A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor alpha2 subunit mRNA expression in both unstimulated or platelet-derived growth factor-stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239-249). Here we report that the collagen gel can signal protein kinase C (PKC)-zeta activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-zeta immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-zeta protein levels or intracellular location was observed. DNA binding activity of nuclear factor kappaB (NF-kappaB), a downstream regulatory target of PKC-zeta, was also increased by fibroblasts grown in collagen gel. The composition of the NF-kappaB/Rel complexes that contained p50, was not changed. The potential role of PKC-zeta in collagen gel-induced mRNA expression of collagen receptor alpha2 subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of alpha2 and MMP-1 mRNA in collagen gel-stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)-inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5' end of PKC-zeta mRNA sequences significantly reduced the collagen lattice-stimulated alpha2 and MMP-1 mRNA levels. Taken together, these data indicate that PKC-zeta, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for alpha2 and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-zeta.

Normal adult human dermal fibroblasts grown in a three-dimensional collagen lattice increase mRNA level of collagen receptor integrin subunit alpha2 (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239- 249.) and DNA binding activity of a nuclear transcription factor, NF-kappaB (Xu, J., and R.A.F. Clark. 1997. J. Cell Biol. 136:473-483.). Here we present evidence that the collagen lattice induced the nuclear translocation of p50, one member of NF-kappaB family, and the degradation of an NF-kappaB inhibitor protein, IkappaB-alpha. The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice. A region located between -549 and -351 bp in the promoter of integrin alpha2 gene conferred the inducibility by three-dimensional collagen lattice. The presence of either SN50 or IkappaB-alpha32, 36, a stable mutant of IkappaB-alpha, abrogated this inducibility, indicating that the activation of integrin alpha2 gene expression was possibly mediated by NF-kappaB through this region. Although there were three DNA-protein binding complexes forming in this region that are sensitive to the inhibition of NF-kappaB nuclear translocation, NF-kappaB was not directly present in the binding complexes. Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested. The involvement of NF-kappaB in reorganization and contraction of three-dimensional collagen lattice, a process that requires the presence of abundant integrin alpha2beta1, was also examined. The inhibition of NF-kappaB activity by SN50 greatly blocked the contraction, suggesting its critical role in not only the induction of integrin alpha2 gene expression by three-dimensional collagen lattice, but also alpha2beta1-mediated tissue-remodeling process.

Erianin has been reported to inhibit tumor activity by suppressing the expression of integrins. It is hypothesized that erianin can inhibit retinal neovascularization in collagen by suppressing the expression of integrins. With an aim to test this hypothesis, the regulation of erianin on collagen-mediated retinal angiogenesis via the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway induced by α2 and β1 integrin-collagen interactions was investigated.

Two collagen receptors, integrins alpha1beta1 and alpha2beta1, can regulate distinct functions in cells. Ligation of alpha1beta1, unlike alpha2beta1, has been shown to result in recruitment of Shc and activation of the Ras/ERK pathway. To identify the downstream signaling molecules activated by alpha2beta1 integrin, we have overexpressed wild-type alpha2, or chimeric alpha2 subunit with alpha1 integrin cytoplasmic domain in human osteosarcoma cells (Saos-2) lacking endogenous alpha2beta1. The chimeric alpha2/alpha1 chain formed a functional heterodimer with beta1. In contrast to alpha2/alpha1 chimera, forced expression of alpha2 integrin resulted in upregulation of alpha1 (I) collagen gene transcription in response to three-dimensional collagen, indicating that the cytoplasmic domain of alpha2 integrin was required for signaling. Furthermore, signals mediated by alpha2beta1 integrin specifically activated the p38alpha isoform, and selective p38 inhibitors blocked upregulation of collagen gene transcription. Dominant negative mutants of Cdc42, MKK3, and MKK4 prevented alpha2beta1 integrin-mediated activation of p38alpha. RhoA had also some inhibitory effect, whereas dominant negative Rac was not effective. Our findings show the isoform-specific activation of p38 by alpha2beta1 integrin ligation and identify Cdc42, MKK3, and MKK4 as possible downstream effectors. These observations reveal a novel signaling mechanism of alpha2beta1 integrin that is distinct from ones previously described for other integrins.

In patients treated for prostate cancer (PCa) with radical prostatectomy (RP), determining the risk of extraprostatic extension (EPE) and nodal involvement (NI) remains crucial for planning nerve-sparing and extended lymphadenectomy. The study aimed to determine proteins that could serve as immunohistochemical markers of locally advanced PCa. To select candidate proteins associated with adverse pathologic features (APF) reverse-phase protein array data of 498 patients was retrieved from The Cancer Genome Atlas. The analysis yielded 6 proteins which were then validated as predictors of APF utilizing immunohistochemistry in a randomly selected retrospective cohort of 53 patients. For univariate and multivariate analysis, logistic regression was used. Positive expression of TfR1 (OR 13.74; p = 0.015), reduced expression of CD49b (OR 10.15; p = 0.013), and PSA (OR 1.29; p = 0.013) constituted independent predictors of EPE, whereas reduced expression of e-cadherin (OR 10.22; p = 0.005), reduced expression of CD49b (OR 24.44; p = 0.017), and PSA (OR 1.18; p = 0.002) were independently associated with NI. Both models achieved high discrimination (AUROC 0.879 and 0.888, respectively). Immunohistochemistry constitutes a straightforward tool that might be easily utilized before RP. Expression of TfR1 and CD49b is associated with EPE, whereas expression of e-cadherin and CD49b is associated with NI. Since following immunohistochemical markers predicts respective APFs independently from PSA, in the future they might supplement existing preoperative nomograms or be implemented in novel tools.

