The Use of 3-D Culture in Peptide Hydrogel for Analysis of Discoidin Domain Receptor 1-Collagen Interaction

The aim of this study is to examine a novel drop culture model using a biologically inspired self-assembling peptide: hydrogel (RAD16-I, also called PuraMatrix), which produces a nanoscale environment similar to native extracellular matrix (ECM) for a cell line weakly adherent to a plastic surface during cell culture. Our work investigates quantitatively analyzing discoidin domain receptor (DDR) 1-mediated protein interactions between collagen type I and matrix metalloproteinase (MMP)-2 or -9, as well as cell invasion, using, as a scaffold, PuraMatrix, a novel peptide hydrogel. Results demonstrate that the dynamic cell culture technique produced a highly stable reharvesting of cells throughout the constructs with HP-75, human pituitary adenoma cell line when compared to the traditional seeding methods. Secretion of MMP via collagen type I was observed quantitatively in the supernatant (EC50; MMP-2, 50.4 ng/ml: MMP-9, 57.6 ng/ml). In PuraMatrix gel impregnated with 50 ng/ml of collagen type I, transfection of the vector encoding full-length DDR1 or siRNA targeting DDR1 up- or downregulated respectively secretion of MMP-2 and -9, and cell invasion. Our results show that incorporation of this peptide with each ECM component provides a more permissive environment to elucidate ECM to cell signal interaction.Key Words: biological scaffolds, cell invasion, ionic self-complementary peptides, nano-fiber hydrogels, pituitary adenoma     

A putative role for Discoidin Domain Receptor 1 in cancer chemoresistance

ABSTRACT

The Discoidin Domain Receptor 1 (DDR1) receptor tyrosine kinase performs pleiotropic functions in the control of cell adhesion, proliferation, survival, migration, and invasion. Aberrant DDR1 function as a consequence of either mutations or increased expression has been associated with various human diseases including cancer. Pharmacological inhibition of DDR1 results in significant therapeutic benefit in several pre-clinical cancer models. Here, we discuss the potential implication of DDR1-dependent pro-survival functions in the development of cancer resistance to chemotherapeutic regimens and speculate on the molecular mechanisms that might mediate such important feature.     

Interaction of Discoidin Domain Receptor 1 with a 14-3-3-Beclin-1-Akt1 Complex Modulates Glioblastoma Therapy Sensitivity

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Discoidin Domain Receptor 1 Contributes to Tumorigenesis through Modulation of TGFBI Expression

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.     

Discoidin Domain Receptor 1 Protein Is a Novel Modulator of Megakaryocyte-Collagen Interactions

Growing evidence demonstrates that extracellular matrices regulate many aspects of megakaryocyte (MK) development; however, among the different extracellular matrix receptors, integrin α2β1 and glycoprotein VI are the only collagen receptors studied in platelets and MKs. In this study, we demonstrate the expression of the novel collagen receptor discoidin domain receptor 1 (DDR1) by human MKs at both mRNA and protein levels and provide evidence of DDR1 involvement in the regulation of MK motility on type I collagen through a mechanism based on the activity of SHP1 phosphatase and spleen tyrosine kinase (Syk). Specifically, we demonstrated that inhibition of DDR1 binding to type I collagen, preserving the engagement of the other collagen receptors, glycoprotein VI, α2β1, and LAIR-1, determines a decrease in MK migration due to the reduction in SHP1 phosphatase activity and consequent increase in the phosphorylation level of its main substrate Syk. Consistently, inhibition of Syk activity restored MK migration on type I collagen. In conclusion, we report the expression and function of a novel collagen receptor on human MKs, and we point out that an increasing level of complexity is necessary to better understand MK-collagen interactions in the bone marrow environment.     

Discoidin Domain Receptors: Unique Receptor Tyrosine Kinases in Collagen-mediated Signaling

The discoidin domain receptors (DDRs) are receptor tyrosine kinases that recognize collagens as their ligands. DDRs display unique structural features and distinctive activation kinetics, which set them apart from other members of the kinase superfamily. DDRs regulate cell-collagen interactions in normal and pathological conditions and thus are emerging as major sensors of collagen matrices and potential novel therapeutic targets. New structural and biological information has shed light on the molecular mechanisms that regulate DDR signaling, turnover, and function. This minireview provides an overview of these areas of DDR research with the goal of fostering further investigation of these intriguing and unique receptors.     

