Integrin-mediated regulation of neurovascular development,physiology and disease

The mammalian central nervous system (CNS) is comprised of billions of neurons and glia that are intertwined with an elaborate network of blood vessels. These various neural and vascular cell types actively converse with one another to form integrated, multifunctional complexes, termed neurovascular units. Cell-cell communication within neurovascular units promotes normal CNS development and homeostasis, and abnormal regulation of these events leads to a variety of debilitating CNS diseases. This review will summarize (i) cellular and molecular mechanisms that regulate physiological assembly and maintenance of neurovascular units; and (ii) signaling events that induce pathological alterations in neurovascular unit formation and function. An emphasis will be placed on neural-vascular cell adhesion events mediated by integrins and their extracellular matrix (ECM) ligands. I will highlight the role of a specific adhesion and signaling axis involving αvβ8 integrin, latent transforming growth factor β’s (TGFβ’s), and canonical TGFβ receptors. Possible functional links between components of this axis and other signal transduction cascades implicated in neurovascular development and disease will be discussed. In summary, comprehensively understanding the pathways that regulate bidirectional neural-vascular cell contact and communication will provide new insights into the mechanisms of neurovascular unit development, physiology and disease.     

Loss of the TGFβ-Activating Integrin αvβ8 on Dendritic Cells Protects Mice from Chronic Intestinal Parasitic Infection via Control of Type 2 Immunity

Chronic intestinal parasite infection is a major global health problem, but mechanisms that promote chronicity are poorly understood. Here we describe a novel cellular and molecular pathway involved in the development of chronic intestinal parasite infection. We show that, early during development of chronic infection with the murine intestinal parasite Trichuris muris, TGFβ signalling in CD4+ T-cells is induced and that antibody-mediated inhibition of TGFβ function results in protection from infection. Mechanistically, we find that enhanced TGFβ signalling in CD4+ T-cells during infection involves expression of the TGFβ-activating integrin αvβ8 by dendritic cells (DCs), which we have previously shown is highly expressed by a subset of DCs in the intestine. Importantly, mice lacking integrin αvβ8 on DCs were completely resistant to chronic infection with T. muris, indicating an important functional role for integrin αvβ8-mediated TGFβ activation in promoting chronic infection. Protection from infection was dependent on CD4+ T-cells, but appeared independent of Foxp3+ Tregs. Instead, mice lacking integrin αvβ8 expression on DCs displayed an early increase in production of the protective type 2 cytokine IL-13 by CD4+ T-cells, and inhibition of this increase by crossing mice to IL-4 knockout mice restored parasite infection. Our results therefore provide novel insights into how type 2 immunity is controlled in the intestine, and may help contribute to development of new therapies aimed at promoting expulsion of gut helminths.     

Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor–specific angiogenic signaling

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin family with diverse functions in epithelial cells, including cell migration, cell contact maturation, and tight junction formation. In endothelial cells, JAM-A has been implicated in basic fibroblast growth factor (bFGF)-regulated angiogenesis through incompletely understood mechanisms. In this paper, we identify tetraspanin CD9 as novel binding partner for JAM-A in endothelial cells. CD9 acts as scaffold and assembles a ternary JAM-A-CD9-αvβ3 integrin complex from which JAM-A is released upon bFGF stimulation. CD9 interacts predominantly with monomeric JAM-A, which suggests that bFGF induces signaling by triggering JAM-A dimerization. Among the two vitronectin receptors, αvβ3 and αvβ5 integrin, which have been shown to cooperate during angiogenic signaling with bFGF and vascular endothelial growth factor (VEGF), respectively, CD9 links JAM-A specifically to αvβ3 integrin. In line with this, knockdown of CD9 blocks bFGF- but not VEGF-induced ERK1/2 activation. JAM-A or CD9 knockdown impairs endothelial cell migration and tube formation. Our findings indicate that CD9 incorporates monomeric JAM-A into a complex with αvβ3 integrin, which responds to bFGF stimulation by JAM-A release to regulate mitogen-activated protein kinase (MAPK) activation, endothelial cell migration, and angiogenesis. The data also provide new mechanistic insights into the cooperativity between bFGF and αvβ3 integrin during angiogenic signaling.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.     

A Role for the αvβ3 Integrin in the Transmigration of Monocytes

The β2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin αvβ3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking β3 integrins revealed weak migratory ability, whereas monocytes expressing β3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains αL, β2, αv, or IAP, a protein functionally associated with αvβ3 integrin. Transfection of β3 integrin chain cDNA into monocytes lacking β3 integrins resulted in expression of the αvβ3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in αLβ2-dependent locomotion on recombinant ICAM-1 which was enhanced by αvβ3 integrin occupancy. Antibodies against IAP were able to revert this αvβ3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of αvβ3 integrin could decrease monocyte binding to ICAM-1.In conclusion, we show that αvβ3 integrin modulates αLβ2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.     

