Soluble E-selectin enhances intercellular adhesion molecule-1 (ICAM-1) expression in human tumor cell lines

E-selectin mediates neovascularization via its soluble form, while its membrane-bound form initiates binding of tumor cells to vascular endothelium. Therefore, it was studied whether soluble E-selectin regulates further adhesion molecules on tumor cells. In tumor cells but not in related nonmalignant cells, intercellular adhesion molecule (ICAM)-1 expression was strikingly increased from 5 to 68% positive cells by in vitro inoculation of a recombinant E-selectin-IgG1 within 24 h, as analyzed by flow cytometry. The absence of changes in the expression of vascular cell adhesion molecule, integrin ligands (CD11a, CD18, integrin alpha 4), and sialyl-Lewis X indicates a specific effect of soluble E-selectin on ICAM-1. A cell adhesion assay revealed that the enhanced adhesion on T-cells to tumor cells mediated by soluble E-selectin-induced ICAM-1 expression was at a maximum after a 12-h incubation period. Therefore, ICAM-1 regulation on tumor cells might be a mechanism of immune escape.     

Expression of leukocyte fucosyltransferases regulates binding to E-selectin: relationship to previously implicated carbohydrate epitopes

E-selectin is a carbohydrate-binding endothelial cell adhesion molecule that reportedly interacts with several related sialylated and fucosylated carbohydrates. The activity of leukocyte alpha1,3-fucosyltransferases (FucT-IV or FucT-VII) is an essential step in the synthesis of E-selectin ligands. Using a panel of stably transfected hemopoietic cell lines, we have investigated the role of alpha1,3-fucosyltransferases in generating E-selectin ligands, and the relationship between adhesion to E-selectin and expression of mAb-defined carbohydrates. Expression of FucT-VII was always sufficient for binding to E- and P-selectin, while the ability of FucT-IV to construct E-selectin ligands varied among different cell types. Furthermore, FucT-IV was unable to support any binding to P-selectin in a lymphoid cell line, even when expressed at levels equivalent to those in myeloid cells. FucT-IV expression generated high levels of surface Le(x)/CD15 and CDw65, whereas expression of FucT-VII correlated with a subset of mAb-defined sialyl Lewis X (sLex)-like structures. FucT-IV-associated epitopes were present on both binding and nonbinding cells, whereas all cells that expressed FucT-VII-associated epitopes bound E-selectin. However, treatment of HL60 cells with neuraminidase destroyed FucT-VII-associated epitopes at a faster rate than E-selectin binding sites. Surface expression of a subset of mAb-defined sLex-like carbohydrates is therefore a good marker for high levels of FucT-VII activity, but these carbohydrates are not themselves required for recognition of E-selectin.     

Expression of E-selectin, sialyl Lewis X, and macrophage inflammatory protein-1alpha by colonic epithelial cells in ulcerative colitis

The pathogenic significance of cell adhesion molecules (CAMs) in ulcerative colitis (UC) is largely unknown. Colonic expression of E-selectin, sialyl Lewis X (sLe(x)), and macrophage inflammatory protein-1x (MIP-1alpha) as well as serum concentrations of E-selectin and MIP-1alpha in UC were studied. Thirty patients with UC, 10 patients with irritable bowel syndrome, and 10 healthy subjects were included. Colonic biopsies were stained immunohistochemically, and blood concentrations were measured with an ELISA technique. Soluble E-selectin did not correlate with diagnosis or disease activity. MIP-1alpha was below the detection limit. Epithelial cells expressed all three molecules, both on surface membranes and intracellularly. sLe(x) staining was weaker (P = 0.0002) and MIP-1alpha staining stronger (P = 0.014) in UC patients than in controls. Leukocyte MIP-alpha staining correlated with diagnosis (P = 0.021), sLe(x) staining (P = 0.023), and colonoscopy (P = 0.018). It is shown that E-selectin, sLe(x), and MIP-1alpha are synthesized and expressed by epithelial cells, indicating that CAMs are not only involved in leukocyte extravasation and migration, but also in the interaction between leukocytes and colonic epithelium. This knowledge might contribute to the development of improved treatments in UC.     

