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PubMed2001

Beta1 integrins show specific association with CD98 protein in low density membranes.

BACKGROUND: The CD98 (4F2, FRP-1) is a widely expressed cell surface protein heterodimer composed of a glycosylated heavy chain and a non-glycosylated light chain. Originally described as a T cell activation antigen, it was later shown to function in amino acid transport, cell fusion and homotypic cell aggregation. Several lines of evidence suggest its functional interaction with integrins but the biochemical basis for this interaction has been unclear. RESULTS: We demonstrate that CD98 constitutively and specifically associates with beta1 integrins (alpha2beta1,alpha3beta1, alpha5beta1 and alpha6beta1), but minimally with alpha4beta1. Integrin-CD98 association was established by reciprocal immunoprecipitation experiments, and confirmed by CD98-induced clustering of alpha3beta1 but not alpha4beta1 on the surface of rhabdomyosarcoma cells. Integrin-CD98 association is independent of the alpha subunit cytoplasmic tail, is maintained in alpha3beta1 ligand-interaction deficient mutants, and is not inhibited by EDTA. Within the CD98 heavy chain, a C109S mutation (but not a C330S mutation) caused a loss of beta1 integrin association. The same C109S mutation also caused a loss of CD98 light chain association. Importantly, CD98 associated selectively with beta1 integrins present in low density "light membrane" fractions on a sucrose gradient. CD98 was not present in dense fractions that contained the majority of beta1 integrins. Notably, the C109S mutant of CD98, that did not associate with beta1 integrins, showed also a reduced localization into light membrane fractions. CONCLUSIONS: We demonstrate that CD98 association with beta1 integrins is specific, occurs in the context of low density membranes, and may require the CD98 light chain.

3T3 Cells↗

PubMed2002

A new type of antimetastatic peptide derived from fibronectin.

PURPOSE: We found previously that fibronectin (FN) has a cryptic functional site (YTIYVIAL sequence within the 14th type III repeat) opposing cell adhesion to extracellular matrix. A 22-mer FN peptide containing this site, termed FNIII14, inhibits beta1 integrin-mediated adhesion without binding to integrins. The present study shows that FNIII14 has the potential to prevent lymphoma cell metastasis. EXPERIMENTAL DESIGN: Antimetastatic effect of FNIII14 has been evaluated through in vitro or in vivo experiments. RESULTS: FNIII14 inhibited the integrin alpha4beta1-mediated B lymphoma Ramos cell adhesion to VCAM-1 on venule endothelial cells, as well as to FN. Murine T lymphoma L5178Y-ML25 cells, which are known to metastasize to liver and spleen, preferentially adhered to vitronectin (VN) and migrated toward VN concentration gradients. FNIII14 abrogated both the integrin alphavbeta3-mediated adhesion and migration of L5178Y-ML25 cells. Inhibition of the alphavbeta3mediated L5178Y-ML25 cell adhesion by FNIII14 was reversed by phenylarsine oxide, a protein tyrosine phosphatase inhibitor. In addition, FNIII14 abrogated the VN-stimulated tyrosine phosphorylation of intracellular signaling proteins, including focal adhesion kinase (p125(FAK)) and paxillin, suggesting that such a diversity of FNIII14 effects might be because of the negative regulation of p125(FAK) and paxillin tyrosine phosphorylation, which has been involved in adhesion signals transduced by different integrins. The in vivo experiment using a murine metastasis model showed that FNIII14 would inhibit liver and spleen metastases of L5178Y-ML25 cells at a dose much lower than that of RGDS. CONCLUSIONS: FNIII14 might be applicable as a new type of antimetastatic agent distinct from integrin-binding peptides.

Animals↗

PubMed2002

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions.

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.

Animals↗

PubMed2002

Pulmonary eosinophilia in a murine model of allergic inflammation is attenuated by small molecule alpha4beta1 antagonists.

