Arginine-glycine-aspartic acid binding leading to molecular stabilization between integrin alpha v beta 3 and its ligand.

Cell adhesion is characterized by an integrin-mediated ligand binding event followed by reorganization of the actin-cytoskeleton leading to cell spreading and/or migration. In this report we examine the role of integrin alpha v beta 3 in mediating cell attachment to vitronectin or a RGD-containing peptide in the presence of cytochalasin B to prevent actin polymerization. Under these conditions cell attachment to a RGD-containing peptide can be dissociated by excess soluble ligand whereas cells attached to vitronectin cannot. These results suggest that alpha v beta 3-mediated cell attachment to vitronectin results in a highly stabilized interaction that is independent of the actin-cytoskeleton. To investigate the molecular nature of this interaction alpha v beta 3 was purified to homogeneity, and its binding properties toward various ligands were measured in a solid-phase receptor assay. The data indicate that alpha v beta 3 binds to vitronectin or fibronectin in a nondissociable manner whereas a RGD-containing peptide derived from vitronectin binds specifically but is completely dissociable with a Kd of 9.4 x 10(-7) M. Moreover, chemical modification of alpha v beta 3 with limited glutaraldehyde treatment allowed vitronectin to bind in a RGD-dependent and dissociable manner, suggesting that receptor conformational changes or specific amino acid residues proximal to the ligand binding site(s) are involved in the stabilization event. Thus, in the absence of cytoskeletal proteins or other cellular components, integrin alpha v beta 3-ligand binding involves recognition of the RGD sequence leading to a highly stabilized protein-protein association.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain

Integrin-mediated cell attachment and growth factor stimulation often act synergistically on cell proliferation, differentiation, migration, and survival. Some of these synergistic effects depend on the physical interaction of integrins with growth factor receptors. Here we examine the nature of the physical interaction between the alpha(v)beta(3) integrin and two receptor tyrosine kinases (RTKs), the platelet-derived growth factor receptor beta (PDGF-Rbeta) and the vascular endothelial growth factor receptor 2 (VEGF-R2, also known as KDR and flk-1). Both of these RTKs associate with the alpha(v)beta(3) integrin but do not associate with beta(1) integrins. Furthermore, growth factor stimulation of these RTKs promotes increased cell proliferation and migration when cells are attached to the alpha(v)beta(3) ligand, vitronectin. We show that alpha(v)beta(3) in which the beta(3) cytoplasmic domain is deleted or replaced with the beta(1) cytoplasmic domain coimmunoprecipitates with PDGF-Rbeta and VEGF-R2. The beta(3) extracellular domain alone was sufficient for the PDGF-Rbeta association whereas the VEGF-R2 association required the presence of the alpha(v) subunit. Activation of the RTKs by their ligands was not required for them to associate with the integrin. Cell migration to PDGF was enhanced in the cells transfected with the chimeric subunit containing the beta(3) extracellular domain but not when that domain came from the beta(1) subunit. These results show that the interactions that lead to the association of the alpha(v)beta(3) integrin with PDGF-Rbeta and VEGF-R2 and enhancement of RTK activity take place outside the cell.     

The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

Distinct ligand-binding modes for integrin alpha(v)beta(3)-mediated adhesion to fibronectin versus vitronectin.

Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including alpha(v)beta(3) can bind to multiple ligands. The binding of alpha(v)beta(3) to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the alpha(v)beta(3)-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the alpha(v)beta(3)-vitronectin bond required phosphorylation of the beta(3) cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of beta(3) controlled by Tyr-747 and Tyr-759. In contrast, alpha(v)beta(3)-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of alpha(v)beta(3).     

Adhesion of a chicken myeloblast cell line to fibrinogen and vitronectin through a beta 1-class integrin.

The adhesive interactions of circulating blood cells are tightly regulated, receptor-mediated events. To establish a model for studies on regulation of cell adhesion, we have examined the adhesive properties of the HD11 chick myeloblast cell line. Function-perturbing antibodies were used to show that integrins containing the beta 1 subunit mediate HD11 cell attachment to several distinct extracellular matrix proteins, specifically fibronectin, collagen, vitronectin, and fibrinogen. This is the first evidence that an integrin heterodimer in the beta 1 family functions as a receptor for fibrinogen. While the alpha v beta 1 heterodimer has been shown to function as a vitronectin receptor on some cells, this heterodimer could not be detected on HD11 cells. Instead, results suggest that the beta 1 subunit associates with different, unidentified alpha subunit(s) to form receptors for vitronectin and fibrinogen. Results using function-blocking antibodies also demonstrate that on these cells, additional receptors for vitronectin are formed by alpha v beta 3 and alpha v associated with an unidentified 100-kD beta subunit. The adhesive interactions of HD11 cells with these extracellular matrix ligands were shown to be regulated by lipopolysaccharide treatment, making the HD11 cell line attractive for studies of mechanisms regulating cell adhesion. In contrast to primary macrophage which rapidly exhibit enhanced adhesion to laminin and collagen upon activation, activated HD11 cells exhibited reduced adhesion to most extracellular matrix constituents.     