Alcohol ingestion disrupts alveolar epithelial barrier function by activation of macrophage-derived transforming growth factor beta1

Background

Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Experimentally, alcohol-induced oxidative stress increases the expression of transforming growth factor β1 (TGFβ1) in the lung; however, we do not know the precise contribution of various alveolar cells in this process. In the present study, we focused on cell-cell interactions between alveolar macrophages and epithelial cells and the potential mechanisms by which TGFβ1 may become activated in the alveolar space of the alcoholic lung.

Methods

Primary alveolar macrophages and epithelial cells were isolated from control- and alcohol-fed Sprague–Dawley rats. Expression of TGFβ1 and the epithelial integrin αvβ6 were examined by real time PCR and either immunocytochemistry or flow cytometry. Alveolar epithelial cells were cultured on transwell supports in the presence of macrophage cell lysate from control- or alcohol-fed rats or in the presence of viable macrophages ± alcohol. Epithelial barrier function was assessed by transepithelial resistance (TER) and paracellular flux of Texas Red dextran.

Results

TGFβ1 expression was increased in alveolar macrophages from alcohol-fed rats, and TGFβ1 protein was predominantly membrane-bound. Importantly, alveolar macrophage cellular lysate from alcohol-fed rats decreased TER and increased paracellular dextran flux in primary alveolar epithelial cell monolayers as compared to the lysates from control-fed rats. Alcohol-induced epithelial barrier dysfunction was prevented by anti-TGFβ1 antibody treatment, indicating the presence of bioactive TGFβ1 in the macrophage lysate. In addition, co-culturing macrophages and epithelial cells in the presence of alcohol decreased epithelial barrier function, which also was prevented by anti-TGFβ1 and anti-αvβ6 treatment. In parallel, chronic alcohol ingestion in vivo, or direct treatment with active TGFβ1 in vitro, increased the expression of αvβ6 integrin, which is known to activate TGFβ1, in alveolar epithelial cells.

Conclusions

Taken together, these data suggest that interactions between alveolar epithelial cells and macrophages contribute to the alcohol-mediated disruption of epithelial barrier function via the expression and activation of TGFβ1 at points of cell-cell contact.     

Saccharomyces boulardii Improves Intestinal Epithelial Cell Restitution by Inhibiting αvβ5 Integrin Activation State

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn''s disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.     

Localization of αυβ6 Integrin-TGF-β1/Smad3, mTOR and PPARγ in Experimental Colorectal Fibrosis

A simultaneous action of several pro-fibrotic mediators appears relevant in the development of fibrosis. There are evidences that transforming growth factor-β (TGF-β)/Smad3 pathway forms with αvβ6 integrin, mammalian target of Rapamycin (mTOR) and peroxisome proliferator-activated receptor-γ (PPARγ) a complex signalling network with extensive crosstalk and strong effects on fibrosis development. The present study evaluated the expression of TGFβ, Smad3, αvβ6 integrin, mTOR and PPARγ in 2, 4, 6-trinitrobenzenesulphonic acid (TNBS)-induced colorectal fibrosis in Smad3 wild-type (WT) and null mice. Smad3 WT mice treated with TNBS developed a marked colorectal fibrosis and showed a concomitant up-regulation of TGFβ, Smad3, αvβ6 and mTOR and a reduction of PPARγ expression. On the other hand, Smad3 Null mice similarly treated with TNBS did not develop fibrosis and showed a very low or even absent expression of TGFβ, Smad3, αvβ6 and mTOR and a marked over-expression of PPARγ. At the same time the expression of α-smooth muscle actin (a marker of activated myofibroblasts), collagen I-III and connective tissue growth factor (a downstream effector of TGFβ/Smad3-induced extracellular matrix proteins) were up-regulated in Smad3 WT mice treated with TNBS compared to Null TNBS-treated mice. These preliminary results suggest a possible interaction between these pro-fibrotic molecules in the development of intestinal fibrosis.Key words: intestinal fibrosis, integrins, TGF-β, SMAD, mTOR, PPAR, IBD     

Spatial-temporal reorganization of activated integrins

Integrin receptors play important roles in cell adhesion and tumor metastasis. The coupling of mechanical sensing and biochemical ligation is known to collectively regulate the activation of integrin receptors. Recently, oligomerization of activated integrins has been considered as the primordial signature of cytoskeletal remodeling and the initiation of various downstream signals, such as focal and fibrillar adhesions. However, spatio-temporal reorganization of activated integrins and associated proteins remains poorly understood. Here, we summarized the recent discovery of sequential biophysical events of integrin activation during early adhesion formation. Using the cyclic Arg-Gly-Asp (RGD) peptide as a mobile ligand on supported lipid membranes, a series of previously unreported events were observed following integrin αvβ3 clustering and cell spreading, including a long-range lateral translocation of the integrin clusters. With initial clustering, localized actin polymerization occurred in a Src family kinase dependent manner. Clustering of liganded integrins recruits various adaptor proteins and serves as a reaction core for mechanobiological activities. In addition, there are future possibilities to investigate the role of other synergetic interactions with the activated integrin receptors.     

Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell–matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell–vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin αvβ5, and that active PAK4 induces accelerated integrin αvβ5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin αvβ5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion.     

