Site-specific N-glycan profiles of α5 ß1 integrin from rat liver.

BACKGROUND INFORMATION: Like most other cell surface proteins, α5 ß1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. RESULTS: Here, we have established the first comprehensive site-specific glycan map of α5 ß1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of ß1 integrin. The extensive glycan information that results from our study was projected onto a map of α5 ß1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5 ß1 integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis. CONCLUSION: We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5 ß1 integrin activity and endocytic trafficking. SIGNIFICANCE: Glycosylation of α5 ß1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5 ß1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of "native" glycoproteins.

Human mesenchymal stem cells promote tumor growth via MAPK pathway and metastasis by epithelial mesenchymal transition and integrin α5 in hepatocellular carcinoma.

Mesenchymal stem cells (MSCs) appear to be a potential vehicle for anticancer drugs due to their excellent tumor tropism ability. However, the interactions between MSCs and hepatocellular carcinoma (HCC) are quite controversial and the underlying mechanisms are ambiguous. In this study, an investigation was conducted into the effect of human MSCs (hMSCs) on tumor proliferation and metastasis both in xenograft and orthotopic models. It was discovered that hMSCs could promote tumor growth though activating mitogen-activated protein kinase (MAPK) signaling pathway and promote metastasis by epithelial mesenchymal transition (EMT) in vivo. To test whether hMSCs could induce immunosuppressive effects, the expression of the Natural killer (NK) cell marker CD56 was measured by immunohistochemical staining and the expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured by qRT-PCR. It was found out that CD56 expression significantly decreased, while TNF-α and IL-6 expression increased in the hMSCs-treated tissues. Mechanistically, RNA sequencing was performed, which led to a discovery that integrin α5 (ITGA5) was over-expressed in hMSCs-treated HCC. ITGA5 siRNAs blocked the hMSCs-induced migration and invasion of HCC, while over-expression of ITGA5 promoted the migration and invasion ability in HCC-hMSCs, indicating that the expression of ITGA5 is associated with hMSCs-induced tumor metastasis. These findings suggest that hMSCs may play a vital role in HCC proliferation and metastasis and could be identified as a putative therapeutic target in HCC.

Dioscin, a potent ITGA5 inhibitor, reduces the synthesis of collagen against liver fibrosis: Insights from SILAC-based proteomics analysis.

Inhibiting collagen generation is one effective method to treat liver fibrosis. Dioscin showed protective effect against liver fibrosis in our previous studies, and in the present work, SILAC-based proteomics was employed to test the underlying mechanism. A total of 121 differentially expressed proteins caused by dioscin in LX-2 cells were found, and dioscin significantly decreased the expression levels of FN, FAK1, ITGA5, p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, Col1a1, Col1a2, Col2a1, Col5a1, Col6a1, and increased 2ABB level in vivo and in vitro. Thus, we elucidated that dioscin specifically suppressed collagen synthesis through modulating PI3K/Akt pathway. In addition, we found that dioscin directly targeted with ITGA5 by molecular docking assay. SiRNA and overexpression transfection tests showed that ITGA5 siRNA plus dioscin slightly altered the effect of ITGA5 siRNA, and ITGA5 DNA transfection reversed the inhibitory effect of dioscin on collagen expressions via PI3K/Akt pathway. Our data explicated that dioscin should be considered as a novel and potent ITGA5 inhibitor to suppress collagen synthesis, which can also be developed as an effective food and healthcare product against hepatic fibrosis.

Integrin αv ß8 Adopts a High Affinity State for Soluble Ligands Under Physiological Conditions.

It has been proposed that integrins adopt a low affinity conformation under physiological conditions. Integrin can either be activated through cytoplasm or by binding of cations such as Mn2+ to the head domain. The cytoplasmic activation pathway, that is, inside-out signaling has been regarded as the physiological pathway for integrin activation. Integrin ß8 is important for neuron vascular development. However, due to the highly divergent cytoplasmic domain, this integrin probably does not rely on inside-out signaling for affinity regulation. We therefore hypothesized that the ß8 integrin uniquely assumes a constitutively high affinity state under physiological conditions. We discovered that ß8 indeed exhibited high binding to soluble vitronectin in the presence of Ca2+ and the ligand binding could not be further enhanced by addition of Mn2+ . The lower ectodomain stalk of the integrin, which is comprised by the integrin epidermal growth factor-like (I-EGF) domains and ßTD domain, is critical for this high affinity conformation. In addition, we found that unlike other integrins, Mg2+ at low concentration inhibited ß8 ligand binding. Mutagenesis studies indicated that ß8 integrin possesses a unique cation binding site which might contribute to the ligand binding affinity. Our study showed that both the ß8 lower ectodomain stalk and the head domain play an important role in its high affinity state under physiological conditions. J. Cell. Biochem. 118: 2044-2052, 2017. © 2016 Wiley Periodicals, Inc.

