1 and simultaneous proteolytic activation of PAR-1.

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We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na The ubiquitous plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) 4 plays central roles in cellular pH and volume homeostasis, cell migration, proliferation, and survival, and increased NHE1 activity contributes to ischemia-reperfusion injury as well as tumor growth and proliferation (1, 2). Hence, the ability to selectively block NHE1, although of high clinical relevance, is hampered by a lack of detailed understanding of NHE1 structure and mechanism of ion translocation. Hydropathy analyses and accessibility studies indicate that NHE1 has 12 transmembrane (TM) segments and a large Cterminal cytoplasmic region (3). Cysteine accessibility studies suggest the presence of two small re-entrant loops between TM IV and TM V (intracellular loop (IL) II) and TM VIII and TM IX (IL IV), respectively, and a larger re-entrant loop between TM IX and TM X (extracellular loop V) (1, 3). Portions of IL II and IL IV are located within the membrane and accessible from either side of the membrane, suggesting that they may form structures lining an aqueous pore and could be involved in ion translocation by NHE1 (3). Extracellular loop V is also interesting in this regard, because it resembles the Ploops found in voltage-gated ion channels (1, 3). These putative re-entrant loops are highly conserved among several NHE1 homologs, consistent with the notion that they are critical for NHE1 function (3-5).A number of regions within the NHE1 protein have been implicated in inhibitor binding, e.g. TM IV and TM IX (6 -11); however, the mechanism(s) of interaction between NHE1 and its commonly used inhibitors, amiloride and benzoyl guanidine type compounds, remain to be fully elucidated.Using a comparative approach based on chimeras generated using human NHE1 (hNHE1) and two NHE1 homologs (flounder paNHE1 and Amphiuma tridactylum NHE1) with high sequence homology to hNHE1 yet markedly different inhibitor profiles (4, 5), we previously obtained novel information on the regions of NHE1 important for inhibitor binding and ion transport (12). These studies confirmed that TM IV plays a central role in inhibitor binding (12) as suggested by earlier point mutation studies (6 -11). Moreover, we demonstrated that regions in TM X-XI and/or IL V and extracellular loop VI are important determinants of inhibitor sensitivity (12).The three-dimensional structure of NHE1 is unknown; however, the structure of the distantly related bacterial (Esch-

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