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PubMed2006

Integrin alpha3beta1 interacts with I1PP2A/lanp and phosphatase PP1.

Integrin alpha3beta1 is a receptor for the extracellular matrix component laminin 5. To elucidate possible signaling pathways induced by integrin alpha3beta1, we looked for proteins that interact with the cytoplasmic part of the alpha3A integrin subunit. We identified several multifunctional proteins by affinity chromatography and subsequent MALDI-TOF-MS and focused on the inhibitor 1 of serine/threonine phosphatase PP2A (I1PP2A, synonym: lanp) which also plays a role during the development of the mouse cerebellum. I1PP2A/lanp colocalizes with the alpha3A integrin subunit in differentiated PC12 cells in the cell body and in neurites as well as in Purkinje cells of mouse cerebellum. Overexpression of GFP-I1PP2A/lanp in PC12 cells leads to markedly reduced neurite length on laminin 5 after induction with nerve growth factor. By affinity chromatography the protein phosphatase PP1 can also be identified as a alpha3A/cyto-binding protein. PP1 and integrin alpha3beta1 can be pulled down by GST-I1PP2A/lanp from cell lysates of differentiated and undifferentiated PC12 cells. The phosphatase binds to the cytoplasmic membrane-proximal conserved GFFKR motif of the alpha integrin subunit, whereas I1PP2A/lanp requires a longer sequence for binding. PP1 but not PP2A is able to dephosphorylate precipitated integrin alpha3beta1 in vitro. Furthermore, PP1 releases phosphate from T1046 of phosphopeptides that mimic the phosphorylation consensus sequence in the cytoplasmic part of the alpha3A integrin subunit. These data suggest that I1PP2A/lanp forms a complex with PP1 and the alpha3A integrin subunit and might possibly regulate the phosphorylation status of integrin alpha3beta1 and/or integrin downstream targets.

Animals↗

PubMed1998

Separate functions of alpha2beta1 and alpha3beta1 integrins in the metastatic process of human gastric carcinoma.

The expression of alpha2beta1 and alpha3beta1 integrins was studied immunohistochemically in 110 resected human gastric carcinomas. Formalin-fixed and paraffin-embedded samples were serially sectioned and stained with monoclonal antibodies specifically against the alpha2 and alpha3 subunits of beta1 integrins. Approximately 27% of the tumors expressed alpha2beta1 integrin, and 20% expressed alpha3beta1 integrin. The expression of alpha2beta1 integrin was associated with lymph node and liver metastases (P < 0.05 and P < 0.05, respectively), and the expression of alpha3beta1 integrin was associated with liver and peritoneal metastases (P < 0.005 and P < 0.0005, respectively). A multivariate analysis by the logistic regression model revealed that the expression of alpha2beta1 and/or alpha3beta1 integrins was an independent factor related to liver metastasis, and that the expression of alpha3beta1 integrin was as strong an influence on the formation of peritoneal metastases as the depth of invasion. The expression of alpha3beta1 integrin also correlated with increased invasiveness of the tumor; however, there was no correlation between alpha2beta1 integrin expression and the invasiveness. These results suggest that alpha2beta1 and alpha3beta1 integrins have separate influences on the metastatic properties of cancer cells.

Extracellular Matrix Proteins↗

PubMed2005

A specific microdomain ("glycosynapse 3") controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9.

Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349-34358), later termed "glycosynapse 3" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin alpha3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the alpha3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of alpha3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.

Antigens, CD↗

PubMed2005

Potentiation of the ligand-binding activity of integrin alpha3beta1 via association with tetraspanin CD151.

