Model of integrin-mediated cell adhesion strengthening

Cell adhesion to extracellular matrix components involves integrin binding, receptor clustering, and recruitment of cytoskeletal elements, leading to the formation of discrete adhesive structures (focal adhesions). A force balance, macroscopic-to-microscopic model of these adhesive events is presented in the context of experimentally measured parameters. Integrin bond force, bond numbers, and distribution along the contact area strongly modulated the resulting adhesive force. Furthermore, focal adhesion assembly enhanced adhesion strength by 30% over integrin clustering alone. Predicted values are in excellent agreement with experimental results. This model provides a simple framework to systematically analyze the contributions of different adhesive parameters to overall adhesion strength.     

(Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion

We have prepared protein-peptide conjugates composed of bovine serum albumin (BSA) derivatized with short peptides containing the Arg-Gly-Asp (RGD) sequence derived from the adhesion site of fibronectin. The RGD-BSA conjugates were used to coat tissue culture plastic surfaces which then served as substrata in cell adhesion experiments. Our results indicate that the efficiency of adhesion to RGD-BSA-coated surfaces is highly dependent on the valency of the (RGD)n-BSA conjugates. For example, on surfaces with approximately equal amounts of RGD ligand, CHO cells adhered virtually 100% to the (RGD)n-BSA (n = 20.8) conjugate and not at all to the (RGD)n-BSA (n = 3.5) conjugate. Adhesion on (RGD)n-BSA-coated substrata and on fibronectin- or vitronectin-coated substrata was also examined in terms of the relationship between cell adhesion and the intermolecular distances of adsorbed proteins. It was observed that for substrata coated with relatively compact, symmetric molecules, such as RGD-BSA or vitronectin, adhesion dropped off sharply as intermolecular distances increased; by contrast, for fibronectin, a large asymmetric molecule, adhesion declined more gradually as intermolecular distances increased. Finally, we have examined the role of different cell-surface receptors in the process of adhesion to RGD-BSA substrata. Interestingly, competition and blocking experiments with antibodies and with soluble competing proteins suggest that it is the vitronectin receptor rather than the fibronectin receptor which mediates adhesion to RGD-BSA.     

Irisin supports integrin-mediated cell adhesion of lymphocytes

Irisin, a myokine released from skeletal muscle, has recently been found to act as a ligand for the integrins αVβ5, αVβ1, and α5β1 expressed on mesenchymal cells, thereby playing an important role in the metabolic remodeling of the bone, skeletal muscle and adipose tissues. Although the immune-modulatory effects of irisin in chronic inflammation have been documented, its interactions with lymphocytic integrins have yet to be elucidated. Here, we show that irisin supports the cell adhesion of human and mouse lymphocytes. Cell adhesion assays using a panel of inhibitory antibodies to integrins have shown that irisin-mediated lymphocyte adhesion involves multiple integrins including not only α4β1 and α5β1, but also leukocyte-specific αLβ2 and α4β7. Importantly, mouse lymphocytic TK-1 cells that lack the expression of β1 integrins have exhibited αLβ2- and α4β7-mediated cell adhesion to irisin. Irisin has also been demonstrated to bind to purified recombinant integrin αLβ2 and α4β7 proteins. Thus, irisin represents a novel ligand for integrin αLβ2 and α4β7, capable of supporting lymphocyte cell adhesion independently of β1 integrins. These results suggest that irisin may play an important role in regulating lymphocyte adhesion and migration in the inflamed vasculature.     

Redox regulation of beta-actin during integrin-mediated cell adhesion

Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion.     

Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event

Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four- to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not. Neither the number of cell surface FnRs nor the receptor affinity, as measured by 125I-fibronectin and 125I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand-receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectin-dependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.     