Inhibition of alpha4beta1/vascular cell adhesion molecule-1 (VCAM-1) interactions have therapeutic potential in treating allergic airway disease because of the importance of these adhesion molecules in the trafficking of eosinophils, lymphocytes, and monocytes. We examined several small molecule inhibitors of alpha4beta1/VCAM-1 interactions with in vitro potencies (IC(50) values) ranging from 0.52 nM (CP-664511; 3-[3-(1-[2-[3-methoxy-4-(3-O-tolyl-ureido)phenyl]-acetylamino]-3-methyl-butyl)isoxazol-5-yl]-propionic acid) to 38.5 nM (CP-609643; 3-[3-methyl-1-[2-[4-(3-O-tolyl-ureido)-phenyl]-acetylamino]-butyl)-isoxazol-5-yl]-propionic acid). The same compounds were evaluated in vivo using a murine model of ovalbumin-induced pulmonary eosinophilia. In this model, systemic administration of antibodies against alpha4 reduced bronchoalveolar lavage (BAL) eosinophilia approximately 60%. Small molecule alpha4beta1 antagonists were administered by intratracheal instillation and demonstrated dose-dependent inhibition of BAL eosinophil numbers and achieved a maximum inhibition of approximately 60%. In general, the rank order of potency for these compounds in vitro was consistent with that observed in vivo, which confirms that their efficacy is likely via blockade of alpha4beta1/VCAM-1 interactions. The most potent compound, CP-664511, also inhibited BAL eosinophilia following s.c. administration (1-10 mg/kg, s.c.). These data support the utility of small molecule alpha4beta1 antagonists in the treatment of relevant diseases, such as asthma.

Animals↗

PubMed2006

Highly constrained bicyclic VLA-4 antagonists.

VLA-4 is implicated in several inflammatory and autoimmune disease states. A series of cyclic beta-amino acids (beta-aa) was studied as VLA-4 antagonists. Binding affinity was highly dependent on the dihedral angle (phi) between the amino and the carboxyl termini of the beta-aa. Compound 5 m where the beta-aa is embedded in a bicycle possesses the most preferred phi (120 degrees). It is a potent and bioavailable VLA-4 antagonist (VCAM-Ig alpha4beta1 IC50 = 54 nM, rat po F = 49%).

Biological Availability↗

PubMed1997

Activation of beta1 integrins on CML progenitors reveals cooperation between beta1 integrins and CD44 in the regulation of adhesion and proliferation.

Adhesion of normal colony-forming cells (CFC) to bone marrow (BM) stroma and the extracellular matrix (ECM) component fibronectin (FN) depends at least in part on the alpha4beta1 and alpha5beta1 integrins and the CD44 receptor. Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal CFC is associated with inhibition of their proliferation. In contrast to normal CFC, chronic myelogenous leukemia (CML) Ph+ CFC adhere significantly less to either stroma or FN. CML Ph+ CFC proliferation is also not inhibited by coculture with stroma or FN. However, equal numbers of alpha4, alpha5, and beta1 integrins and CD44 are present on CML and normal CD34+ cells. We have previously demonstrated that beta1-dependent adhesion to and subsequent proliferation inhibition by FN can be restored when CML Ph+ CFC are incubated with the beta1 integrin activating antibody, 8A2, and demonstrated a role for the alpha5beta1 integrin in this phenomenon. Since the integrin alpha4beta1 and the proteoglycan form of CD44 may cooperate in establishing normal CFC adhesion to FN, we examined if treatment of CML Ph+ CFC with 8A2 also restores the cooperativity between beta1 integrins and CD44. We demonstrate that 8A2 induces adhesion of CML Ph+ CFC not only to intact FN but also to alpha4beta1, alpha5beta1, and proteoglycan binding fragments of FN. 8A2-induced adhesion to these fragments and peptides also results in a significant inhibition of the proliferation of CML Ph+ CFC. Addition of antibodies to either the alpha5, alpha4, or beta1 integrins, antibodies against the CD44 receptor, or removal of chondroitin sulfate glycosaminoglycans from the surface of CML CD34+ HLA-DR+ cells significantly reduced the 8A2-induced adhesion to and adhesion-mediated inhibition of proliferation by FN. These studies demonstrate that activation of beta1 integrins on CML Ph+ CFC not only results in upregulation of beta1 integrin-dependent adhesion and adhesion-mediated inhibition of proliferation, but also in the restoration of cooperation between beta1 integrins and CD44. These studies suggest that decreased beta1 integrin avidity may also affect the function of the proteoglycan adhesion receptor CD44, both of which may contribute to the abnormal circulation and expansion of malignant progenitors in CML.