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the alpha chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the alpha, beta, and gamma chains. We have determined the crystal structure at 2.8-A resolution of the LG1-3 region of the laminin alpha2 chain (alpha 2LG1-3). The three LG domains adopt typical beta-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing gamma chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the beta-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin alpha chains, suggesting that these surfaces may harbor the integrin binding site. The alpha 2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.

Mutations in laminin-alpha2 cause a severe congenital muscular dystrophy, called MDC1A. The two main receptors that interact with laminin-alpha2 are dystroglycan and alpha7beta1 integrin. We have previously shown in mouse models for MDC1A that muscle-specific overexpression of a miniaturized form of agrin (mini-agrin), which binds to dystroglycan but not to alpha7beta1 integrin, substantially ameliorates the disease (Moll, J., P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, and M.A. Ruegg. 2001. Nature. 413:302-307; Bentzinger, C.F., P. Barzaghi, S. Lin, and M.A. Ruegg. 2005. Matrix Biol. 24:326-332.). Now we show that late-onset expression of mini-agrin still prolongs life span and improves overall health, although not to the same extent as early expression. Furthermore, a chimeric protein containing the dystroglycan-binding domain of perlecan has the same activities as mini-agrin in ameliorating the disease. Finally, expression of full-length agrin also slows down the disease. These experiments are conceptual proof that linking the basement membrane to dystroglycan by specifically designed molecules or by endogenous ligands, could be a means to counteract MDC1A at a progressed stage of the disease, and thus opens new possibilities for the development of treatment options for this muscular dystrophy.

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in alpha2beta1. Furthermore, the mutation E336A did neither prevent EV1 induced and alpha2beta1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on alpha2beta1 clustering, but do not require the conformational regulation of the receptor.

Clathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins beta1, alpha1, alpha2, and alpha3 but not alpha5 or alphav chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin beta1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge. By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg2+ or Mn2+ but not Ca2+. Adhesion was partially inhibited by an antibody recognizing beta1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The beta1 integrin antibody immunoprecipitated two proteins with molecular mass approximately 110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a beta1 integrin on chondrocytes interacts with chondroadherin. To identify the alpha integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified alpha2beta1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit alpha2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since alpha2beta1 also is a receptor for collagen type II, we tested the ability of different antibodies against the alpha2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the alpha2beta1 integrin. Although alpha2beta1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.

During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin alpha2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects.

We earlier found that a rat monoclonal antibody (mAb) RE2 can induce rapid death of murine activated, but not resting, lymphocytes and lymphocyte cell lines, in a complement-independent manner, a cell death differing from typical apoptosis or necrosis. We here found that this cell death is independent of pathways involving Fas, caspase, and phosphoinositide-3 kinase. With the advantage of producing human B cell line transfectants with stable expression of human/mouse xeno-chimeric MHC class I genes, we found that RE2 epitope resides on the murine class I alpha2 domain. However, the alpha3 domain plays a key role in transducing the death signal, which mediates extensive aggregation of the MHC class I-integrin-actin filament system, giving rise to membrane blebs and pores. In mouse models with T/NKT cell activation-associated fulminant hepatitis, administration of mAb RE2 almost completely inhibited the development of liver cell injuries. Taken collectively, this form of cell death may be involved in homeostatic immune regulation, and induction of this form of cell death using the mAbs may be potentially therapeutic for subjects with immunological diseases mediated by activated lymphocytes.

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin beta1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin beta1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin beta1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin beta1, alpha4 and alpha6 or beta1, alpha6, alpha2, and alpha5, respectively.

During human prostate cancer progression, the integrin alpha6beta1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. We previously identified a novel structural variant of the alpha6 integrin called alpha6p. This variant was produced on the cell surface and was missing the beta-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of alpha6p. We also showed that addition of uPA in the culture media of cells that do not produce alpha6p, resulted in a dose-dependent alpha6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using alpha2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly. Further, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not alpha2-antiplasmin. Finally, the alpha6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the beta-barrel ligand-binding domain of the integrin while preserving its heterodimer association.

Integrin alpha 2 (ITGA2) promotes cancer metastasis through selective adhesion to ECM proteins; however, the specific contribution of integrin glycosylation remains uncertain. We provide evidence that ITGA2 is a highly glycosylated transmembrane protein expressed in ovarian cancer tissue and cell lines. In-depth glycoproteomics identified predominant N- and O-glycosylation sites harboring substantially divergent ITGA2 glycosylation profiles. Generated putative ITGA2 N-glycosite mutants halted collagen and laminin binding and cells lacking N-glycosylated ITGA2 were marginally adherent to collagen, likely associated with its enhanced proteasome degradation through poly-ubiquitination. Proteomic and enrichment pathway analysis revealed increased cellular apoptosis and collagen organization in non-glycosylated ITGA2 mutant cells. Moreover, we provide evidence that ITGA2-specific sialylation is involved in selective cell-ECM binding. These results highlight the importance of glycans in regulating ITGA2 stability and ligand binding capacity which in turn modulates downstream focal adhesion and promotes cell survival in a collagen environment.

The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.

Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2β1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10(-10) M and 1.5×10(-10) M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2β1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2β1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy.

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.