Calcium-dependent self-association of the C-type lectin domain of versican

Versican is a large (1-2 x 10(6) Da) chondroitin-sulfate proteoglycan that can form large aggregates by means of interaction with hyaluronan and also binds to a series of other extracellular matrix proteins, chemokines and cell-surface molecules. Versican is a multifunctional molecule with roles in cell adhesion, matrix assembly, cell migration and proliferation. Characterization of the binding interactions mediated by the various domains of versican is a first step towards understanding the functions of versican and interacting molecules in the extracellular matrix. In this study we investigated a recombinant construct corresponding to the C-type lectin domain of versican and demonstrated a calcium-dependent self-association of this region by blot overlay and plasmon surface resonance assays. Electron microscopy provided further evidence of the relevance of the binding reaction by demonstrating a mixture of monomers, dimers and complex aggregates of recombinant versican C-type lectin domain. This binding reaction could contribute to the ability of versican to organize formation of the proteoglycan extracellular matrix by inducing binding of individual versican molecules or by modulating binding reactions to other matrix components.     

Versican protects cells from oxidative stress-induced apoptosis.

Oxidant injury plays a critical role in the degenerative changes that are characterized by a decline in parenchymal cell numbers and viability, and occur with aging and in the etiology of many diseases. The extracellular proteoglycan versican is widely distributed in the extracellular matrix surrounding the cells. This study examines whether versican plays a role in protecting cells from free radical-induced apoptosis. Stable expression of versican or its C-terminal domain significantly decreased H(2)O(2)-induced cellular apoptosis. Cells in adherent monolayer were more resistant to H(2)O(2)-induced apoptosis than cells cultured in suspension. While vigorous trypsinization caused integrin cleavage and rendered the cells more susceptible to H(2)O(2)-induced damages, expression of versican or its C-terminal domain enhanced cell attachment and expression of beta1 integrin and fibronectin. Enhanced cell-matrix interaction by addition of manganese (MnCl(2)) to cultures also significantly diminished H(2)O(2)-induced apoptosis. The results suggest that versican plays an important role in reducing oxidant injury through an enhancement of cell-matrix interaction.     

G3 domains of aggrecan and PG-M/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction

The extracellular matrix plays a critical role in maintaining tissue integrity. Among the matrix molecules, the large aggregating chondroitin sulfate proteoglycans are the major structural molecules and are the primary contributors to the stability for some tissues such as cartilage. The notable exceptions are nanomelic cartilage and arthritic cartilage: the former contains a point mutation leading to a stop codon before translating to the C-terminal G3 domain; the latter contains a large proportion of aggrecan from which the G3 domain has been cleaved. These phenomena suggest that the G3 domain may be important in cartilage stability. Here, we demonstrated for the first time that the G3 domains of aggrecan and another proteoglycan, PG-M/versican, formed intermolecular disulfide bonds, and all subdomains were involved. Further studies indicated that each of the 10 cysteine residues of the aggrecan G3 domain could potentially form intermolecular disulfide bonds in vitro. The disulfide bonds were disrupted in the presence of reducing reagent beta-mercaptoethanol and dithiothreitol. As a result, normal chondrocyte-matrix interaction was disrupted, and the structure of the extracellular matrix was altered. Furthermore, disruption of disulfide bonds also reduced the role of PG-M/versican G3 domain in mediating cell adhesion. Our study provides strong evidence of the importance of proteoglycan interactions through intermolecular disulfide bonds in cartilage firmness and cell-matrix stability.     

The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.     

Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.     

Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells

Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.     

The interacting binding domains of the beta(4) integrin and calcium-activated chloride channels (CLCAs) in metastasis

CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta4 integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta4 integrin. Endothelial CLCAs share a common beta4-binding motif (beta4BM) in their 90- and 35-kDa subunits of the sequence F(S/N)R(I/L/V)(S/T)S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta4BMs from the 90- and 35-kDa CLCA subunits bind to the beta4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta1 and beta3 served as negative controls. beta4BM fusion proteins competitively blocked the beta4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta4BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta4 integrin. The corresponding CLCA-binding domain of the beta4 integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta4 SDL (amino acids 184-203). A chimeric beta4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta4 activation and outside-in signaling is discussed in reference to our previous report that beta4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.     