N-Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5ß1 to α6ß4.

N-Glycosylation of integrin α5ß1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3-5,10-14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3-5,10-14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5ß1 or α6ß4; i.e., the loss of site-11 switched EGFR-α5ß1 to EGFR-α6ß4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5ß1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR.

Nuclear monomeric integrin αv in cancer cells is a coactivator regulated by thyroid hormone.

Thyroid hormone induces tumor cell and blood vessel cell proliferation via a cell surface receptor on heterodimeric integrin αvß3. We investigated the role of thyroid hormone-induced internalization of nuclear integrin αv monomer. Physiological concentration of thyroxine (free T4, 10(-10) M), but not 3,5,3'-triiodo-l-thyronine (T3), induced cellular internalization and nuclear translocation of integrin αv monomer in human non-small-cell lung cancer (H522) and ovarian carcinoma (OVCAR-3) cells. T4 did not complex with integrin αv monomer during its internalization. The αv monomer was phosphorylated by activated ERK1/2 when it heterodimerized with integrin ß3 in vitro. Nuclear αv complexed with transcriptional coactivator proteins, p300 and STAT1, and with corepressor proteins, NCoR and SMRT. Nuclear αv monomer in T4-exposed cells, but not integrin ß3, bound to promoters of specific genes that have important roles in cancer cells, including estrogen receptor-α, cyclooxygenase-2, hypoxia-inducible factor-1α, and thyroid hormone receptor ß1 in chromatin immunoprecipitation assay. In summary, monomeric αv is a novel coactivator regulated from the cell surface by thyroid hormone for the expression of genes involved in tumorigenesis and angiogenesis. This study also offers a mechanism for modulation of gene expression by thyroid hormone that is adjunctive to the nuclear hormone receptor (TR)-T3 pathway.

Regulation of endodermal differentiation of human embryonic stem cells through integrin-ECM interactions.

Many cellular responses during development are regulated by interactions between integrin receptors and extracellular matrix proteins (ECMPs). Although the majority of recent studies in human embryonic stem cell (hESC) differentiation have focused on the role of growth factors, such as FGF, TGFß, and WNT, relatively little is known about the role of ECMP-integrin signaling in this process. Moreover, current strategies to direct hESC differentiation into various lineages are inefficient and have yet to produce functionally mature cells in vitro. This suggests that additional factors, such as ECMPs, are required for the efficient differentiation of hESCs. Using a high-throughput multifactorial cellular array technology, we investigated the effect of hundreds of ECMP combinations and concentrations on differentiation of several hPSC lines to definitive endoderm (DE), an early embryonic cell population fated to give rise to internal organs such as the lung, liver, pancreas, stomach, and intestine. From this screen we identified fibronectin (FN) and vitronectin (VTN) as ECMP components that promoted DE differentiation. Analysis of integrin expression revealed that differentiation toward DE led to an increase in FN-binding integrin α5 (ITGA5) and VTN-binding integrin αV (ITGAV). Conditional short hairpin RNA-mediated knockdown of ITGA5 and ITGAV disrupted hESC differentiation toward DE. Finally, fluorescence-based cell sorting for ITGA5 and ITGAV significantly enriched cells with gene expression signatures associated with DE, demonstrating that these cell surface proteins permit isolation and enrichment of DE from hESCs. These data provide evidence that FN and VTN promote endoderm differentiation of hESCs through interaction with ITGA5 and ITGAV, and that ECMP-integrin interactions are required for hESC differentiation into functionally mature cells.

Rv2468c, a novel Mycobacterium tuberculosis protein that costimulates human CD4+ T cells through VLA-5.

Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5ß1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.