Expression of Discoidin Domain Receptor 2 (DDR2) Extracellular Domain in Pichia Pastoris and Functional Analysis in Synovial Fibroblasts and NIT3T3 Cells

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained, After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II. Wei Zhang and Tianbing Ding equally contributed to this work.     

Interaction Between Umami Peptide and Taste Receptor T1R1/T1R3

The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. The homology models of the ligand binding domains of the human umami receptor have been constructed based on crystallographic structures of the taste receptor of the central nervous system. Furthermore, the molecular simulations of the ligand binding domain show that the likely conformation was that T1R1 protein exists in the closed conformation, and T1R3 in the open conformation in the heterodimer. The molecular docking study of T1R1 and T1R3 in complex with four peptides, including Lys–Gly–Asp–Glu–Ser–Leu–Leu–Ala, Ser–Glu–Glu, G1u–Ser, and Asp–Glu–Ser, displayed that the amino acid residue of SER146 and Glu277 in T1R3 may play great roles in the synergism of umami taste. This docking result further validated the robustness of the model. In the paper, binding of umami peptide and the T1R1/T1R3 receptor was first described and the interaction is the base of umami activity theory.     

Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn²¹¹ residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved ²¹¹NDS N-glycosylation motif, but not other N-glycosylation sites (Asn²⁶⁰, Asn³⁷¹, and Asn³⁹⁴), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr⁴⁸⁴, Tyr⁵²⁰, Tyr⁷⁹², and Tyr⁷⁹⁷. The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved ²¹¹NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.     

盘状结构域受体2胞外区的可溶性表达、纯化和功能鉴定

盘状结构域受体2（discoidin domain receptor 2,DDR2）是一种与肿瘤细胞转移相关的蛋白酷氨酸激酶,其配体为纤维性胶原,胶原对DDR2的活化上调细胞中基质金属蛋白酶1（MMP－1）的表达。为研究DDR2在类风温性关节炎(rteumatoid arthritis,RA）软骨破坏和肿瘤转移中的作用,尝试了在大肠杆菌中表达一段DDR2胞外区（命名DB）,并进行了可溶性部分的纯化和功能鉴定,以备将来用作DDR2的特异性阻断剂。获得了一株表达GST－DB融合蛋白的大肠杆菌克隆;其表达的蛋白质中可溶性部分约占全部融合蛋白的13％;经GST融合蛋白特异性亲和珠纯化后,获得了纯度约86.1%的可溶性GST－DB融合蛋白;竞争结合抑制实验显示,GST－DB具有阻断Ⅱ型胶原和细胞表面天然DDR2受体结合的功能;细胞实验表明,GST－DB有抑制Ⅱ型胶原刺激下的类风湿性关节炎滑膜细胞和NIH3T3细胞分泌MMP－1的作用。以上结果提示,表达的融保蛋白GST－DB具有抑制天然DDR2功能的作用;DDR2在滑膜细胞和NIH3T3细胞中介导Ⅱ型胶原刺激下的MMP－1的分泌。     

Structural Determinants for the Interaction of Formyl Peptide Receptor 2 with Peptide Ligands

Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-281^7.32 is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-281^7.32 might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D281^7.32G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides.     

核受体辅活化子PNRC与孤儿核受体SF1的相互作用有赖于SF1的AF-2功能结构域

核受体辅活化子PNRC(proline richnuclearreceptorcoregulatoryprotein ,富含脯氨酸的核受体辅调节蛋白 )可通过含SH3结合模体的PNRC2 78 30 0区域与孤儿核受体类固醇生成因子 1(steroido genicfactor 1,SF1)相互作用 .激活功能 2 (activationfunction 2 ,AF 2 )结构域在核受体配体依赖性转录激活中发挥了重要作用 ,为探讨AF 2结构域在SF1转录激活中的作用机制 ,采用酵母双杂合分析、缺失突变技术和瞬时转染等研究方法考察了AF 2结构域对SF1反式激活功能及SF1与PNRC相互作用的影响 .SF1的反式激活功能有赖于AF 2结构域 ,其机制是SF1AF 2结构域的突变严重影响了SF1与PNRC的有效相互作用 ,并消除了PNRC对SF1反式激活功能的辅激活作用 .结果表明 ,SF1与PNRC的相互作用有赖于AF 2的功能结构域     