CD151, one of the tetraspanins, forms a stable complex with integrin alpha3beta1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin alpha3beta1-CD151 complexes, was capable of dissociating CD151 from integrin alpha3beta1, thereby allowing us to deplete CD151 from purified integrin alpha3beta1-CD151 complexes. The CD151-free integrin alpha3beta1 thus obtained showed a significant reduction in its ability to bind to laminin-10/11, a high-affinity ligand for integrin alpha3beta1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated beta1-containing integrins. These results raised the possibility that the association of integrin alpha3beta1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin alpha3beta1 was reassociated with purified CD151. 8C3-induced dissociation of CD151 from integrin alpha3beta1 was also demonstrated on the surface of living cells by fluorescent resonance energy transfer imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10/11-coated substrates. CD151 knock-down by RNA interference also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligand-binding activity of integrin alpha3beta1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context.

Animals↗

PubMed2000

Expression of the alpha2beta1 and alpha3beta1 integrins at the surface of mesothelial cells: a potential attachment site of endometrial cells.

OBJECTIVE: To localize alpha2beta1 and alpha3beta1 integrins in the cell membrane of peritoneal mesothelium in vivo and in vitro. DESIGN: Descriptive study using confocal and two-photon laser-scanning microscopy. SETTING: University-based laboratory. PATIENT(S): Women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Peritoneal biopsies (n = 9) and mesothelial monolayer cultures (n = 4) were incubated with antibodies to the alpha2 and alpha3 subunits and to the intact alpha2beta1 and alpha3beta1 integrins. Specimens were examined with laser-scanning microscopy. RESULT(S): The alpha2 and alpha3 subunits and the intact alpha2beta1 and alpha3beta1 integrins were identified at the base of the mesothelial cells (i.e., toward the basement membrane). There was also expression of the alpha2 and alpha3 subunits and the intact alpha2beta1 and alpha3beta1 integrins at the cell surface (i.e., toward the peritoneal cavity). CONCLUSION(S): The resolution of the confocal and two-photon laser-scanning microscope enabled localization of integrins in mesothelial cells. The presence of alpha2beta1 (collagen-laminin receptor) and alpha3beta1 integrins (collagen-laminin-fibronectin receptor) at the base of mesothelial cells suggests a role for these molecules in adhesion to the basement membrane. The presence of these molecules at the cell surface suggests a potential locus for cell adhesion in such processes as endometriosis and cancer metastasis.

Adult↗

PubMed1999

H-Ras is a negative regulator of alpha3beta1 integrin expression in ECV304 endothelial cells.

We have examined the role of Ras in integrin expression in ECV304 endothelial cells. Among the integrins examined in stable ECV304 transfectants expressing dominant active H-Ras (DAR-ECV), expression of alpha3beta1 integrin showed a prominent reduction in all the DAR-ECV clones when compared to the parental ECV304 cells. This implies that H-Ras negatively regulates the expression of alpha3beta1 integrin in ECV304 cells. When treated with inhibitors of the Ras downstream pathway (LY294002, PD98059, SB203580), the expression of alpha3beta1 integrin was up-regulated most significantly by LY294002, suggesting that among the downstream pathways of Ras, phosphatidylinositol 3-kinase is a major determinant. With the application of blocking antibody to alpha3beta1 integrin (2 - 2 x 10(4) nM), migration of ECV304 cells was enhanced to maximal (18%) at 20 nM. These results suggest that migration of endothelial cells could be modulated by H-Ras via alteration of the expression levels of alpha3beta1 integrin.

Antibodies↗

PubMed2003

Characterization of the ligand-binding specificities of integrin alpha3beta1 and alpha6beta1 using a panel of purified laminin isoforms containing distinct alpha chains.

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.

Animals↗

PubMed2001

Immunohistochemical distribution of CD9, heparin binding epidermal growth factor-like growth factor, and integrin alpha3beta1 in normal human tissues.