Expression of human H-type alpha1,2-fucosyltransferase encoding for blood group H(O) antigen in Chinese hamster ovary cells. Evidence for preferential fucosylation and truncation of polylactosamine sequences

The human H(O) blood group is specified by the structure Fucalpha1-2Galbeta1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:beta-D-galactoside alpha1, 2-fucosyltransferase (alpha1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(alpha1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(alpha1, 2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(alpha1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(alpha1,2)FT cells binding to UEA-I contained polylactosamine [3Galbeta1-4GlcNAcbeta1-]n with the terminal sequence Fucalpha1-2Galbeta1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(alpha1, 2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type alpha1, 2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type alpha1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

Sialosyl-fucosyl poly-LacNAc without sialosyl-Lex epitope in myeloid cell line HL60 was shown to be the ligand for E-selectin-dependent adhesion, particularly under dynamic flow conditions, in our previous study (Handa K, Stroud MR, Hakomori S, Biochemistry 36, 12412-12420, 1997). HL60 cells express only fucosyl-transferase (FT) IV and VII. X3NeuAcVII3FucnLc10, a representative component showing E-selectin-dependent binding under dynamic flow conditions, is not alpha 1-->3 fucosylated at the penultimate GlcNAc catalyzed by FT-VII, but is alpha 1-->3 fucosylated at the internal GlcNAc catalyzed by FT-IV. VI3NeuAcnLc6 is converted to VI3NeuAcIII3FucnLc6 by FT-IV, but is also converted to VI3NeuAcV3FucnLc6 by FT-VII. Thus, penultimate fucosylation catalyzed by FT-VII is not restricted for nLc6 backbone, but is highly restricted for nLc10 backbone. The cooperative effect of FT-IV and FT-VII for synthesis of poly-LacNAc having sialosyl-Lex with internal fucosylation may be blocked or highly restricted in poly-LacNAc having more than two LacNAc units, because preferential alpha 1-->3 fucosylation by FT-IV takes place at internal GlcNAc, inhibiting penultimate fucosylation by FT-VII.     

Incidence of ligament lesions and associated degenerative changes in the elderly wrist

As the carbohydrate lacto-N-fucopentaose III (CD15 antigen or X-determinant) and its sialylated derivative sialyl-Lewis X are involved in the adhesion of cells rolling along the surface of endothelial cells, experiments were done to study the presence of these molecules on human eosinophils from patients with the idiopathic hypereosinophilic syndrome. Normal-density eosinophils from some patients showed higher levels of expression for lacto-N-fucopentaose III than light-density eosinophils. In contrast, sialyl-Lewis X was highly expressed by light-density eosinophils. Activation of normal-density eosinophils with calcium ionophore A23187 resulted in increased expression of these molecules for a short time. Monoclonal antibodies to these carbohydrates stimulated eosinophils to secrete eosinophil cationic protein, but not eosinophil peroxidase, and acted as costimulatory signals for C3b-induced degranulation of eosinophil cationic protein. It was suggested that CD15 and sialyl-Lewis X might contribute to eosinophil-mediated tissue injury in patients with the idiopathic hypereosinophilic syndrome.     

IL-12 promotes the adhesion of NK cells to endothelial selectins under flow conditions

This study examined the adhesive interactions of peripheral blood NK cells with P- and E-selectin and analyzed the effect of IL-12 on the binding of NK cells to these selectins. P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on most resting and IL-12-activated NK cells. However, the percentage of resting NK cells bound to P-selectin-IgG was 15%, and that of activated NK cells bound to P-selectin-IgG was 65%. Furthermore, the number of IL-12-activated NK cells bound to P-selectin-transfected Chinese hamster ovary cells was significantly higher than that of resting NK cells under flow conditions. These interactions were abolished by the incubation of these NK cells with anti-PSGL-1 (PL-1) mAb. Thus, PSGL-1/P-selectin interaction is important in the binding of resting and activated NK cells to P-selectin. NK cells express sialyl-Lewis(x) (sLe(x)) structure recognized by anti-sLe(x) mAb (KM-93), and IL-12 activation of NK cells increased the mean fluorescence intensity of KM-93-reactive NK cells. Adhesion of IL-12-activated NK cells to E-selectin-transfected Chinese hamster ovary cells was stronger than that of resting NK cells under flow conditions. These interactions were reduced markedly by incubation with anti-sLe(x) mAb. Thus, sLe(x) is the major ligand of resting and activated NK cells for E-selectin. These findings indicate that IL-12 stimulation of NK cells promotes their adhesion activity to endothelial selectins.     

