Ultrastructural localization of integrin subunits beta4 and alpha3 within the migrating epithelial tongue of in vivo human wounds

Subsequent to wounding, keratinocytes must quickly restore barrier function. In vitro wound models have served to elucidate mechanisms of epithelial closure and key roles for integrins alpha6beta4 and alpha3beta1. To extrapolate in vitro data to in vivo human tissues, we used ultrathin cryomicrotomy to simultaneously observe tissue ultrastructure and immunogold localization in unwounded skin and acute human cutaneous wounds. Localization of the beta4 integrin subunit in unwounded skin shows dominant hemidesmosomal association and minor basal keratinocyte lateral filopodic cell-cell expression. After wounding, beta4 dominantly localized to cytokeratin-rich regions (trailing edge hemidesmosomes) and minor association with lamellipodia (leading edge). beta4 colocalizes with alpha3 within filopodia juxtaposed to wound matrix, and increased concentrations of beta4 were found in cytoplasmic vesicles within basal keratinocytes of the migrating tongue. alpha3 integrin subunit dominantly localized to filopodia within basal keratinocyte lateral cell-cell interfaces in unwounded skin and both cell-cell and cell-matrix filopodic interactions in wounded skin. This study indicates that beta4 interacts with the extracellular environment through both stable and transient interactions and may be managed through a different endosomal trafficking pathway than alpha3. alpha3 integrin, despite its ability to respond to alternate ligands after wounding, does so through a single structure, the filopodia.     

Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis

Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.     

The crystal structure of calcium- and integrin-binding protein 1: insights into redox regulated functions

Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the alpha(IIb) subunit of the platelet-specific integrin alpha(Iib)beta(3). Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin alpha(Iib)beta(3). We report the structure of human CIB1 to a resolution of 2.3 A, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to alpha(IIb) binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts

The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.     

RGD targeted poly(L-glutamic acid)-cystamine-(Gd-DO3A) conjugate for detecting angiogenesis biomarker alpha(v) beta3 integrin with MRT, mapping

Cyclic Arg-Gly-Asp-D-Phe-Lys [c(RGDfK)] targeted poly(L-glutamic acid) (PGA)-(Gd-DO3A) conjugate with a biodegradable cystamine spacer was prepared and evaluated for in vivo detection of an angiogenesis biomarker, alpha(v)beta3 integrin, in neoplastic tissues with T1 mapping, a quantitative magnetic resonance imaging (MRI) technique. The binding activity of the c(RGDfK) containing conjugate was investigated using in vitro vitronectin assay with human prostate carcinoma DU145 cell line and Kaposi's sarcoma SLK cell line. The peptide c(RGDfK) and PGA-cystamine-(Gd-DO3A) conjugate were used as controls. The binding affinity of polymer bound c(RGDfK) was slightly lower than free c(RGDfK) peptide. The RGD targeted conjugate had higher binding affinity to the DU145 cells than the SLK cells, which was consistent to free c(RGDfK). The imaging of alpha(v)beta3 integrin with targeted PGA-cystamine-(Gd-DO3A) was evaluated in nude mice bearing DU145 and SLK xenografts at a dose of 5 micromol-Gd/kg. The targeted conjugate demonstrated higher in vivo binding affinity to the DU145 xenografts than the SLK xenografts, resulting in a significant decrease of T1 values of water protons in the periphery of the DU145 tumors as shown in the MR T1 maps. No significant decrease of T1 values was observed in the SLK tumor with the targeted conjugate and in both tumors with the non-targeted conjugate. The targeted polymeric Gd(III) chelate conjugate with a degradable spacer has the potential to be a new paradigm for safe and effective probes in molecular imaging with quantitative MR T1 mapping.     

Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.     

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface.In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.     

Tumor angiogenic endothelial cell targeting by a novel integrin-targeted nanoparticle

Angiogenesis is an important process in cancer growth and metastasis. During the tumor angiogenic process, endothelial cells express various cell surface receptors which can be utilized for molecular imaging and targeted drug delivery. One such protein receptor of interest is the integrin alphav beta3. Our group is involved in the development of molecular imaging probes and drug delivery systems targeting alphav beta3. Based on extensive lead optimization study with the integrin antagonist compounds, we have developed a new generation of integrin alphav beta3 compound (IA) which has superior binding affinity to alphav beta3. Utilizing this IA as a targeting agent, we have developed a novel integrin-targeted nanoparticle (ITNP) system for targ alphav beta3 was observed. These ITNPs also were rapidly taken up by cells that express alphav beta3. The ITNPs accumulated in the angiogenic vessels, after systemic administration in a murine squamous cell carcinoma model. This novel intergrin targeted ITNP platform will likely have an application in targeted delivery of drugs and genes in vivo and can also be used for molecular imaging.     

Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2

Collagen plays a critical role in hemostasis by promoting adhesion and activation of platelets at sites of vessel injury. In the present model of platelet-collagen interaction, adhesion is mediated via the inside-out regulation of integrin alpha2beta1 and activation through the glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex. The present study extends this model by demonstrating that engagement of alpha2beta1 by an integrin-specific sequence from within collagen or by collagen itself generates tyrosine kinase-based intracellular signals that lead to formation of filopodia and lamellipodia in the absence of the GPVI-FcR gamma-chain complex. The same events do not occur in platelet suspensions. alpha2beta1 activation of adherent platelets stimulates tyrosine phosphorylation of many of the proteins in the GPVI-FcR gamma-chain cascade, including Src, Syk, SLP-76, and PLCgamma2 as well as plasma membrane calcium ATPase and focal adhesion kinase. alpha2beta1-mediated spreading is dramatically inhibited in the presence of the Src kinase inhibitor PP2 and in PLCgamma2-deficient platelets. Spreading is abolished by chelation of intracellular Ca2+. Demonstration that adhesion of platelets to collagen via alpha2beta1 generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of beta1-deficient thrombi and why loss of the GPVI-FcR gamma-chain complex has a relatively minor effect on bleeding.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Hierarchy of ADAM12 binding to integrins in tumor cells

ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADAM12. However, when alpha9beta1 integrin is not expressed--as in many carcinoma cells--other members of the beta1 integrin family can replace its ligand binding activity. In attachment assays, the recombinant disintegrin domain of ADAM12 only supported alpha9 integrin-dependent tumor cell attachment, whereas full-length ADAM12 supported attachment via alpha9 integrin and other integrin receptors. Cells that attached to full-length ADAM12 in an alpha9 integrin-dependent manner also attached to ADAM12 in which the putative alpha9beta1 integrin-binding motif in the disintegrin domain had been mutated. This attachment was mediated through use of an alternate beta1 integrin. We also found that cell spreading in response to ADAM12 is dependent on the apparent level of integrin activation. Binding of cells to ADAM12 via the alpha9beta1 integrin was Mn(2+)-independent and resulted in attachment of cells with a rounded morphology; attachment of cells with a spread morphology required further activation of the alpha9beta1 integrin. We demonstrated that phosphoinositide-3-kinase appears to be central in regulating alpha9beta1 integrin cell spreading activity in response to ADAM12.     

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha5beta1 integrin (alpha5beta1), but not by one against anti-alphavbeta3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha5beta1 is involved in this adhesion.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

Effect of altered glycosylation on the structure of the I-like domain of beta1 integrin: a molecular dynamics study

Glycosylation plays an important role in the regulation of integrin function. Molecular mechanisms underlying the effects of altered glycosylation on beta1 integrin structure and function are still largely unknown. In this study, we used a molecular modeling approach to study the effects of altered glycosylation, with alpha2-6 sialic acid and without alpha2-6 sialic acid, on the structure of the I-like domain of the beta1 integrin. Our results demonstrated that altered glycosylation affected the interactions between oligosaccharides and the I-like domain, which in turn changed the accessibility of the specificity-determining loop for ligand binding. Altered glycosylation caused significant conformational changes for most of the key functional regions of the I-like domain of beta1 integrin, including the metal ion-dependent adhesion site that contains a DLSYS motif, and other critical residues for ligand binding (Asn-224, Glu-229, Asp-233, Asp-267, and Asp-295). In addition, altered glycosylation caused significant movement of the alpha1 and alpha7 helices, which are important for the activation of beta1 integrin. The results from this study offered molecular mechanisms for the experimental observations that variant glycosylation regulates integrin function.     