Phosphotyrosine signalling as a regulator of neural crest cell adhesion and motility

We demonstrate that neural crest cell-cell adhesion, cell-substrate adhesion, and ultimately cell motility, are highly dependent on the balanced action of tyrosine kinases and tyrosine phosphatases. Neural crest cell migration on fibronectin is diminished in the presence of the tyrosine phosphatase inhibitor vanadate or tyrosine kinase inhibitor herbimycin A, while cadherin-rich cell-cell adhesions are significantly increased. In contrast, cells treated with the kinase inhibitor genistein have decreased motility, rearrange rapidly and reversibly into a pavement-like monolayer, but have no increase in cadherin interactions. Genistein-sensitive tyrosine kinases may therefore abrogate a latent sensitivity of neural crest cells to contact-mediated inhibition of movement. Furthermore, we show that the activity of herbimycin A-sensitive kinases is necessary for focal adhesion formation in these cells. Moreover, the size and distribution of these adhesions are acutely sensitive to the actions of tyrosine phosphatases and genistein-sensitive kinases. We propose that in migrating neural crest cells there is a balance in phosphotyrosine signalling which minimises both cell-cell adhesion and contact inhibition of movement, while enhancing dynamic cell-substrate interactions and thus the conditions for motility.     

P-selectin activates integrin-mediated colon carcinoma cell adhesion to fibronectin

During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized P-selectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes. We hypothesize that selectin binding may regulate subsequent integrin-mediated steps in metastasis. Using a model system of cultured Colo 320 human colon adenocarcinoma cells incubated with soluble P-selectin-IgG chimeric protein, we have found that P-selectin can stimulate activation of the alpha(5)beta(1) integrin resulting in a specific increase of adhesion and spreading of these cells on fibronectin substrates. P-selectin binding also induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3-K). PI3-K inhibitors blocked P-selectin-mediated integrin activation, cell attachment, and cell spreading. Inhibition of p38 MAPK activation blocked cell spreading, but not cell attachment. P-selectin binding also resulted in formation of a signaling complex containing PI3-K and p38 MAPK. These results suggest that P-selectin binding to tumor cells can activate alpha(5)beta(1) integrin via PI3-K and p38 MAPK signaling pathways leading to increased cell adhesion. We propose that P-selectin ligands are important tumor cell signaling molecules that modulate integrin-mediated cell adhesion in the metastatic process.     

Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of β1 integrin at the cell surface but had no effect on total cellular β1 integrin, indicating that VAMP3 is required for trafficking of β1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.     

Vinculin--a dynamic regulator of cell adhesion

The ability of cells to tightly adhere to one another and to the extracellular matrix is fundamentally important in numerous biological processes, including embryogenesis, wound healing and maintenance of tissue integrity. Vinculin, a protein localized at the cytoplasmic face of cell-matrix and cell-cell adhesions, is required for strong cell adhesion. Two new crystal structures reveal that vinculin exhibits a high degree of structural plasticity upon ligand binding that might promote rapid changes in cell adhesion.     

Acidic pH as a physiological regulator of human cathepsin L activity.

Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ.mol-1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.     

JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration

Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.     

The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism

Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.     

Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator

Glycosylation is one of the most abundant posttranslational modification reactions, and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structure (glycan) are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation and tumor invasion. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar chains to various complex carbohydrates such as glycoproteins, glycolipids and proteoglycans. Functional glycomics, which uses sugar remodeling by glycosyltransferases, is a promising tool for the characterization of glycan functions. Here, we will focus on the positive and negative regulation of biological functions of integrins by the remodeling of N-glycans with N-acetylglucosaminyltransferase III (GnT-III) and N-acetylglucosaminyltransferase V (GnT-V), which catalyze branched N-glycan formations, bisecting GlcNAc and β1,6 GlcNAc, respectively. Typically, integrins are modified by GnT-III, which inhibits cell migration and cancer metastasis. In contrast, integrins modified by GnT-V promote cell migration and cancer invasion.     

Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator

Glycosylation is one of the most abundant posttranslational modification reactions, and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structure (glycan) are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation and tumor invasion. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar chains to various complex carbohydrates such as glycoproteins, glycolipids and proteoglycans. Functional glycomics, which uses sugar remodeling by glycosyltransferases, is a promising tool for the characterization of glycan functions. Here, we will focus on the positive and negative regulation of biological functions of integrins by the remodeling of N-glycans with N-acetylglucosaminyltransferase III (GnT-III) and N-acetylglucosaminyltransferase V (GnT-V), which catalyze branched N-glycan formations, bisecting GlcNAc and β1,6 GlcNAc, respectively. Typically, integrins are modified by GnT-III, which inhibits cell migration and cancer metastasis. In contrast, integrins modified by GnT-V promote cell migration and cancer invasion.Key words: integrin, E-cadherin, GnT-III, GnT-V, N-glycosylation, glycosyltransferaseProtein glycosylation encompasses N-glycans, O-glycans and Glycosaminoglycans. N-glycans are linked to asparagine residues of proteins, which is a specific subset residing in the Asn-X-Ser/Thr motif, whereas O-glycans are attached to a subset of serines and threonines (Fig. 1).¹ An increasing body of evidence indicates that glycans in glycoproteins are involved in the regulation of cellular functions including cell-cell communication and signal transduction.^2,3 In fact, most receptors on the cell surface are N-glycosylated—integrins and epithelial growth factor receptors; and transforming growth factor β receptors. Here, we focus mainly on the modification of N-glycans of integrin α3β1 and α5β1 to address the important roles of N-glycans in cell adhesion and migration.Open in a separate windowFigure 1Two major types of protein glycosylation. N-glycans are covalently linked to asparagine (Asn) residue of proteins, specifically the Asn-X-Ser/Thr motif. In contrast, O-glycans are attached to a subset of glycosidically linked hydroxyl groups of the amino acids serine (Ser) and threonine (Thr).Previous studies indicate that the presence of the appropriate oligosaccharide can modulate integrin activation. When human fibroblasts were cultured in the presence of l-deoxymannojirimycin, an inhibitor of α-mannosidase II, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared at the cell surface, and fibronectin (FN)-dependent adhesion was greatly reduced.⁴ In addition, the treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost GlcNAc and asparagine residues of N-glycans from N-linked glycoproteins, resulted in the blockage of α5β1 binding to FN and the inherent association of both subunits,⁵ suggesting that N-glycosylation is essential for functional integrin α5β1. The production of glycoprotein glycans is catalyzed by various glycosyltransferases. N-Acetylglucosaminyltransferase III (GnT-III) transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to a β1, 4 mannose in N-glycans to form a “bisecting” GlcNAc linkage, as shown in Figure 2. Bisecting GlcNAc linkage is found in various hybrid and complex N-glycans. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways. Introduction of a bisecting GlcNAc suppresses further processing and elongation of N-glycans catalyzed by N-acetylglucosaminyltransferase V (GnT-V), which is strongly associated with cancer metastasis, since GnT-V cannot utilize the bisected oligosaccharide as a substrate.^6–8 It has also been reported that GnT-V activity and β1, 6 branched N-glycan levels are increased in highly metastatic tumor cell lines.^9,10 When NIH3T3 cells were transformed with the oncogenic Ras gene, cell spreading on FN was greatly enhanced due to an increase in β1, 6 GlcNAc branched tri- and tetra-antennary oligosaccharides in α5β1 integrins.⁹ Similarly, the characterization of N-glycans of integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that β1, 6 GlcNAc branched structures were expressed at high levels in metastatic cells compared with non-metastatic cells.¹⁰ Cancer metastasis was consistently, and significantly, suppressed in GnT-V knockout mice.¹¹Open in a separate windowFigure 2Glycosylation reactions catalyzed by the action of glycosyltransferase GnT-III and GnT-V. The remodeled N-glycans regulate cell adhesion and migration. Enhanced expression of GnT-V in epithelial cells results in a loss of cell-cell adhesion, increasing integrin-mediated cell migration. In contrast, overexpression of GnT-III strengthens cell-cell interaction and downregulates integrin-mediated cell migration, which may contribute to the suppression of cancer metastasis. The β1,6GlcNAc branching is preferentially modified by polylactosamine and other sugar motifs such as sialyl Lewis X, which also contribute to promotion of cancer metastasis. It is worth mentioning that GnT-III could be proposed as an antagonistic of GnT-V, since GnT-V cannot utilize the bisected oligosaccharide as a substrate.To explore the possible mechanisms involved in increased β1, six branched N-glycans on cancer cells, Guo et al. found that cell migration toward FN and invasion through the matrigel were both substantially stimulated in cells in which the expression of GnT-V was induced.