Steven K. Akiyama

Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function

Integrin receptors mediate cell adhesion, signal transduction, and cytoskeletal organization. How a single transmembrane receptor can fulfill multiple functions was clarified by comparing roles of receptor occupancy and aggregation. Integrin occupancy by monovalent ligand induced receptor redistribution, but minimal tyrosine phosphorylation signaling or cytoskeletal protein redistribution. Aggregation of integrins by noninhibitory monoclonal antibodies on beads induced intracellular accumulations of pp125FAK and tensin, as well as phosphorylation, but no accumulation of other cytoskeletal proteins such as talin. Combining antibody-mediated clustering with monovalent ligand occupancy induced accumulation of seven cytoskeletal proteins, including alpha-actinin, talin, and F-actin, thereby mimicking multivalent interactions with fibronectin or polyvalent peptides. Integrins therefore mediate a complex repertoire of functions through the distinct effects of receptor aggregation, receptor occupancy, or both together.

Fibronectin and integrins in invasion and metastasis

SummaryThe adhesive glycoprotein fibronectin and integrin receptors appear to play important roles in the progression of metastatic disease. Fibronectin is a multifunctional extracellular glycoprotein that has at least two independent cell adhesion regions with different receptor specificities. The cell adhesive region in the central portion of fibronectin is comprised of at least two minimal amino acid sequences - an Arg-Gly-Asp (RGD) sequence and a Pro-His-Ser-Arg-Asn (PHSRN) sequence - which function in synergy. Another cell adhesive region is located near the carboxy-terminus in the alternatively spliced IIICS module. The critical minimal sequences for this region are Leu-Asp-Val (LDV) and Arg-Glu-Asp-Val (REDV) which function in an additive rather than synergistic fashion. Integrins are heterodimeric, transmembrane cell adhesion receptors for fibronectin and other extracellular matrix molecules. Several different integrins bind to fibronectin. The α5β1 fibronectin-specific integrin binds to the central RGD/PHSRN site. The α4β1 integrin binds to the IIICS site. Fibronectin-integrin interactions are important in tumor cell migration, invasion, and metastasis. In addition to promoting cell adhesion to the extracellular matrix, these proteins may also function in chemotaxis and control of proliferation. Peptide and antibody inhibitors of fibronectin and integrin functions have been shown to be effective inhibitors of metastasis, and are potentially important reagents for the study and control of cancer.

Localization of integrin receptors for fibronectin, collagen, and laminin in human skin: variable expression in basal and squamous cell carcinomas

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Identification of a novel anti‐integrin monoclonal antibody that recognises a ligand‐induced binding site epitope on the β1 subunit

Integrins are the major family of receptors involved in the adhesive interactions of cells with extracellular matrix macromolecules. Although it is known that integrins can exist in active or inactive states, the molecular mechanisms by which integrin activity is modulated are poorly understood. A novel anti‐integrin monoclonal antibody, 12G10, that enhances α5β1‐fibronectin interactions has been identified. 12G10 binds to the β1 subunit and appears to recognise a region of the subunit that contains the epitopes of several previously described activating or inhibitory monoclonal antibodies. However, unlike other activating anti‐β1 antibodies, the binding of 12G10 to α5β1 is increased in the presence of ligands (fibronectin fragment or RGD peptide). This is the first report for the β1 integrin family of an antibody that recognises a ligand‐induced binding site, and further emphasises the functional importance of a specific region of the β1 subunit in regulating integrin‐ligand interactions.

The inhibitory anti-beta1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy. Evidence for allosteric inhibition of integrin function

Integrin-ligand binding causes conformational changes in the integrin, as evidenced by the increased expression of epitopes known as ligand-induced binding sites. Some monoclonal antibodies (mAbs) that recognize ligand-induced binding sites stimulate ligand binding, possibly by stabilizing the ligand-occupied conformation of the integrin. Here we have investigated the effect of ligand recognition by α5β1 on the binding of a mAb that inhibits β1 integrin function (mAb 13). Ligand (fibronectin fragment or GRGDS peptide) decreased the binding of mAb 13 to α5β1. Analysis of this inhibition showed that at high ligand concentrations, approximately 50% of the total integrin bound mAb 13 with >50-fold lower affinity than in the absence of ligand. The concentration of ligand required for half-maximal inhibition of antibody binding was independent of antibody concentration, suggesting that ligand acts as an allosteric inhibitor of mAb 13 binding. Hence, ligand and mAb 13 did not appear to compete directly for binding to α5β1. The stimulatory anti-β1 mAb 9EG7 was found to increase the maximum level of ligand binding ∼2-fold, indicating that up to 50% of the total integrin could not bind ligand without 9EG7 stimulation. Analysis of mAb 13 binding in the presence of 9EG7 and ligand (i.e. maximal ligand occupancy) demonstrated that essentially all of the integrin bound mAb 13 with very low or zero affinity. Our results demonstrate that mAb 13 recognizes an epitope that is dramatically attenuated in the ligand-occupied form of α5β1. Hence, since mAb 13 preferentially recognizes the unoccupied conformation of the integrin, the antibody may inhibit ligand binding by stabilizing the unoccupied state of α5β1. In addition, we present evidence that the binding of mAb 13 to ligand-occupied α5β1 may also induce a conformational change in the integrin, resulting in the displacement of ligand.