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is involved in the activation of endothelial integrins

We previously reported that mouse orthologue of puromycin insensitive leucyl-specific aminopeptidase (mPILSAP) played an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs) (Miyashita et al., 2002. Blood 99:3241-3249). Here, we examined the mechanism as to how mPILSAP regulates the migration of ECs. Cell adhesion through integrins plays a crucial role in cell migration, and ECs use at least type-1 collagen receptor integrin alpha2beta1, fibronectin receptor alpha5beta1, and vitronectin receptors alphavbeta3 and alphavbeta5. mPILSAP antisense oligodeoxynucleotide (AS-ODN) or leucinethiol (LT), a leucyl-aminopeptidase inhibitor, did not affect the attachment but did significantly inhibit the spreading of cells of the murine endothelial cell line MSS31 when they were plated on vitronectin-, fibronectin-, or type-1 collagen, although they did not affect the expression of integrin alpha2, alpha5, alphav, beta1, beta3, and beta5 subunits on the cell surface. AS-ODN and LT also inhibited the tyrosine phosphorylation of FAK when cells were plated on vitronectin, fibronectin, or type-1 collagen. This inhibition of cell spreading and of tyrosine phosphorylation of FAK could be negated by Mg(2+). These results suggest that mPILSAP is involved in the activation of endothelial integrins.     

Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation

Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.     

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Alpha v beta 3 and alpha v beta 5 integrins and their role in muscle precursor cell adhesion

BACKGROUND INFORMATION: Functional adaptation of skeletal muscle is a requirement for different muscle groups (e.g. craniofacial, ocular and limb) to undergo site-specific changes. Such tissue remodelling depends on dynamic interactions between muscle cells and their extracellular matrix, via participation of multifunctional molecules such as integrins. In view of data suggesting a role in fundamental muscle biology and muscle development in other systems, the present study has focused on expression and function of alpha v integrins, in cultured adult human craniofacial muscle (masseter) precursor cells and myotubes, and the predominantly fibroblastic IC (interstitial cells) population. RESULTS AND CONCLUSIONS: Flow-cytometric phenotyping and immunofluorescence phenotyping show that alpha v, alpha v beta 3 and alpha v beta 5 are expressed in all mononuclear cells (muscle precursors and IC) seeded on muscle extracellular molecules such as gelatin, VN (vitronectin) and FN (fibronectin). In this system, blockade of alpha v activity using a function-perturbing antibody abrogates cell migration on VN and FN. alpha v integrins act predominantly as VN receptors as cell-substrate attachment is diminished when alpha v neutralizing agents are introduced into cultures seeded on VN, and this inhibition is reversible; these integrins also appear to be minor FN receptors. These results demonstrate that the alpha v subset of integrins present on both myogenic precursors and IC is an essential cohort of VN and, to a lesser extent, FN receptors mediating cell adhesion and, either directly or indirectly, arbiters of cell motility.     

Receptor tyrosine kinase signaling required for integrin alpha v beta 5- directed cell motility but not adhesion on vitronectin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.     

ERK1 associates with alpha(v)beta 3 integrin and regulates cell spreading on vitronectin

Kinases that associate with integrins are likely to mediate the assembly/disassembly of cell:matrix junctions during cell migration. Here we show that ERK1 associates with alpha(v)beta(3) integrin following the addition of platelet-derived growth factor to serum-starved Swiss or NIH 3T3 fibroblasts in an interaction that is mediated by the central region of the beta(3) integrin cytodomain. alpha(v)beta(3).ERK1 association occurred prior to focal complex formation and was seen to initiate in small punctate complexes primarily in the peripheral regions of the plasma membrane. Expression of a dominant negative mutant of ERK1 (but not ERK2) significantly reduced the spreading of cells on vitronectin, whereas cell spreading on fibronectin was unaffected by inhibition of ERK1. In contrast, inhibition of ERK activation by PD98059 had no effect on the platelet-derived growth factor-regulated Rab4-dependent flux of alpha(v)beta(3) integrin from early endosomes to the plasma membrane, an event that is also necessary for cells to spread efficiently on vitronectin. We propose that alpha(v)beta(3) integrin must recycle to the plasma membrane via the Rab4 pathway and recruit active ERK1 in order to function efficiently.     