Antigens, CD↗

PubMed2003

Rac2 specificity in macrophage integrin signaling: potential role for Syk kinase.

Herein we report that, despite the similarity of Rac2 to Rac1 (92% amino acid identity), macrophages derived from Rac2-/- mice, which continue to express Rac1, display a marked defect in alphavbeta3/alphavbeta5 and alpha4beta1 integrin-directed migration measured on vitronectin and fibronectin fragments (FN-H296), respectively. In contrast, mouse embryo fibroblasts derived from the Rac2 knockout mice utilize Rac1 for migration via alphavbeta3/alphavbeta5 and alpha4beta1. The genetic reconstitution of bone marrow-derived macrophages (BMM) with Rac2 restores the integrin-dependent migration of Rac2-deficient macrophages on vitronectin (VN) and FN-H296. The levels of GTP-Rac2 generated upon specific integrin engagement in wild type macrophages parallels the phenotypic defect observed in Rac2-deficient macrophages; i.e. FN-H296, alpha4beta1 > VN, alphavbeta3/alphavbeta5 > FN-CH271, alpha5beta1 > intact FN. In a COS7 cell system, the expression of Syk kinase alone is sufficient to convert the alpha4beta1 migration response to Rac2 dependence. Therefore, we present the first evidence that the alpha4beta1 receptor in blood cells has evolved a Syk-Rac2 signaling axis to transmit signals required for integrin-directed migration suggesting that Syk kinase in part encodes myeloid Rac2 specificity in vivo.

Animals↗

PubMed2000

Fibronectin-mononuclear cell interactions regulate type 1 helper T cell cytokine network in tolerant transplant recipients.

Fibronectin (FN), expressed primarily by macrophages, endothelial cells, and smooth muscle cells, represents an integral feature of the rejection response in transplant recipients. Here we demonstrate a unique pattern of cellular FN expression in rat recipients of cardiac allografts rendered tolerant in an infectious manner with either nondepleting CD4 mAb or regulatory spleen cells. Unlike in rejecting controls, cellular FN in tolerant hosts was restricted to the graft vessels and no vascular cell adhesion molecule-1 or intercellular adhesion molecule-1 expression could be found, supporting the role of FN in leukocyte sequestration at the graft site. The lack of myocardial FN in tolerant rats, despite dense macrophage infiltration, correlated with profound depression of Th1 (interleukin-2 and interferon-gamma) cytokines. Treatment with CD4-depleting mAb prevented tolerance induction and restored myocardial expression of FN in parallel with marked increase in the expression of interleukin-2 and interferon-gamma mRNA/protein. Furthermore, connective segment-1 peptide-facilitated adjunctive blockade of FN-alpha4beta1 interactions in recipients conditioned with CD4 depleting mAb, significantly depressed intragraft expression of interleukin-2 and interferon-gamma mRNA/protein. Hence, the lack of FN associated with infiltrating leukocytes plays an important role in the maintenance of tolerance in transplant recipients by depressing local expression of Th1 cytokines that otherwise facilitate acute graft rejection.

Animals↗

PubMed2000

CD99 (E2) up-regulates alpha4beta1-dependent T cell adhesion to inflamed vascular endothelium under flow conditions.

CD99/E2 is an integral transmembrane protein which forms, together with Xga, a distinct family whose genes are located in the pseudoautosomal region. The number of T cells that firmly bound to vascular endothelial cells under physiological shear stress increased 2-14-fold upon CD99 stimulation, and bound cells became much more resistant to detachment forces and spread. T cell arrest occurred within 1 min and was dependent on the alpha4beta1-VCAM-1 pathway. In contrast, the alphaLbeta2-ICAM-1 pathway remained unactivated. This was observed with T cell lines and with activated peripheral blood lymphocytes, and was limited within the resting peripheral CD4+ T cells to the memory subset, while virgin cells were unaffected. This discloses a stepwise regulation of the T cell extravasation cascade.

12E7 Antigen↗

PubMed2005

Alpha4beta1 and alpha4beta7 CD4 T cell numbers increase and CLA CD4 T cell numbers decrease in systemic sclerosis.