A Mechanism for Regulation of Melanoma Invasion LIGATION OF α6β1 INTEGRIN BY LAMININ G PEPTIDES

Invasion of LOX human melanoma cells involves extracellular matrix (ECM) degradation and formation of cell surface invadopodia. Here we show that the ligation of α6β1 by two peptides derived from the COOH-terminal globular domain of laminin-1 α1 chain (laminin G peptides), designated AG-10 (NPWHSIYITRFG) and AG-32 (TWYKIAFQRNRK), and antibodies against α6 and β1 integrins promoted invasiveness. AG-10 and AG-32 inhibited cell adhesion on laminin, and the antibodies blocked cell adhesion on immobilized AG-10 and AG-32, suggesting that the peptides interact primarily with α6β1 integrin. These soluble peptides and integrin antibodies induced invasiveness by causing an 2-3-fold increase in ECM degradation and invadopodial activity independently of adhesion activity of integrins that were prebound to ECM. The induced ECM degradation and invasion was associated with an increased surface expression of the 170-kDa membrane-bound gelatinase, seprase, as well as its intense localization at invadopodia but not at focal adhesions. However, the total expression levels of seprase, gelatinase A and β1 integrins were not altered. We suggest that laminin G peptides act on the α6β1 integrin signaling of invasion by stimulating invadopodial activities, which is distinct from their direct effects on cell adhesion on immobilized ECM.

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N‐terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel ‘MGGQSES’ motif. RD2 binds to the N‐terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface‐exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells.

We have previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the alpha(5)beta(1) integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK-dependent manner. Here, we identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 significantly decreases the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc. Moreover, nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. We have also found P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell-surface nucleolin, PI3-K and p38 MAPK. Using siRNA approaches, we have found that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/PI3-K signaling complex require nucleolin expression. These results show that nucleolin (or a nucleolin-like protein) is a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on fibronectin substrates.

Altered processing of integrin receptors during keratinocyte activation.

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.

Latex beads as probes of cell surface-extracellular matrix interactions during chondrogenesis: evidence for a role for amino-terminal heparin-binding domain of fibronectin

Fibronectin-rich mesenchymal condensations form at sites of incipient chondrogenesis in the developing vertebrate limb, and in cultures of limb bud mesenchyme. We have used 6 microns polystyrene latex beads coated with various substances as probes for adhesive interactions that may mediate the formation of these condensations. Beads coated with heparin, chondroitin sulfate, or poly L-lysine, that were mixed with limb bud mesenchymal cells were centripetally conveyed into fibronectin-rich regions of cell condensation over a period of several days. Beads coated with dextran sulfate remained uniformly dispersed throughout the cultures during the same period. A monoclonal antibody directed against the amino-terminal heparin-binding domain of fibronectin completely inhibited accumulation of heparin-coated beads at condensing foci, but monoclonal antibodies directed against the collagen- or cell-binding domains of fibronectin were not inhibitory. Accumulation of chondroitin sulfate- or poly L-lysine-coated beads at condensing foci was unaffected by the antibody against the fibronectin amino terminus. Peptides with the sequence arg-gly-asp-ser or gly-arg-gly-asp-ser, which inhibit adhesive interactions mediated by the integrin-binding domain of fibronectin, had no effect on conveyance or accumulation of heparin-coated beads, but the peptide with the sequence gly-arg-gly, a repeated motif in the amino-terminal heparin-binding domain was completely inhibitory. These findings indicate that the amino-terminal heparin-binding domain of fibronectin can, within a tissue microenvironment, interact adhesively with heparin-like components on the surfaces of polystyrene beads, and by implication, on mesenchymal cells themselves. This interaction may therefore be a component of the condensation-forming mechanism in chondrogenic mesenchyme.