Inhibition of FGF-2-mediated chemotaxis of murine brain capillary endothelial cells by cyclic RGDfV peptide through blocking the redistribution of c-Src into focal adhesions

The alpha(v)beta(3) integrin is essential for fibroblast growth factor (FGF)-induced angiogenesis in vivo. However, the role of this integrin in FGF-2-mediated cellular responses by cultured endothelial cells is largely unknown. Cyclic RGDfV (cRGDfV) peptide is widely used to inhibit the binding of alpha(v)beta(3) integrin to vitronectin. To investigate the role of this integrin in FGF-2-mediated cellular responses, we used immortalized murine brain capillary endothelial cells, denoted IBE cells. Because IBE cells proliferate and migrate in response to FGF-2-treatment, when cultured on fibronectin-coated surface, we first examined the inhibitory activity of this peptide on the binding of alpha(v)beta(3) integrin to fibronectin as well as vitronectin. Solid phase binding assay revealed that cRGDfV peptide strongly inhibited the binding of purified alpha(v)beta(3) integrin to vitonectin- and fibronectin-coated plastic surfaces at a concentration of 50 microM. cRGDfV peptide at 50 microM inhibited spreading as well as adhesion of IBE cells on vitronectin-coated plastic surface but not on fibronectin. On fibronectin-coated substrata, cRGDfV at 50 microM attenuated FGF-2-mediated chemotaxis, but not FGF-2-induced proliferation, of IBE cells. We have previously demonstrated that mitogen-activated protein kinase (MAPK) activation within focal adhesions through c-Src activity was involved in FGF-2-induced chemotaxis of IBE cells. Treatment of cells with cRGDfV peptide was associated with reduced c-Src activity without tyrosine dephosphorylation. Immunofluorescent staining showed that cRGDfV inhibited redistribution of c-Src into focal adhesions. MAPK activation by FGF-2 within focal adhesions was also attenuated in the presence of cRGDfV peptide. Our results indicated that cRGDfV peptide inhibited redistribution of c-Src into focal adhesions, leading to impaired MAPK activation within focal adhesions and motility in FGF-2-treated endothelial cells.     

Characterization of the oligosaccharide component of alpha3beta1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin alpha3beta1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, alpha3beta1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the alpha3 and beta1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In alpha3beta1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of alpha3beta1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex7HexNAc6FucSia4 was present. In a direct ligand binding assay, desialylated alpha3beta1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, alpha3beta1 integrin was shown to take part in T24 cell migration on fibronectin: anti-alpha3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of beta1,6 branched complex type glycans in this process. Our data show that alpha3beta1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.     

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.     

Three-dimensional structure of (1,4)-beta-D-mannan mannanohydrolase from tomato fruit

The three-dimensional crystal structure of tomato (Lycopersicon esculentum) beta-mannanase 4a (LeMAN4a) has been determined to 1.5 A resolution. The enzyme adopts the (beta/alpha)(8) fold common to the members of glycohydrolase family GH5. The structure is comparable with those of the homologous Trichoderma reesei and Thermomonospora fusca beta-mannanases: There is a conserved three-stranded beta-sheet located near the N terminus that stacks against the central beta-barrel at the end opposite the active site. Three noncanonical beta-helices surround the active site. Similar helices are found in T. reesei but not T. fusca beta-mannanase. By analogy with other beta-mannanases, the catalytic acid/base residue is E204 and the nucleophile residue is E318. The active site cleft of L. esculentum beta-mannanase most closely resembles that of the T. reesei isozyme. A model of substrate binding in LeMAN4a is proposed in which the mannosyl residue occupying the -1 subsite of the enzyme adopts the (1)S(5) skew-boat conformation.     

The human heart and rat brain IIA Na+ channels interact with different molecular regions of the beta1 subunit

The alpha subunit of voltage-gated Na(+) channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory beta(1), but not the beta(2) subunit. In the present study, we used beta(1)/beta(2) chimeras to identify molecular regions within the beta(1) subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na(+) channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the beta(1)/beta(2) chimeras with rat brain IIA channels. In agreement with previous studies, the beta(1) extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the beta(1) subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the beta(1) subunit was required. An exchange of the beta(1) membrane anchor by the corresponding beta(2) subunit region almost completely abolished the effects of the beta(1) subunit on hH1, suggesting that the beta(1) membrane anchor plays a crucial role for the modulation of the cardiac Na(+) channel isoform. It is concluded that the beta(1) subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.