Identification of immunoreactive regions of homology between soluble epidermal growth factor receptor and α5-integrin.

Proteins encoded by the epidermal growth factor receptor (EGFR/HER1/ERBB1) gene are being studied as diagnostic, prognostic, and theragnostic biomarkers for numerous human cancers. The clinical application of these tissue/tumor biomarkers has been limited, in part, by discordant results observed for epidermal growth factor receptor (EGFR) expression using different immunological reagents. Previous studies have used EGFR-directed antibodies that cannot distinguish between full-length and soluble EGFR (sEGFR) expression. We have generated and characterized an anti-sEGFR polyclonal antiserum directed against a 31-mer peptide (residues 604-634) located within the unique 78-amino acid carboxy-terminal sequence of sEGFR. Here, we use this antibody to demonstrate that sEGFR is coexpressed with EGFR in a number of carcinoma-derived cell lines. In addition, we show that a second protein of ~140 kDa (p140) also is detected by this antibody. Rigorous biochemical characterization identifies this second protein to be α5-integrin. We show that a 26-amino acid peptide in the calf domain of α5-integrin (residues 710-735) is 35% identical in sequence with a 31-mer carboxy-terminal sEGFR peptide and exhibits an approximately 5-fold lower affinity for anti-sEGFR than the homologous 31-mer sEGFR peptide does. We conclude that the carboxy terminus of sEGFR and the calf-1 domain of α5-integrin share a region of sequence identity, which results in their mutual immunological reactivity with anti-sEGFR. We also demonstrate that anti-sEGFR promotes three-dimensional tissue cohesion and compaction in vitro, further suggesting a functional link between sEGFR and α5-integrin and a role of the calf-1 domain in cell adhesion. These results have implications for the study of both EGFR and sEGFR as cancer biomarkers and also provide new insight into the mechanisms of interaction between cell surface EGFR isoforms and integrins in complex processes such as cell adhesion and survival signaling.

Characterization of glycoprotein E C-end of West Nile virus and evaluation of its interaction force with alphaVbeta3 integrin as putative cellular receptor.

Recombinant polypeptide containing the 260-466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E(260-466)) was constructed. Immunochemical similarity between the E(260-466) and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E(260-466). Polypeptide E(260-466) induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E(260-466) with one of the proposed cell receptors of WNV that average E(260-466)-alphaVbeta3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and alphaVbeta3 integrin) during virus entry.

Concurrent suppression of integrin alpha5, radixin, and RhoA phenocopies the effects of miR-31 on metastasis.

miR-31 inhibits breast cancer metastasis via the pleiotropic suppression of a cohort of prometastatic target genes that include integrin alpha(5) (ITGA5), radixin (RDX), and RhoA. We previously showed that the concomitant overexpression of ITGA5, RDX, and RhoA was capable of overriding the antimetastatic effects of ectopically expressed miR-31 in vivo. However, these prior studies failed to investigate whether the combined suppression of the endogenous mRNAs encoding these three proteins recapitulated the in vivo consequences of miR-31 expression on metastasis. We show here that short hairpin RNA-mediated concurrent downregulation of ITGA5, RDX, and RhoA is sufficient to phenocopy the full spectrum of described influences of miR-31 on metastasis in vivo, including the effects of this microRNA (miRNA) on local invasion, early post-intravasation events, and metastatic colonization. These findings provide mechanistic insights into the metastatic process and have implications about the importance of pleiotropy for the biological actions of miRNAs.

Role of ß5-integrin in epithelial-mesenchymal transition in response to TGF-ß.

Epithelial-mesenchymal-transition (EMT) in response to TGFß contributes to normal development, wound healing and tumor progression. The present study provides evidence for a critical role of ß5-integrin in the TGFß-induced EMT and the tumorigenic potential of carcinoma cells. We show that the αvß-integrin subunits are upregulated during the TGFß-induced EMT and this response requires Smad transcription factors. Depletion of αv-integrin by siRNA blocked the EMT response whereas knock-down of ß1-integrin had no effect. Importantly, depletion of ß5-integrin blocked the TGFß-induced EMT impairing adhesion to cell-matrix and integrin signaling, but did not change expression of E-cadherin and TGFß-target genes. Accordingly, the EMT process and integrin signaling were blocked by cRGD peptide interfering with cell-matrix adhesion or by inhibition of focal adhesion kinase, indicating the importance of ß5-integrin-mediated adhesions in EMT. Finally, depletion of ß5-integrin significantly reduced invasiveness of breast carcinoma cells. Thus, the ß5-integrin adhesions contribute to the TGFß-induced EMT and the tumorigenic potential of carcinoma cells.