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha5beta1 integrin (alpha5beta1), but not by one against anti-alphavbeta3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha5beta1 is involved in this adhesion.     

Phospholipase Cgamma binds alpha1beta1 integrin and modulates alpha1beta1 integrin-specific adhesion.

Integrin adhesion receptors have been implicated in bidirectional signal transduction. The dynamic regulation of integrin affinity and avidity as well as post-ligand effects involved in outside-in signaling depends on the interaction of integrins with cytoskeletal and signaling proteins. In this study, we attempted to identify cytoplasmic binding partners of alpha(1)beta(1) integrin. We were able to show that cell adhesion to alpha(1)beta(1)-specific substrates results in the association of phospholipase Cgamma (PLCgamma) with the alpha(1)beta(1) integrin independent of PLCgamma tyrosine phosphorylation. Using peptide-binding assays, the membrane proximal sequences within the alpha(1)beta(1) integrin subunits were identified as binding sites for PLCgamma. In particular, the conserved sequence of beta(1) subunit binds the enzyme very efficiently. Because purified PLCgamma also binds the integrin peptides, binding seems to be direct. Inhibition of PLC by leads to reduced cell adhesion on alpha(1)beta(1)-specific substrates. Cells lacking the conserved domain of the alpha(1) subunit fail to respond to the PLC inhibition, indicating that this domain is necessary for PLC-dependent adhesion modulation of alpha(1)beta(1) integrin.     

14-3-3 regulation of cell spreading and migration requires a functional amphipathic groove

The 14-3-3 proteins associate with many cellular proteins that participate in the regulation of various cellular events including apoptosis, the cell cycle, spreading, and migration. We have previously described that 14-3-3beta binds the beta1-integrin and overexpression of 14-3-3beta promoted increased cell spreading and migration (Han et al. [2001] Oncogene 20: 346-357). In this study, we find that mutation of Ser 60 of 14-3-3beta, outside of the amphipathic groove which is involved in 14-3-3 protein interactions with other ligands, abolished its interaction with integrin. Surprisingly, this mutant retained its ability to promote cell spreading, suggesting that 14-3-3beta interaction with the beta1-integrin is not required for its regulation of cell adhesion. We next showed that mutations of several critical residues in the amphipathic groove did not affect 14-3-3beta interaction with the beta1-integrin. As expected, these mutants disrupted their association with the phosphoserine dependent ligands Raf and Cas. Analysis of the groove mutant LF (mutation of Arg129Tyr130 to Leu and Phe) indicated that, unlike wild type 14-3-3beta, it could not stimulate cell spreading or migration, suggesting that a functional amphipathic groove is required for 14-3-3 regulation of cell adhesion and migration. Consistent with this, cells expressing the LF mutant exhibited a delay in F-actin organization compared to cells expressing wild type or the S60A mutant (Ser 60 to Ala mutation) upon cell adhesion to fibronectin (FN). Taken together, these studies identified a novel binding site on 14-3-3 for integrin beta1 and showed that a functional amphipathic groove, rather than its interaction with integrin beta1, is required for 14-3-3 regulation of cell spreading and migration.     

Alpha L-integrin I domain cyclic peptide antagonist selectively inhibits T cell adhesion to pancreatic islet microvascular endothelium

Insulitis is a hallmark feature of autoimmune diabetes that ultimately results in islet beta-cell destruction. We examined integrin requirements and specific inhibition of integrin structure in T cell and monocyte adhesion to pancreatic islet endothelium. Examination of cell surface integrin expression on WEHI 7.1 T cells revealed prominent expression of beta-, beta(1)-, alpha(L)-integrins, and low expression of alpha(M)-integrins; whereas WEHI 274.1 monocytes showed significant staining for beta(2)-, beta(1)-, alpha(M)-molecules and no expression of alpha(L)-molecules. Unstimulated islet endothelium showed constitutive levels of ICAM-1 counter-ligand expression with minimal VCAM-1 expression; however, TNF-alpha stimulation increased cell surface density of both molecules. TNF-alpha increased T cell and monocyte rolling and adhesion under hydrodynamic flow conditions. Administration of a cyclic peptide competitor for the alpha(L)-integrin I domain binding sites (cyclo1,12-PenITDGEATDSGC) blocked T cell adhesion without inhibiting monocyte adhesion. Examination of T cell rolling revealed that cLAB.L treatment increased the average rolling velocity on activated endothelium and significantly decreased the fraction of T cells rolling at < or =50 microm/s, suggesting that cLAB.L treatment interferes with signal activation events required for the conversion of T cell rolling to firm adhesion. These data demonstrate for the first time that cyclic peptide antagonists against alpha(L)-integrin I domain attenuate T cell recruitment to islet endothelium.     