High affinity binding of latent matrix metalloproteinase-9 to the alpha2(IV) chain of collagen IV

Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular matrix degradation. Previously, we showed the surface association of pro-MMP-9 in human breast epithelial MCF10A cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity (Kd approximately 22 nM) to MCF10A cells and other cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in zymogen activation and is not followed by ligand internalization, even after complex formation with tissue inhibitor of metalloproteinase-1 (TIMP-1). A 190-kDa cell surface protein was identified by ligand blot analysis and affinity purification with immobilized pro-MMP-9. Microsequencing and immunoblot analysis revealed that the 190-kDa protein is the alpha2(IV) chain of collagen IV. Specific pro-MMP-9 surface binding was competed with purified alpha2(IV) and was significantly reduced after treatment of the cells with active MMP-9 before the binding assay since alpha2(IV) is hydrolyzed by MMP-9. A pro-MMP-9.TIMP-1 complex and MMP-9 bind to alpha2(IV), suggesting that neither the C-terminal nor the N-terminal domain of the enzyme is directly involved in alpha2(IV) binding. The closely related pro-MMP-2 exhibits a weaker affinity for alpha2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the binding of alpha2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with alpha2(IV), the proenzyme does not bind to triple-helical collagen IV. These studies suggest a unique interaction between pro-MMP-9 and alpha2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network.     

Stimulation of extracellular matrix components in the normal brain by invading glioma cells

Malignant gliomas are characterized by an extensive invasion of tumor cells into the normal brain parenchyma. A substantial amount of data indicates that cell movement in general is regulated by specific interactions between extracellular matrix components and specific cell-surface receptors. In the present work, multicellular spheroids from 4 human glioma cell lines (U-373Mg, A-172Mg, U-251Mg and HF-66) were confronted with normal rat brain cell aggregates in vitro, which resulted in a progressive invasion of tumor cells into the brain aggregates. The co-cultures were then sectioned and immuno-stained for specific extracellular matrix components (laminin, fibronectin and collagen type IV) and for specific cell-surface receptors which bind to these components (integrins beta1, beta4, alpha3, alpha6). In addition, flow-cytometric measurements and Northern blot analyses showed expression of several different integrins within the cell lines. The alpha3 subunit was expressed strongly in all cell lines. Whereas the beta1 subunit was expressed weakly in exponentially growing monolayer cultures, it showed a pronounced expression in multicellular spheroids, indicating that the integrin expression may vary depending on the micro-environment within a tumor. Furthermore, normal brain tissue was able to produce laminin when confronted with the glioma cells, which also was observed for fibronectin and collagen type IV. The relevance of our observations to the in vivo situation was investigated further by immuno-staining 5 human glioma biopsy samples for laminin. In some areas of the tumors, specific deposits of laminin were observed. In conclusion, we have shown that normal brain tissue has the ability to produce extracellular matrix components, such as laminin, collagen type IV and fibronectin, when confronted with invading glioma cells. Our results show that the glioma cells express specific integrins which can interact with these extracellular matrix components. Such interactions may facilitate tumor cell migration and invasion.     

Tick-borne encephalitis virus (TBEV)-specific RT-PCR for characterization of natural foci of TBE and for other applications

Human cytomegalovirus infection of human umbilical vein endothelial cells reduces the ability of these cells to bind to fibronectin, collagen type IV and laminin. This suppression requires active virus, since UV-inactivated virus did not alter the binding ability of these cells to adhere to fibronectin, collagen type IV, and laminin. In an attempt to elucidate the molecular mechanism of this altered interaction, the surface expression of alpha 5 beta 1, alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins on cytomegalovirus-infected endothelial cells was examined using attachment inhibition assay and flow cytometric analysis. The results presented here show that infection with human cytomegalovirus selectively alters the expression of integrin on human endothelial cells, with the ability to induce downregulation of alpha 5 beta 1 and alpha 2 beta 1 (p = 0.001) and p = 0.03, respectively), while significantly upregulating alpha 6 beta 1 (p = 0.03), and marginally upregulating alpha 3 beta 1 (p = 0.05).     