¹² Increased branched sugar chains inhibited the clustering of integrin α5β1 and the organization of F-actin into extended microfilaments in cells plated on FN-coated plates, which supports the hypothesis that the degree of adhesion of cells to their extracellular matrix (ECM) substrate is a critical factor in regulating the rate of cell migration, i.e., migration is maximal under conditions of intermediate levels of cell adhesion.¹³ Conversely, GnT-V null mouse embryonic fibroblasts (MEF) displayed enhanced cell adhesion to, and spreading on, FN-coated plates with the concomitant inhibition of cell migration. The restoration of GnT-V cDNA in the null MEF reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes.In contrast to GnT-V, the overexpression of GnT-III resulted in an inhibition of α5β1 integrin-mediatedcell spreading and migration, and the phosphorylation of the focal adhesion kinase.¹⁴ The affinity of the binding of integrin α5β1 to FN was significantly reduced as a result of the introduction of a bisecting GlcNAc to the α5 subunit. In addition, overexpression of GnT-III in highly metastatic melanoma cells reduced β1, six branching in cell-surface N-glycans and increased bisected N-glycans.¹⁵ Therefore, GnT-III has been proposed as an antagonistic of GnT-V, thereby contributing to the suppression of cancer metastasis. In fact, the opposing effects of GnT-III and GnT-V have been observed for the same target protein, integrin α3β1.¹⁶ GnT-V stimulates α3β1 integrin-mediated cell migration, while overexpression of GnT-III inhibits GnT-V-induced cell migration. The modification of the α3 subunit by GnT-III supersedes modification by GnT-V. As a result, GnT-III inhibits GnT-V-induced cell migration. These results strongly suggest that remodeling of glycosyltransferase-modified N-glycan structures either positively or negatively modulates cell adhesion and migration.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. The increased sialylation of the β1 integrin subunit was correlated with a decreased adhesiveness and metastatic potential.^17–19 On the other hand, the enzymatic removal of α2, eight-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN,²⁰ supporting the observation that the N-glycans of α and β integrin subunits play distinct roles in cell-ECM interactions.²¹ Collectively, these findings suggest that the interaction of integrin α5β1 with FN is dependent on N-glycosylation and the processing status of N-glycans.Although alteration of the oligosaccharide portion on integrin α5β1 could affect cis- and trans-interactions caused by GnT-III, ST6GalI and GnT-V, as described above, the molecular mechanism remains unclear. Considering integrin α5β1 contains 26 potential N-linked glycosylation sites (14 in the α subunit and 12 in the β subunit), the determination of those crucial N-glycosylation sites for its biological function is, therefore, quite important for an understanding of the underlying mechanism. We sequentially mutated either one or a combination of asparagine residues in the putative N-glycosylation sites of glutamine residues, and found that N-glycosylation on the β-propeller domain of the α5 subunit (in particular sites number 3–5) is essential for its hetero-dimer formation and its biological functions such as cell spreading and cell migration, as well as for the proper folding of the α5 subunit.²² On the other hand, N-glycans on β1 integrin also play important roles in the regulation of its biological functions^23,24 (and our unpublished data). Very recently, we also found that GnT-III specifically modifies one of the important glycosylation sites, which results in functional regulation (unpublished data). We postulate that these important sites may participate in supramolecular complex formation on the cell surface, which controls intracellular signal transduction.It also is worth noting that N-glycans regulate cell-ECM association as well as cell-cell adhesion. Overexpression of GnT-III slowed E-cadherin turnover, resulting in increased E-cadherin expression on the surface of B16 melanoma cells.²⁵ E-cadherin engagement at cell-cell contacts is known to suppress cell migration, and that effect has been best described in the context of tumorigenesis.²⁶ Conversely, the disruption of E-cadherin-mediated cell adhesion appears to be a central event in the transition from non-invasive to invasive carcinomas. Interestingly, we recently found that E-cadherin-mediated cell-cell interaction upregulated GnT-III expression,^27,28 suggesting that regulation of GnT-III and E-cadherin expression may exist as a positive feedback loop. Taken together, the overexpression of GnT-III inhibits cell migration by at least two mechanisms: an enhancement in cell-cell adhesion and a downregulation of cell-ECM adhesion (Fig. 2).Indeed, glycosylation defects in humans and their links to disease have shown that the mammalian glycome contains a significant amount of biological information.²⁹ The mammalian glycome repertoire is estimated to be between hundreds and thousands of glycan structures and could be larger than its proteome counterpart. Nevertheless, characterization of the biological functions of each glycan could one day make a significant contribution to the diagnosis and treatment of disease.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