Intestinal epithelial CD98: an oligomeric and multifunctional protein

The intestinal epithelial cell-surface molecule, CD98 is a type II membrane glycoprotein. Molecular orientation studies have demonstrated that the C-terminal tail of human CD98 (hCD98), which contains a PDZ-binding domain, is extracellular. In intestinal epithelial cells, CD98 is covalently linked to an amino-acid transporter with which it forms a heterodimer. This heterodimer associates with beta(1)-integrin and intercellular adhesion molecular 1 (ICAM-1) to form a macromolecular complex in the basolateral membranes of polarized intestinal epithelial cells. This review focuses on the multifunctional roles of CD98, including involvement in extracellular signaling, adhesion/polarity, and amino-acid transporter expression in intestinal epithelia. A role for CD98 in intestinal inflammation, such as Intestinal Bowel Disease (IBD), is also proposed.     

An interaction between alpha-actinin and the beta 1 integrin subunit in vitro

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.     

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.     

C-terminal modification of osteopontin inhibits interaction with the αVβ3-integrin

Osteopontin (OPN) is a multifunctional phosphorylated protein containing the integrin binding sequence Arg-Gly-Asp through which it interacts with several integrin receptors, such as the α(V)β(3)-integrin. OPN exists in many different isoforms differing in phosphorylation status that are likely to interact differently with integrins. The C-terminal region of OPN is particularly well conserved among mammalian species, which suggests an important functional role of this region. In this study, we show that modification of the extreme C terminus of OPN plays an important regulatory role for the interaction with the α(V)β(3)-integrin. It is demonstrated that highly phosphorylated OPN has a much reduced capability to promote cell adhesion via the α(V)β(3)-integrin compared with lesser phosphorylated forms. The cell attachment promoted by highly phosphorylated OPN could be greatly increased by both dephosphorylation and proteolytic removal of the C terminus. Using recombinantly expressed OPN containing a tag in the N or C terminus, it is shown that a modification in the C-terminal part significantly reduces the adhesion of cells to OPN via the α(V)β(3)-integrin, whereas modification of the N terminus does not influence the binding. The inhibited binding of the α(V)β(3)-integrin to OPN could be restored by proteolytic removal of the C terminus by thrombin and plasmin. These data illustrate a novel mechanism regulating the interaction of OPN and the α(V)β(3)-integrin by modification of the highly conserved C-terminal region of the protein.     

Critical role of the fifth domain of E-cadherin for heterophilic adhesion with alpha E beta 7, but not for homophilic adhesion

The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.     

Retinoic acid upregulates beta(1)-integrin in vascular smooth muscle cells and alters adhesion to fibronectin

Retinoic acid has an established physiological role in differentiation, development, and cellular growth. This study investigated the action of all-trans retinoic acid (ATRA) on vascular integrins, cell-surface receptors that control growth and remodeling of blood vessels. The beta(1)-integrin subunit mRNA and protein was induced after treatment with ATRA in two different rat vascular smooth muscle cell lines. To relate this result to the in vivo state, the aortas from adult rats fed with therapeutic doses of ATRA were examined for beta(1)-integrin protein. A significant upregulation of the integrin subunit was observed in vivo. To assess if this increase contributed to physiological changes in cellular function, cells treated with ATRA were tested for alterations in adhesion to extracellular matrix proteins. The cells exposed to the retinoid were seen to adhere more strongly to fibronectin, via the beta(1)-integrin. These results showed that modulation of vascular integrins by ATRA in adult rats contributes to functional changes that can cause remodeling of blood vessels.     

Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry

Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with alphaV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin alphaVbeta5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin alpha5beta1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind alpha5beta1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a "click-to-fit" mechanism that involves the cooperative binding of heparan sulfate and alpha5beta1 integrin by the AAV2 capsids.