The tetra-membrane-spanning protein CD9 forms a complex with a membrane-anchored heparin binding epidermal growth factor-like growth factor (HB-EGF) and integrin alpha3beta1 in some human and monkey cell lines. We show here the immunohistochemical distribution of CD9, HB-EGF, and integrin alpha3beta1 in normal human tissues. Distribution of CD9, HB-EGF, and integrin alpha3beta1 was similar in various tissues, including transitional epithelium, squamous epithelium, thyroid follicular epithelium, adrenal cortex, testis, smooth muscle, and stromal fibrous tissue. However, distribution of the three proteins did not coincide in some tissues, such as lung, liver, kidney, gastric and intestinal epithelium, pancreas, salivary gland, and ovary. In striated muscle, including cardiac muscle, CD9 was present not in the muscle cells themselves but in the endomysium and perimysium, whereas HB-EGF was distributed in the muscle cells themselves. CD9 was distributed in the myelin, but HB-EGF was found in the axon of the peripheral and central nervous systems. Coincident distribution of integrin alpha3beta1 with others was not observed in muscles and neural tissues. In conclusion, there is a possibility of complex formation and functional cooperation of CD9 with HB-EGF and/or integrin alpha3beta1 in several tissues.

Antigens, CD↗

PubMed2004

Quantification of thrombospondin-1 secretion and expression of alphavbeta3 and alpha3beta1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells.

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface alphavbeta3 and alpha3beta1 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of alphavbeta3 and alpha3beta1 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of alphavbeta3 and alpha3beta1 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/l x 10(6) cells/24 h (mean +/- SD = 626 +/- 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 microg. Seven of eight non-glioma cell lines secreted less than 100 ng of TSP-1. All glioma cell lines expressed alpha3beta1 integrin and syndecan-1, and seven of 10 glioma cell lines expressed alphavbeta3 integrin. Treatment of the glioma cell lines with TGF-beta2 did not change the expression of alphavbeta3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with alphavbeta3 and alpha3beta1 integrins, and syndecan-1.

Brain Neoplasms↗

PubMed2006

Cooperation between Myc and YY1 provides novel silencing transcriptional targets of alpha3beta1-integrin in tumour cells.

We show that human osteosarcoma cells (Saos-2) have downregulation of alpha3beta1-integrin compared to normal bone cells; this was further described in human osteosarcomas and in a primary murine sarcoma. The alpha3 gene was silenced in Saos-2 cells causing a low expression of alpha3beta1-integrin and reduction in collagen attachment with increasing migratory capacity. Chromatin immunoprecipitation assay performed on alpha3 promoter established that Myc and Yin Yang protein (YY1) cooperate in tandem to downregulate the alpha3 gene. This silencing mechanism involves the binding of Myc and YY1 to DNA and formation of complexes among Myc/Max, YY1, CREB-binding protein and deacetylation activity. The promoter containing deletions of E-boxes or YY1 cassettes failed to downregulate the transcription of a reporter gene as well as the inhibition of deacetylation activity. Overexpression of both Myc and YY1 was necessary to determine the alpha3-integrin promoter downregulation in normal osteoblasts. This downregulation of alpha3beta1-integrin can contribute to the acquisition of a more aggressive phenotype. YY1 regulated negatively the Myc activity through a direct interaction with the Myc/Max and deacetylase complexes. This represents a novel silencing mechanism with broad implications in the transcription machinery of tumours.

Animals↗

PubMed1997

An all-D amino acid peptide model of alpha1(IV)531-543 from type IV collagen binds the alpha3beta1 integrin and mediates tumor cell adhesion, spreading, and motility.