Regulation of integrin-mediated T cell adhesion by the transmembrane protein tyrosine phosphatase CD45.

The transmembrane protein tyrosine phosphatase CD45 is required for Ag receptor signal transduction in lymphocytes. Recently, a role for CD45 in the regulation of macrophage adhesion has been demonstrated as well. To investigate further the role of CD45 in the regulation of adhesion, we examined integrin-mediated adhesion to fibronectin of two T cell lines and their CD45-deficient variants. The absence of CD45 correlated with enhanced adhesion to fibronectin via integrin alpha5beta1 (VLA-5), but not alpha4beta1 (VLA-4) in both cell lines. Adhesion returned to normal levels upon transfection of wild-type CD45 into the CD45-deficient lines. Transfection of chimeric or mutant molecules expressing some, but not all, CD45 domains and activities demonstrated that both the transmembrane domain and the tyrosine phosphatase activity of CD45 were required for regulation of integrin-dependent adhesion, but the highly glycosylated extracellular domain was dispensable. In contrast, only a catalytically active CD45 cytoplasmic domain was required for TCR signaling. Transfectants that restored normal levels of adhesion to fibronectin coimmunoprecipitated with the transmembrane protein known as CD45-associated protein. These studies demonstrate a novel role for CD45 in adhesion regulation and suggest a possible function for its association with CD45-associated protein.     

Somatostatin as a physiological regulator of pulsatile growth hormone secretion

Somatostatin plays an important role in the regulation of the episodic and ultradian rhythm of growth hormone (GH) secretion. Passive immunization of rats with specific antibodies to the 14 and 28 amino acid sequences caused a significant GH elevation. The fact that somatostatin antiserum was unable to block episodic GH surges indicates that this hormone's release must be regulated by a dual mechanism. Indeed, GH-releasing factor (GRF) seems to be instrumental in the maintenance of pulsatile GH secretion. Moreover, exogenous GRF induced a further GH increase predominantly during the period of active secretion. Neutralization of endogenous somatostatin eliminated this time-dependent effect, indicating that this peptide blocks periodical spontaneous GH release. Food deprivation and changes in glucose homeostasis virtually obliterate the ultradian GH rhythm. In this context, peripheral somatostatin seems to play an important role. Also the central GRF/somatostatin interplay is responsible for a short-loop feedback control on pituitary somatotrops.     

Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8

Blood vessel formation requires endothelial cell interactions with the extracellular matrix through cell surface receptors, and signaling events that control endothelial cell adhesion, migration, and lumen formation. Laminin-8 (alpha4beta1gamma1) is present in all basement membranes of blood vessels in fetal and adult tissues, but despite its importance in vessel formation, its role in endothelial cell adhesion and migration remains undefined. We examined adhesion and migration of HMEC-1 human microvascular endothelial cells on laminin-8 with an emphasis on the integrin-mediated signaling events, as compared with those on laminin-10/11 and fibronectin. We found that laminin-8 was less potent in HMEC-1 cell adhesion than laminin-1, laminin-10/11, and fibronectin, and mediated cell adhesion through alpha6beta1 integrin. Despite its weak cell-adhesive activity, laminin-8 was as potent as laminin-10/11 in promoting cell migration. Cells adhering to laminin-8 displayed streaks of thin actin filaments and formed lamellipodia at the leading edge of the cells, as observed with cells adhering to laminin-10/11, while cells on fibronectin showed thick actin stress fibers and large focal adhesions. Pull-down assays of GTP-loaded Rho, Rac, and Cdc42 demonstrated that Rac, but not Rho or Cdc42, was preferentially activated on laminin-8 and laminin-10/11, when compared with fibronectin. Furthermore, a dominant-negative mutant of Rac suppressed cell spreading, lamellipodial formation, and migration on laminin-8, but not on fibronectin. These results, taken together, indicate that Rac is activated during endothelial cell adhesion to laminin-8, and is pivotal for alpha6beta1 integrin-mediated cell spreading and migration on laminin-8.     

Signal-transducing adaptor protein-2 regulates integrin-mediated T cell adhesion through protein degradation of focal adhesion kinase

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology- and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP-2-deficient splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, and that STAP-2-deficient T cells contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP-2 induces a dramatic decrease in the protein contents of FAK and integrin-mediated T cell adhesion to fibronectin in Jurkat T cells via the degradation of FAK. Regarding the mechanism for this effect, we found that STAP-2 associates with FAK and enhances its degradation, proteasome inhibitors block FAK degradation, and STAP-2 recruits an endogenous E3 ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin-mediated signaling in T cells via STAP-2, which directly interacts with and degrades FAK.