Alphav-integrin utilization in human beta-cell adhesion, spreading, and motility

The role of individual integrins in human beta-cell development and function is largely unknown. This study describes the contribution of alpha(v)-integrins to human beta-cell adhesion, spreading, and motility. Developmental differences in alpha(v)-integrin utilization are addressed by comparing the responses of adult and fetal beta-cells, and vitronectin is used as a substrate based on its unique pattern of expression in the developing pancreas. Fetal and adult beta-cells attached equally to vitronectin and integrin alpha(v)beta(5) was found to support the adhesion of both mature and immature beta-cell populations. Fetal beta-cells were also observed to spread and migrate on vitronectin, and integrin alpha(v)beta(1) was found to be essential for these responses. In contrast to their fetal counterparts, adult beta-cells failed to either spread or migrate and this deficit was associated with a marked down-regulation of alpha(v)beta(1) expression in adult islet preparations. The integrin alpha(v)beta(3) was not found to support significant beta-cell attachment or migration. Based on our findings, we conclude that integrins alpha(v)beta(5) and alpha(v)beta(1) are important mediators of human beta-cell adhesion and motility, respectively. By supporting fetal beta-cell migration, alpha(v)beta(1) could play an important role in early motile processes required for islet neogenesis.     

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.     

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line

We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. alpha3beta1, alpha5beta1, alpha6beta1/beta4, alpha8beta1 and alpha(v)beta1/beta5/beta6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, alpha2beta1, alpha(v)beta6 and alpha6beta1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas alpha(v)beta5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.     

Ligand-dependent activation of integrin alpha vbeta 3

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(v)beta(3). In this study, we examined the activation of alpha(v)beta(3) resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected alpha(v)beta(3), adhesion to vitronectin required tyrosine phosphorylation of the beta(3) subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without beta(3)-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.     

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen Matrices

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.     

Protein kinase B/Akt acts via glycogen synthase kinase 3 to regulate recycling of alpha v beta 3 and alpha 5 beta 1 integrins

Protein kinase B (PKB)/Akt is known to promote cell migration, and this may contribute to the enhanced invasiveness of malignant cells. To elucidate potential mechanisms by which PKB/Akt promotes the migration phenotype, we have investigated its role in the endosomal transport and recycling of integrins. Whereas the internalization of alpha v beta 3 and alpha 5 beta 1 integrins and their transport to the recycling compartment were independent of PKB/Akt, the return of these integrins (but not internalized transferrin) to the plasma membrane was regulated by phosphatidylinositol 3-kinases and PKB/Akt. The blockade of integrin recycling and cell spreading on integrin ligands effected by inhibition of PKB/Akt was reversed by inhibition of glycogen synthase kinase 3 (GSK-3). Moreover, expression of nonphosphorylatable active GSK-3 beta mutant GSK-3 beta-A9 suppressed recycling of alpha 5 beta 1 and alpha v beta 3 and reduced cell spreading on ligands for these integrins, indicating that PKB/Akt promotes integrin recycling by phosphorylating and inactivating GSK-3. We propose that the ability of PKB/Akt to act via GSK-3 to promote the recycling of matrix receptors represents a key mechanism whereby integrin function and cell migration can be regulated by growth factors.     

Amphiphysin1 inhibits vitronectin-mediated cell adhesion,spreading, and migration in vitro

To investigate the regulatory mechanism of cell adhesion, we have searched for cellular inhibitory factors which prevent cell adhesion. The brain cytosol was found to inhibit the adhesion of various transformed cells to the substratum. An inhibitory 120-kDa protein was purified by sequential column chromatography. Peptide sequencing revealed that the protein is identical to amphiphysin1. GST-amphiphysin1 suppressed the attachment of HeLa cells to the plate when cells were cultured in the serum-containing medium. Vitronectin, a major cell-adhesive protein in serum and a ligand to alpha(v)beta3 integrin, was responsible for this cell attachment, and the vitronectin action was blocked by GST-amphiphysin1. GST-amphiphysin1 also inhibited the vitronectin-mediated spreading and migration of malignant melanoma cells. Furthermore, GST-amphiphysin1 bound directly to vitronectin. These findings point to the interesting possibility that amphiphysin1 could be a useful tool to inhibit cell-adhesive vitronectin.