Type IV collagen promotes integrin-mediated cell adhesion, spreading, and motility. Several regions within the triple-helical domain of type IV collagen have been identified as tumor cellular recognition sites. Among these regions, the alpha1(IV)531-543 sequence, designated L-Hep-III, promotes integrin-mediated tumor cell adhesion and directly binds to the alpha3beta1 integrin [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. We have presently compared the activities of the all-d enantiomeric peptide model of alpha1(IV)531-543, designated D-Hep-III, with L-Hep-III, for promoting the adhesion, spreading, and motility of metastatic melanoma and breast carcinoma cells. D-Hep-III was found to support melanoma and breast carcinoma cell adhesion, spreading, and motility in a dose-dependent fashion similar to that of L-Hep-III. The adhesions of melanoma and breast carcinoma cells to both type IV collagen and fibronectin were effectively inhibited by L-Hep-III and D-Hep-III. Melanoma cell invasion of the basement membrane was also inhibited by D-Hep-III. Characterization of the cell surface receptor for D-Hep-III was acheived via cell adhesion assays and affinity chromatography using monoclonal antibodies against integrin subunits. Immunoprecipitation analysis following EDTA elution from a D-Hep-III affinity column indicated that D-Hep-III binds to the alpha3beta1 integrin but not to the alpha2 or alpha6 integrin subunits. In summary, these studies demonstrate that an all-D model of the alpha1(IV)531-543 sequence mimics the biological activities of the all-L peptide. D-Hep-III is the first all-D peptide that has been shown to promote tumor cell adhesion, spreading, and migration, inhibit tumor cell adhesion and migration on type IV collagen and invasion of the basement membrane, and bind directly to an integrin. Due to the resistance to proteolysis, all-D receptor-binding peptides such as D-Hep-III have great potential for in vivo studies and as therapeutic agents.

Antibodies, Monoclonal↗

PubMed1999

Cellular localization of alpha3beta1 integrin isoforms in association with myofibrillogenesis during cardiac myocyte development in culture.

The cellular localization of alpha3beta1 integrin isoforms was examined in cultured neonatal myocytes at selected times during development using double immunofluorescence assays. The distribution of alpha3A subunits began as diffuse and patternless, but as the cells matured, the distribution assumed a sarcomeric banding pattern, and alpha3A appeared to be localized in costameres - sarcolemmal regions adjacent to the Z-disks. Alpha-actinin, a component of the Z-disk, was localized in the same intracellular regions. Temporal analysis of the incorporation of the alpha3A subunit and other myofibrillar proteins into sarcomeres revealed that alpha3A was integrated into sarcomeres following incorporation of alpha-actinin and myosin heavy chain (MHC) but prior to that of desmin. This suggests that alpha3A integrins are incorporated into a pre-existing myofibrillar structure, and it is unlikely that alpha3A integrins participate in the initial assembly of myofibrillar proteins. The alpha3B, beta1A and beta1D subunits were also localized in costameres, where they formed alpha3Abeta1A, alpha3Abeta1D and alpha3Bbeta1A heterodimers. The alpha3Bbeta1D heterodimer, however, was not found in cardiac myocytes. The antisera raised against the cytoplasmic domains of alpha3A, alpha3B, beta1A and beta1D caused disruption of sarcomere structure. Thus, the myofibril-extracellular matrix linkages mediated by isoforms of alpha3beta1 integrin may play a crucial role in the stabilization of myofibril assembly and in the maintenance of sarcomere structure. Co-immunoprecipitation experiments revealed that beta1A, but not beta1D, interacts with the Nck signaling protein, suggesting that Nck participates in downstream signaling triggered by beta1A and that the beta1A-mediated signaling pathway is distinct from that of beta1D.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Reelin binds alpha3beta1 integrin and inhibits neuronal migration.

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1.

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.

Allantois↗

PubMed1998

Association of the decreased expression of alpha3beta1 integrin with the altered cell: environmental interactions and enhanced soft agar cloning ability of c-myc-overexpressing small cell lung cancer cells.