We studied the expression of adhesion molecules affecting recirculation and homing on peripheral blood CD4(+) T cells of patients with systemic sclerosis (SSc), in order to evaluate whether the distribution of tissue targeted subsets could reflect the participation of internal organs or the extent of cutaneous involvement [i.e. limited cutaneous (lc) and diffuse cutaneous (dc)]. Peripheral blood mononuclear cells (PBMC) from 51 patients with SSc and 19 sex- and age-matched controls were investigated by cytofluorimetric analysis for lymphocyte subpopulations carrying the following surface molecules: CD3, CD4, CLA, alpha4beta7 and alpha4beta1. Standard routine biochemistry and clinical examinations were also performed in all patients. We found that both alpha4beta1(+) and alpha4beta7(+) cells within the CD4(+) T cell population were significantly increased, while CLA(+) CD4(+) T cells were significantly reduced in SSc, compared to healthy donors. Significantly lower absolute numbers of alpha4beta7(+) cells were found in lc- compared to dc-SSc. Patients with oesophageal involvement had high numbers of alpha4beta7(+) cells, while those with nephritis also showed low levels of CLA(+) cells. Lung involvement was related directly to alpha4beta1(+) cell numbers and inversely to alpha4beta7(+) CD4 cell numbers. Taken together, our findings demonstrate that distinct CD4(+) T cell populations with selective homing properties show changes from normal distribution in SSc, and such changes are related to clinical expression and organ involvement in the course of the disease.

Adult↗

PubMed2002

Role of integrins in invasion of endometrial cancer cell lines.

OBJECTIVE: The objective of this study was to determine whether there is a link between integrins and components of the plasminogen system, regarding tumor cell invasion to the artificial extracellular matrix. METHODS: Immunocytochemistry was performed with antibodies directed against integrins alpha2beta1, alpha4beta1, alpha5beta1, alpha6beta1, and alpha(nu)beta3 Cell adhesion was studied by cell attachment to matrix proteins. Integrin-mediated migration of cells into the artificial matrix was studied in an invasion assay. Last, immunoreactivity of the components of the plasminogen system was estimated by ELISA. RESULTS: HEC-1A, Ishikawa, and AN3CA cell lines express the alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 integrins. Adhesion of cells to fibronectin and laminin was inhibited by monoclonal antibodies directed against appropriate integrin subunits. Migration into the matrigel was partially blocked with mAbs to alpha4beta1 as well as alpha5beta1, alpha6beta1 integrins and their combination. No differences in either uPA or PAI-1 immunoreactivity were found after addition of the mAbs. CONCLUSION: Alpha4beta1, alpha5beta1, and alpha6beta1 heterodimers mediate adhesion as well as migration into the artificial matrix matrigel of endometrial tumor cell lines. A cooperative signaling between these integrins and uPA or its inhibitor in modulation of in vitro cell invasion is rather unprobable.

Antibodies, Monoclonal↗

PubMed2006

In vivo correlation between IL-1beta concentration in uterine fluid and integrin expression pattern in infertile women.

PROBLEM: To evaluate the possible correlation between interleukin-1beta (IL-1beta) and integrin expression. METHOD OF STUDY: Endometrium and uterine fluid from 77 infertile women and 24 fertile, healthy control were studied with immunohistochemistry and enzyme-linked immunosorbent assay for the presence of IL-1beta and integrin alphaVbeta3 and alpha4beta1 expression. RESULTS: Highest expression of alphaVbeta3 was found in epithelium and glands in endometria from fertile women. There was no positive correlation between IL-1beta and integrin expression pattern. CONCLUSION: This study confirms the importance of alphaVbeta3 as molecular marker of receptive phase. Also supports the views that synchrony in development of endometrium is necessary for successful nidation. There was however, contrary to results of previous in vitro studies, no positive correlation between IL-1beta and endometrial integrin patter expression.

Adult↗

PubMed1999

A comparison of C3a and C5a-mediated stable adhesion of rolling eosinophils in postcapillary venules and transendothelial migration in vitro and in vivo.