Cbl-mediated ubiquitination of alpha5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation.

Fibroblast growth factor receptor signaling is an important mechanism regulating osteoblast function. To gain an insight into the regulatory role of FGF receptor-2 (FGFR2) signaling in osteoblasts, we investigated integrin-mediated attachment and cell survival in human calvarial osteoblasts expressing activated FGFR2. FGFR2 activation reduced osteoblast attachment on fibronectin. This was associated with reduced expression of the alpha5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restored alpha5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis showed that alpha5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that alpha5 integrin colocalizes with FGFR2 and Cbl at the leading edge in membrane ruffle regions. Transfection with the 70Z-Cbl mutant lacking the RING domain required for Cbl-ubiquitin interaction, or with the G306E Cbl mutant that abolishes the binding ability of Cbl phosphotyrosine-binding domain restored alpha5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in alpha5 integrin proteasome degradation induced by FGFR2 activation. Reduced alpha5 integrin expression was associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of alpha5 integrin rescued cell attachment and corrected both the Bax/Bcl-2 ratio and caspase-3 and caspase-9 activities in FGFR2 mutant osteoblasts. We show that Cbl recruitment induced by FGFR2 activation triggers alpha5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the alpha5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation in osteoblasts, and reveals that a Cbl-dependent mechanism is involved in the coordinated regulation of cell apoptosis induced by alpha5 integrin degradation.

Interaction of human fibrinogen receptor (GPIIb-IIIa) with decorsin.

AIM: To build up the structure of human fibrinogen receptor GPIIb-IIIa, subsequently combined with its antagonist decorsin, and to investigate the interaction between decorsin and its receptor GPIIb-IIIa at the molecular level. METHODS: A three-dimensional (3D) molecular model of human fibrinogen receptor GPIIb-IIIa was generated by InsightII, a distance geometry-based homologous modeling package. The structure of human fibrinogen receptor GPIIb-IIIa was built by the InsightII/Homology module using the corresponding of integrin alphaVbeta3 (PDB filecode 1JV2) as the template. Then the primary structures were optimized by energy minimization. Subsequently the structural model was docked with its antagonist decorsin (PDB filecode 1dec). RESULTS: A good substrate-receptor interaction model was achieved. The interaction sites with decorsin converge at domain 8 (betaA domain of beta3 subunit) of GPIIb-IIIa. The direct interatomic contacts were made between 16 GPIIb/IIIa residues and 10 decorsin amino-acid residues. These included van der Waals contacts, electrostatic interaction, hydrogen bond, and salt bridge. Residues in contact were concentrated in four dispersed regions of human GPIIb-IIIa: the RGD reaction motif (118-132 of GPIIIa), the span from 210 to 213 of GPIIIa, Thr182 residue and Asp251 residue of GPIIIa; and they were distributed over five segments of decorsin: Asp10 residue, Asn18 and Lys19 residues, Arg28 residue, RGD motif, and Asp35-Pro36-Tyr37 segment. CONCLUSION: This complex model plays an important role in development and research of some new drugs, especially a new guided fusion-type fibrinogen receptor antagonist.

A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin.

In a previous study [Alahari, Lee and Juliano (2000) J. Cell Biol. 151, 1141-1154], we have identified a novel protein, nischarin, that specifically interacts with the cytoplasmic tail of the alpha5 integrin subunit. Overexpression of this protein profoundly affects cell migration. To examine the nischarin-alpha5 interaction in detail, and to find the minimal region required for the interaction, several mutants of nischarin and alpha5 were created. The results obtained for the yeast two-hybrid system indicate that a 99-aminoacid region of nischarin (from residues 464 to 562) is indispensable for the interaction. Also, we demonstrate that the membrane proximal region (from residues 1017 to 1030) of the alpha5 cytoplasmic tail is essential for the interaction. To characterize more directly the properties of the interaction between nischarin and alpha5, we performed surface-plasmon resonance studies in which peptides were immobilized on the surface of a sensor chip, and the recombinant nischarin protein fragments were injected. Consistent with the two-hybrid results, recombinant nischarin binds well to immobilized alpha5 peptides. In addition, mutational analysis revealed that residues Tyr(1018) and Lys(1022) are crucial for alpha5-nischarin interactions. These results provide evidence that nischarin is capable of directly and selectively binding to a portion of the alpha5 cytoplasmic domain. Further studies demonstrated that the minimal alpha5 binding region of nischarin does not affect cell migration.

Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V.

The expressions of integrin alpha5, beta1, and alpha6 were studied in H7721 cells by means of flow cytometric and RT-PCR method after transfected with sense and antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V). The transfected cells were characterized by Northern blot. It was found that the order of expression from high to low was beta1>alpha5>alpha6. Transfection of sense GnT-V up-regulated alpha5 and alpha6, but not beta1 subunit, while antisense GnT-V down-regulated alpha5 and beta1, but not alpha6. The alterations of surface integrin subunits were quite compatible with the changes of their mRNAs. Using enzyme-labeled lectin analysis, it was shown that alpha5 subunit contained only C(2)C(2) biantennary N-glycan, which was not regulated by sense and antisense GnT-V. In contrast, beta1 subunit contained both biantennary and tri-/tetra-antennary N-glycans with GlcNAcbeta1,6Manalpha1,6-branch, and the latter was up- and down-regulated by the sense and antisense GnT-V, respectively. Therefore, the amount of biantennary N-glycans on beta1 subunit, but not the integrin protein, was correlated to the cell adhesion to fibronectin and laminin, which was reduced and elevated in the sense and antisense GnT-V-transfected cells, respectively, as we previously reported.

Structure of an integrin-ligand complex deduced from solution x-ray scattering and site-directed mutagenesis.

The structural basis of the interaction of integrin heterodimers with their physiological ligands is poorly understood. We have used solution x-ray scattering to visualize the head region of integrin alpha 5 beta 1 in an inactive (Ca2+-occupied) state, and in complex with a fragment of fibronectin containing the RGD and synergy recognition sequences. Shape reconstructions of the data have been interpreted in terms of appropriate molecular models. The scattering data suggest that the head region undergoes no gross conformational changes upon ligand binding but do lend support to a proposed outward movement of the hybrid domain in the beta subunit. Fibronectin is observed to bind across the top of the head region, which contains an alpha subunit beta-propeller and a beta subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the beta-propeller domain. In support of this suggestion, mutagenesis of a prominent loop region on the side of the propeller identifies two residues (Tyr208 and Ile210) involved in recognition of the synergy region. Our data provide the first view of a complex between an integrin and a macromolecular ligand in solution, at a nominal resolution of approximately 10 A.

Differential regulation of cell adhesive functions by integrin alpha subunit cytoplasmic tails in vivo.

Cell adhesion to fibronectin (FN) is crucial for early vertebrate morphogenesis. In Xenopus gastrulae, several distinct integrin-dependent adhesive behaviors can be identified: adhesion of cells to FN, assembly of FN fibrils, and initiation of cell spreading and migration in response to mesoderm inducing signals. We have taken a chimeric integrin approach to investigate the role of the integrin alpha cytoplasmic tail in the specification of these developmentally significant adhesive functions. Cytoplasmic tail-deleted alpha4 constructs and alpha4-ectodomain/alpha-cytoplasmic tail chimeras were generated and expressed in whole embryos. Normal gastrula cells lack integrin alpha4 and, correspondingly, are unable to adhere to the alpha4 ligand, the V-region of FN. The ability of alpha4 constructs to promote adhesive behaviors was established by placing tissue explants or dissociated cells on an FN V-region fusion protein that lacks the RGD (Arg-Gly-Asp)/synergy sites or treating whole embryos with antibodies that block endogenous integrin-FN interactions. We found that each alpha4 cytoplasmic domain deletion mutant and alpha-tail chimera examined could support cell attachment; however, activin induction-dependent cell spreading, mesoderm cell and explant motility, and the ability to assemble FN matrix on the blastocoel roof varied with specific alpha subunit tail sequences. These data suggest that alpha cytoplasmic tail signaling and changes in integrin activation state can regulate a variety of developmentally significant adhesive behaviors in both space and time.