Endothelial activation in monosodium urate monohydrate crystal-induced inflammation: in vitro and in vivo studies on the roles of tumor necrosis factor alpha and interleukin-1

OBJECTIVE: There is relatively little direct evidence for the roles of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in activating endothelium in vivo. The aim of this study was to use in vitro and in vivo models to investigate the contribution of these cytokines to both E-selectin expression and the recruitment of polymorphonuclear cells (PMN) in monosodium urate monohydrate (MSU) crystal-induced inflammation. METHODS: MSU crystals were incubated with freshly isolated mononuclear cells, after which the harvested supernatants were tested for their ability to induce E-selectin expression during coculture with human umbilical vein endothelial cells. Subsequent experiments were performed with the addition of neutralizing anticytokine antibodies/antisera. The role of TNF alpha was then studied in an MSU crystal-induced monarthritis model, in the presence or absence of anti-TNF alpha (5 mg/kg intravenously). 99mtechnetium (99mTc)-labeled PMN cells and (111)indium (111In)-labeled anti-E-selectin monoclonal antibody (MAb) 1.2B6 were intravenously administered 4 hours after intraarticular injection to quantify PMN recruitment and E-selectin expression in inflamed joints. RESULTS: MSU crystals were a potent stimulus for IL-1 and TNF alpha production by monocytes in vitro, and these cytokines fully accounted for MSU crystal-stimulated, monocyte-mediated endothelial activation. In the MSU crystal-induced monarthritis model, TNF alpha blockade was very effective in suppressing both E-selectin expression and PMN emigration into the inflamed joints, as judged by gamma-camera image analysis and postmortem tissue counting following the intravenous injection of 99mTc-PMN and 111In-anti-E-selectin MAb. CONCLUSION: IL-1 and TNF alpha appear to be the only factors released by monocytes following incubation with MSU crystals, which induce E-selectin expression in vitro. Anti-TNF alpha is effective in suppressing endothelial activation and PMN recruitment in vivo E-selectin imaging can be used to assess the endothelial response to therapy and may prove useful for clinical studies.     

Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli

Adhesion to collagens by most cell types is mediated by the integrins alpha1beta1 and alpha2beta1. Both integrin alpha subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified alpha1beta1 and alpha2beta1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the alpha1 and alpha2 I domains in specific collagen adhesion. We find that introducing the alpha2 I domain into alpha1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (alpha1-2-1beta1) is similar to the adhesion profile conferred by alpha2beta1, not alpha1beta1. The presence of alpha2 or alpha1-2-1 results in preferential binding to collagen I, whereas alpha1 expressing cells bind better to collagen IV. In addition, alpha1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas alpha2 or alpha1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by alpha2beta1 or alpha1-2-1beta1, but not by alpha1, requires the presence of Mn2+ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins.     

Targeting endothelium for gene therapy via receptors up-regulated during angiogenesis and inflammation

Endothelial cells are an attractive target for gene therapy because they are intimately involved in disease processes associated with inflammation and angiogenesis and because endothelial cells are readily accessible to gene therapy vectors via the circulation. Furthermore, specific receptors are up-regulated during angiogenesis or during inflammation. Therefore it should be possible to target the specific populations of endothelial cells involved in each of these disease processes. We have utilized two bispecific antibodies to target entry of an adenovirus vector into endothelial cells expressing a receptor up-regulated during angiogenesis (alpha v integrins) and a receptor up-regulated during inflammation (E-selectin). Both bispecific antibodies contain the anti-FLAG M2 mAb, which binds to a FLAG epitope genetically incorporated into the penton base protein of the vector, AdFLAG. The anti-alpha v-integrin x anti-FLAG bsAb was able to direct binding and entry of AdFLAG into endothelial cells via alpha v integrins. Likewise, the anti-E-selectin x anti-FLAG bsAb was able to direct binding and entry of AdFLAG into tumor-necrosis-factor(TNF)-activated endothelial cells via E-selectin. Endothelial cells not activated with TNF were not efficiently transduced by the AdFLAG/E-selectin bsAb complex. These results demonstrate that bispecific antibodies can be successfully used to target adenovirus to endothelially expressed receptors that are up-regulated during angiogenesis or inflammation.     