The comparative ability of the complement anaphylatoxins C3a and C5a to mediate leukocyte adhesion and transendothelial migration in vivo and in vitro was investigated. Superfusion of IL-1beta-stimulated rabbit mesentery with C3a resulted in a rapid and stable adhesion of rolling eosinophils, but not neutrophils, to postcapillary venules. However, C3a failed to evoke subsequent transmigration of the adherent eosinophils. In contrast, C5a induced both the rapid activation-dependent firm adhesion and transmigration of eosinophils and neutrophils through venular endothelium. C3a induced selective shedding of L-selectin and an increase in alphaMbeta2 integrin expression on eosinophils but not neutrophils, while C5a induced shedding of L-selectin and up-regulation of alphaMbeta2 integrin on both eosinophils and neutrophils. Both C3a- and C5a-dependent adhesion to venular endothelium was blocked by ex vivo treatment of eosinophils with anti-alpha4 and anti-beta2 integrin mAbs. In vitro, both C3a (but not C3a(desArg)) and C5a (including C5a(desArg))-dependent transmigration of eosinophils across IL-1beta-stimulated endothelial monolayer was mediated by alpha4beta1 and alphaMbeta2 integrins. Overall these studies suggest that C3a is eosinophil-specific chemotactic mediator that influences selectively eosinophil adhesion but not transmigration in vivo. C5a in contrast is a complete activator of integrin-dependent adhesion as well as transmigration of eosinophils and neutrophils.

Animals↗

PubMed1999

Inhibition of cell adhesion by antibodies to Arg-Gly-Asp (RGD) in normal immunoglobulin for therapeutic use (intravenous immunoglobulin, IVIg).

Intravenous immunoglobulin (IVIg) therapy is associated with a broad range of immunomodulatory activities. Several of the postulated mechanisms of IVIg action relate to the presence of antibodies to molecules relevant for regulation of the immune response. This article reports that IVIg contains antibodies to the Arg-Gly-Asp (RGD) sequence, and the attachment site of a number of adhesive extracellular matrix proteins, including ligands for beta1, beta3, and beta5 integrins. Anti-RGD antibodies were identified in IVIg by enzyme-linked immunosorbent assay and by using the BIAcore (BIAcore, Uppsala, Sweden) technology. The affinity of anti-RGD antibodies to a synthetic RGD-containing peptide and to fibronectin (Fn) was found to be in the micromolar range. F(ab')2 fragments specific for RGD were purified from IVIg by affinity chromatography. Anti-RGD F(ab')2 antibodies inhibited adenosine diphosphate induced alphaIIb/beta3 integrin-mediated platelet aggregation and the adhesion of activated alpha4beta1 integrin-expressing B cells to Fn. Adhesion of unstimulated platelets to fibrinogen (Fg) involving both the gamma-chain dodecapeptide sequence and the RGD sequence was inhibited by anti-RGD antibodies. In addition, adhesion of thrombin-stimulated platelets to von Willebrand factor or Fg was completely inhibited by affinity-purified anti-RGD antibodies. Our results suggest that the presence of natural IgG antibodies to the RGD motif may contribute to the immunomodulatory and anti-inflammatory effects of therapeutic preparations of normal IgG.

Antibodies↗

PubMed2004

beta1-Integrins mediate enhancement of airway smooth muscle proliferation by collagen and fibronectin.

Airway smooth muscle (ASM) accumulation and enrichment of the extracellular matrix (ECM) with type I collagen and fibronectin are major pathologic features of airway remodeling in asthma. These ECM components confer enhanced ASM proliferation in vitro, but a requirement for specific integrin ECM receptors has not been examined. Here, we examined the mitogen platelet-derived growth factor (PDGF)-BB on beta1-integrin expression on human ASM cells cultured on these ECM substrates and defined the involvement of specific integrins in cell attachment and proliferation using integrin-neutralizing antibodies. PDGF-BB-dependent proliferation was enhanced two- to threefold by monomeric type I collagen or fibronectin and to a lesser extent by vitronectin; other interstitial ECM components (fibrillar type I and III collagen and tenascin-C) had no effect. Except for increased alpha3 expression induced by PDGF-BB and monomeric type I collagen or fibronectin, alpha1, alpha2, alpha4, alpha5, alphav, and alphavbeta3 integrins were unchanged compared with unstimulated cells on plastic. Blocking antibodies revealed alpha2beta1- and alphavbeta3-mediated attachment to monomeric type I collagen, whereas attachment to fibronectin required alpha5beta1. In contrast, enhancement of PDGF-BB-dependent proliferation by either monomeric type I collagen or fibronectin required alpha2beta1, alpha4beta1, and alpha5beta1 integrins. These data suggest multiple beta1-integrins regulate enhanced ASM proliferative responses.