Small cell lung cancer (SCLC) is a highly invasive and metastatic tumor, and the decreased expression of alpha3beta1 integrin may contribute to its virulence. Alpha3beta1 is a critical integrin for pulmonary development and epithelial integrity, and its reduced expression has been linked to the increased malignancy and invasion of other cancers. The amplification of the c-myc oncogene is seen frequently in relapsed SCLC tumors and is associated with a worsened prognosis. In the present study using a model of SCLC tumor progression, overexpression of c-myc in a classic SCLC cell line, NCI H209, enhanced in vitro features of tumorigenesis, altered the relationships between cell and environment, and markedly down-regulated the expression of the alpha3 integrin subunit at both the transcript and protein levels. This inverse relationship between the expression of the alpha3 integrin subunit and c-myc is mimicked by other c-myc-overexpressing SCLC cell lines. Restoring alpha3 expression in the myc-transfected 209 cells reversed the effects of c-myc: alpha3 transfection increased cell:cell adhesion and reduced soft agar cloning without affecting the in vitro doubling time. The diminished soft agar cloning produced by alpha3 transfection was reversed by an antibody that specifically engages alpha3beta1 integrins, P1B5. These results suggest first, that alpha3beta1 integrin mediates homotypic adhesion of SCLC cells, and second, that unengaged alpha3beta1 integrin suppresses the growth of disaggregated SCLC cells. Thus, the down-regulation of the alpha3 integrin subunit may contribute to the enhanced tumorigenicity of c-myc-overexpressing SCLCs by allowing the growth of tumor cells that have reduced contact with ligand-expressing substratum or cells, a condition that occurs during the growth of the primary tumor, tumor invasion, and metastasis.

Agar↗

PubMed2003

Recombinant human laminin-5 domains. Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha3beta1 integrin binding.

Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The alpha3' chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type alpha3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, beta3'and gamma2', produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the alpha3' chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1-3, but not the single chains, supported binding of soluble alpha3beta1 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with alpha3beta1 integrin. The N-glycosylation sites in wild-type alpha3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor alpha3beta1 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4-5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the alpha3'LG1-3 chain showed an increased binding affinity for alpha3beta1 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with alpha3beta1 integrin.

Amino Acid Sequence↗

PubMed2005

alpha3beta1 integrin induced suppression of the Caco-2 epithelial cell IL-1 signaling pathway leading to NF-(kappa)B activation.

Intestinal epithelial cells (IECs) produce several potent cytokines in response to interleukin-1 (IL-1) and may play a role in the inflammatory response. Previously, we determined that treatment of the Caco-2 cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced cytokine secretion and mRNA levels, suggesting that the alpha3beta1 integrin may play a role in the regulation of IEC cytokine responses to IL-1. In this report, treatment of the Caco-2 cells with the anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced levels of NF-kappaB binding activity in nuclear extracts, as determined by EMSA, as well as phosphorylation and degradation of the inhibitor, I(kappa)B(alpha). The anti-integrin antibody treatment was also found to suppress I(kappa)B kinase (IKK) activity and IKK(beta) phosphorylation. Culture of the Caco-2 cells on purified laminin-5, the ligand for the alpha3beta1 integrin, also resulted in suppression of IL-1 induced phosphorylation of I(kappa)B(alpha) and IKK(beta). Together with our previous findings, these results suggest that alpha3beta1 integrin binding results in a suppression of the IL-1 signaling pathway leading to the activation of NF-(kappa)B and ultimately IEC cytokine responses. These studies define a novel regulatory mechanism which may be important in the control of IEC cytokine responses during inflammation.

Antibodies↗

PubMed2006

Deletion of core fucosylation on alpha3beta1 integrin down-regulates its functions.

The core fucosylation (alpha1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently alpha1,6-fucosylation has been further reported to be very crucial by the study of alpha1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on alpha3beta1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in alpha3beta1 integrin-mediated events has been studied by using Fut8(+/+) and Fut8(-/-) embryonic fibroblasts, respectively. We found that the core fucosylation of alpha3beta1 integrin, the major receptor for laminin 5, was abundant in Fut8(+/+) cells but was totally abolished in Fut8(-/-) cells, which was associated with the deficient migration mediated by alpha3beta1 integrin in Fut8(-/-) cells. Moreover integrin-mediated cell signaling was reduced in Fut8(-/-) cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of alpha3beta1 integrin.

Animals↗