CD24 mediates rolling of breast carcinoma cells on P-selectin

P-selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P-selectin glycoprotein ligand-1 (PSGL-1). Certain PSGL-1 negative tumor cell lines can bind P-selectin under static conditions through the GPI-linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL-1- KS breast carcinoma cells attach to and roll on recombinant P-selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL-1+ cells, such as HL-60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol-phospholipase C (PI-PLC) significantly reduced KS cell rolling on P-selectin. The ability of KS cells to roll on P-selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl-Lewis(x) antigen was also necessary for CD24-mediated rolling on P-selectin. CD24 purified from KS cells supported rolling of P-selectin transfectants, but not L-selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P-selectin-dependent manner. Together our data show that CD24 serves as a ligand for P-selectin under physiological flow conditions. Interaction of tumor cells with P-selectin via CD24 may be an important adhesion pathway in cancer metastasis.     

Tumor necrosis factor-induced E-selectin expression on vascular endothelial cells

OBJECTIVE: To determine the tumor necrosis factor (TNF) receptor type involved in induction of E-selectin expression on vascular endothelial cells. DESIGN: Prospective, in vitro repeated-measures analysis of cellular responses. SETTING: Research laboratory in an academic medical center. SUBJECTS: Cultured human umbilical vein endothelial cells. INTERVENTIONS: Human umbilical vein endothelial cells were incubated with recombinant human TNF (rhTNF) to induce the expression of E-selectin on their surfaces. To block rhTNF from binding to receptors, the cells were incubated with monoclonal antibodies against TNF receptors (anti-CD120a and anti-CD120b). TNF-induced E-selectin expression of the endothelial cells, with and without blocking antibodies, was then determined using indirect immunofluorescence and flow cytometry. MEASUREMENTS AND MAIN RESULTS: Blocking of either CD120a or CD120b receptors individually resulted in inhibition of TNF-induced E-selectin expression on human umbilical vein endothelial cells. When both antibodies were added, the inhibition of TNF-induced E-selectin expression was synergistic. Inhibition of E-selectin expression was dependent on both TNF concentrations and antibody concentrations. CONCLUSIONS: Both CD120a and CD120b receptors are involved in TNF-induced E-selectin expression on human umbilical vein endothelial cells. Blocking of both or one receptor type can reduce or totally inhibit expression of E-selectin on human umbilical vein endothelial cells, but the response is dependent on both TNF and antibody concentrations.     

A randomized study of combined zidovudine-lamivudine versus didanosine monotherapy in children with symptomatic therapy-naive HIV-1 infection. The Pediatric AIDS Clinical Trials Group Protocol 300 Study Team

Platelet-derived adherence-inhibiting factor (AIF) has been demonstrated to regulate the neutrophil binding to type IV collagen. In this study, we have examined the effect of AIF on neutrophil adherence to confluently cultured human umbilical vein endothelial cells (EC). AIF inhibited neutrophil adherence to thrombin- or tumor necrosis factor alpha (TNF-alpha)-stimulated EC by 75 or 43%, respectively, but hardly affected neutrophil adherence to resting EC. It is interesting to note that the inhibitory activity of AIF was reduced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of neutrophils. Pretreatment of neutrophils or EC with AIF inhibited neutrophil adherence to thrombin- or TNF-alpha-stimulated EC, suggesting that neutrophils and EC have AIF-binding proteins. Using AIF-Sepharose affinity chromatography, AIF-binding proteins containing L-selectin were isolated from 125I-labeled resting neutrophils. However, L-selectin was markedly decreased in the AIF-binding fraction from fMLP-stimulated neutrophils. With the use of AIF-affinity chromatography, P- and E-selectins were obtained in the AIF-binding fractions from resting, thrombin-, and TNF-alpha-stimulated EC. It is important to note that P- and E-selectin were greatly increased in the AIF-binding fractions from thrombin- and TNF-alpha-stimulated EC, respectively. Furthermore, AIF was able to bind to L-selectin-IgG chimeric protein and inhibit the binding of chimeric protein to thrombin or TNF-alpha-stimulated EC. In addition, AIF inhibited the binding of anti-P- or anti-E-selectin monoclonal antibody to the lysates of thrombin- or TNF-alpha-stimulated EC. Together these observations indicate that AIF could recognize L-, P-, and E-selectins, and modulate neutrophil adherence to EC through interactions with selectin molecules.     

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.