Adult↗

PubMed2004

Structural requirements of MLD-containing disintegrins for functional interaction with alpha 4 beta 1 and alpha 9 beta1 integrins.

Three non-RGD-containing disintegrins, VLO5, EO5, and EC3, belong to the heterodimeric family of these snake venom-derived proteins. They are potent inhibitors of certain leukocyte integrins such as alpha4beta1, alpha4beta7, and alpha9beta1, and act through the MLD motif present in one of their subunits. However, the selectivity of these disintegrins to interact with integrins is related to the amino acid composition of the integrin-binding loop in the MLD-containing subunit. The most important amino acid is that preceding the MLD motif. In vitro experiments in adhesion and ELISA assays revealed that the TMLD-containing disintegrins, VLO5 and EO5, appeared to be very potent inhibitors of human alpha4beta1 and alpha9beta1 and less effective in inhibition of the alpha4beta7 integrin. The reverse effect was observed for the AMLD-containing disintegrin, EC3. The data with native disintegrins were confirmed by experiments with synthetic peptides displaying TMLD and AMLD motifs. The MLD-containing disintegrins showed differential activities to inhibit human and murine alpha4beta1 integrin. EC3 was a weaker inhibitor of human integrin, whereas VLO5 and EO5 less actively inhibited murine alpha4beta1. These data describe a useful set of potent and selective integrin antagonists and suggest conformational requirements of human and mouse integrins for interaction with ligands.

Amino Acid Motifs↗

PubMed2000

Integrin alpha2beta1 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3.

It was previously reported that integrins alpha2beta1, alpha4beta1, and alphaXbeta2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits alpha2, alpha4, and beta2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin alpha2beta1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds alpha2beta1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits alpha4 and beta2 do not seem to be involved in the initial cell binding of either virus.

Antibodies, Monoclonal↗

PubMed1996

Beta1 integrin-mediated activation of focal adhesion kinase and its association with Fyn and Zap-70 in human NK cells.

Adhesive interactions mediated by cell surface receptors have been shown to induce signal transduction pathways that regulate changes in cellular function. We have reported recently that fibronectin (FN) receptors, alpha4beta1 and alpha5beta1 integrins, on NK cells transduce transmembrane signals leading to tyrosine phosphorylation of 60-, 70-, and 120-kDa proteins. In the current study, we have identified a 120-kDa phosphoprotein as the focal adhesion kinase (p125FAK), a structurally unique nonreceptor protein tyrosine kinase that localizes to focal adhesions. Activity of p125FAK was induced by adhesion of NK cells to plastic-immobilized FN, by cross-linking of cell surface-bound FN or FN fragments, FN120 or FN40, with anti-FN mAb, or by cross-linking of alpha4beta1 or alpha5beta1 integrins with alpha-chain-specific Abs. We also observed that enhanced in vitro kinase activity was associated with immunoprecipitates of alpha4beta1 or alpha5beta1 integrins from lysates of FN-adherent NK cells as compared with BSA-treated NK cells. In addition to p125FAK activity, FN-induced kinase activity was also found to be mediated by Fyn, Lyn, and Zap-70, as assessed by in vitro phosphorylation of the immunoprecipitated kinases in the presence of [gamma-32P]ATP. Clustering of FN receptors on NK cells by agonists such as immobilized FN or alpha4- or alpha5-specific Abs also induced association of Fyn and Zap-70 with p125FAK. Our observations indicate that activation and phosphorylation of p125FAK as well as Zap-70 and certain kinases of the src family play an important role in formation of active signaling complexes in response to triggering via beta1 integrins on NK cells. These results also suggest the existence of cross-talk or points of convergence between the beta1 integrin-mediated and other receptor-signaling pathways.

Cell Adhesion↗