Integrin αvβ3 Requirement for Sustained Mitogen-activated Protein Kinase Activity during Angiogenesis

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5–120 min) was refractory to integrin antagonists, whereas the sustained activity (4–20 h) depended on integrin αvβ3, but not β1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained αvβ3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin αvβ3.     

Interactions between Growth Factors and Integrins: Latent Forms of Transforming Growth Factor-β Are Ligands for the Integrin αvβ1

The multipotential cytokine transforming growth factor-β (TGF-β) is secreted in a latent form. Latency results from the noncovalent association of TGF-β with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-β–binding protein (LTBP) produces the most common form of latent TGF-β, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-β. LTBP and the LAP propeptides of TGF-β (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-β function in the ECM, we determined whether latent TGF-β1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits αv and β1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. αvβ1 eluted with EDTA. After purification in the presence of Mn²⁺, a small amount of αvβ5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was αvβ1 dependent. These results establish αvβ1 as a LAP-β1 receptor. Interactions between latent TGF-β and αvβ1 may localize latent TGF-β to the surface of specific cells and may allow the TGF-β1 gene product to initiate signals by both TGF-β receptor and integrin pathways.     

Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes

Transforming growth factor-β (TGFβ) is a dimeric peptide growth factor which regulates cellular differentiation and proliferation during development. Most cells secrete TGFβ as a large latent TGFβ complex containing mature TGFβ, latency associated peptide, and latent TGFβ-binding protein (LTBP)-1. The biological role of LTBP-1 in development remains unclear. Using a polyclonal antiserum specific for LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the mouse embryonic heart. Mature TGFβ protein was required at the onset of the endothelial-mesenchymal transformation to initiate endocardial cushion tissue formation. Double antibody staining showed that LTBP-1 colocalized with TGFβ1 as an extracellular fibrillar structure surrounding the endocardial cushion mesenchymal cells. Immunogold electronmicroscopy showed that LTBP-1 localized to 40–100 nm extracellular fibrillar structure and 5–10-nm microfibrils. The anti–LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This inhibitory effect was reversed by administration of mature TGFβ proteins in culture. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of TGFβ as a large latent TGFβ complex.     

Growth Factor–dependent Activation of αvβ3 Integrin in Normal Epithelial Cells: Implications for Tumor Invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, αvβ3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, αvβ3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. αvβ3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for αvβ3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted αvβ3 enrichment at FCs and impaired adhesion. Accordingly, activation of αvβ3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor–driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

αvβ3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors

We report that αvβ3 integrin strongly affects the innate immune response in epithelial cells. αvβ3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvβ3 integrin. The boosting was exerted specifically by αvβ3 integrin but not by αvβ6 or αvβ8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response.     

β3 integrin–mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner

Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin–dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2–induced Smad signaling through a Cdc42–Src–FAK–ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine.     

Interactions of Soluble Recombinant Integrin αvβ5 with Human Adenoviruses

αv integrins have been identified as coreceptors for adenovirus (Ad) internalization; however, direct interactions of these molecules with Ad have not been demonstrated. We report here the expression of soluble integrin αvβ5, which retains the ability to recognize the Ad penton base as well as vitronectin, an Arg Gly Asp (RGD)-containing extracellular matrix protein. Soluble integrin αvβ5 reacted with seven different Ad serotypes (subgroups A to E) in solid-phase binding assays. The soluble integrin exhibited different levels of binding to each Ad serotype; however, binding to multiple Ad types required the presence of divalent metal cations and was inhibited by a synthetic RGD peptide, indicating that RGD and cation-binding sequences regulate Ad interactions with αvβ5. Incubation of Ad particles with soluble αvβ5 integrin also inhibited subsequent Ad internalization into epithelial cells as well as virus attachment to monocytic cells. These findings suggest that soluble αv integrins or antagonists of these coreceptors could be used to limit infection by multiple Ad types. The generation of soluble αv integrins should also permit further detailed kinetic and structural analysis of Ad interactions with its coreceptors.     

p21-activated Kinase 4 Phosphorylation of Integrin β5 Ser-759 and Ser-762 Regulates Cell Migration

Modulation of integrin αvβ5 regulates vascular permeability, angiogenesis, and tumor dissemination. In addition, we previously found a role for p21-activated kinase 4 (PAK4) in selective regulation of integrin αvβ5-mediated cell motility (Zhang, H., Li, Z., Viklund, E. K., and Strömblad, S. (2002) J. Cell Biol. 158, 1287–1297). This report focuses on the molecular mechanisms of this regulation. We here identified a unique PAK4-binding membrane-proximal integrin β5-SERS-motif involved in controlling cell attachment and migration. We also mapped the integrin β5-binding site within PAK4. We found that PAK4 binding to integrin β5 was not sufficient to promote cell migration, but that PAK4 kinase activity was required for PAK4 promotion of cell motility. Importantly, PAK4 specifically phosphorylated the integrin β5 subunit at Ser-759 and Ser-762 within the β5-SERS-motif. Point mutation of these two serine residues abolished the PAK4-induced cell migration, indicating a functional role for these phosphorylations in migration. Our results may give important leads to the functional regulation of integrin αvβ5, with implications for vascular permeability, angiogenesis, and cancer dissemination.