小鼠前成骨细胞MC3T3-E1在自组装多肽水凝胶支架中的成骨分化

目的研究MC3T3-E1细胞在自组装多肽水凝胶支架上的生长和成骨分化.方法在多肽水凝胶支架RADA16上接种MC3T3-E1细胞,荧光染色观察细胞形态和存活情况;组织化学染色检测MC3T3-E1细胞碱性磷酸酶活性以及细胞外钙质沉积;RT-PCR分析成骨特异性基因的表达.结果 MC3T3-E1细胞在水凝胶支架RADA16上粘附铺展良好,呈纺锤样形态.诱导培养后支架上的细胞有较高水平的碱性磷酸酶表达和矿化基质沉积.此外,骨分化特异性基因骨桥蛋白和骨涎蛋白也有表达,且表达量随培养时间的延长而增多.结论 在自组装水凝胶内MC3T3-E1细胞可向成骨方向分化,并能在凝胶内产生矿化的细胞外基质.     

Live cell measurements of interaction forces and binding kinetics between Discoidin Domain Receptor 1 (DDR1) and collagen I with atomic force microscopy

BackgroundDiscoidin Domain Receptors (DDRs) are membrane-tethered proteins of the receptor tyrosine kinase family, which signal in response to collagen. DDR expression is associated with human diseases, including fibrosis and cancer. The role of DDRs in human pathogenesis is mediated by dysregulated receptor function in response to the collagenous milieu. Thus, understanding DDR-collagen interactions is important for developing novel therapeutic strategies against DDRs.MethodsWe developed a biophysical method to isolate and measure specific interactions between DDR1 and collagen in live cells at the single molecule level using atomic force microscopy. This new method is capable of providing density and kinetics of membrane receptors in live cells.ResultsWe isolated DDR1-collagen interactions and quantified the association and dissociation rates of the DDR1-collagen I complex. We estimated separate binding probabilities of collagen I to DDR and integrin, and by combining kinetic and binding probability data, we were able to estimate the density of receptors in two cancer cell types. We also tested the viability of a DDR1 blocking antibody and determined its efficacy in suppressing DDR1-collagen binding.ConclusionsThe new method shows promise in quantifying receptor-ligand kinetics and receptor density on live cells.General significanceThe new approach is applicable to other receptor-ligand systems and allows the determination of critical parameters at the single cell/single molecule level – in particular, the direct determination of kinetic and density differences of receptors in different cell types. This capability should prove to be useful in cancer research and drug design.     

Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links,Insertions, or Deletions in the Extracellular Juxtamembrane Region: MECHANISTIC IMPLICATIONS*

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.     

兔关节软骨细胞体外三维培养条件的优化

实验使用海藻酸钠水凝胶作为细胞支架.模拟软骨细胞体内生长的三维环境,研究了体外三维培养条件下,不同浓度的胎牛血清(FBS)和硒酸复合液(ITS)体系对兔透明软骨细胞(hyaline cartilage)的牛长、增殖和细胞外基质分泌活动的影响.结果 表明,三维模式培养21天,透明软骨细胞仍然具有较好的增殖活性.在硒酸复合液及低浓度血清时,细胞未去分化,保持分泌Ⅱ型胶原(Collagen Ⅱ)和软骨聚集蛋白聚糖(Aggrecan)的能力,与之比较,高浓度胎牛血清(10%)培养条件下,在21天开始细胞去分化,即硒酸复合液在一定的血清浓度下有助于维持软骨细胞生长、增殖,避免了软骨细胞受高浓度血清影响而去分化.     

3DIANA: 3D Domain Interaction Analysis: A Toolbox for Quaternary Structure Modeling

Electron microscopy (EM) is experiencing a revolution with the advent of a new generation of Direct Electron Detectors, enabling a broad range of large and flexible structures to be resolved well below 1 nm resolution. Although EM techniques are evolving to the point of directly obtaining structural data at near-atomic resolution, for many molecules the attainable resolution might not be enough to propose high-resolution structural models. However, accessing information on atomic coordinates is a necessary step toward a deeper understanding of the molecular mechanisms that allow proteins to perform specific tasks. For that reason, methods for the integration of EM three-dimensional maps with x-ray and NMR structural data are being developed, a modeling task that is normally referred to as fitting, resulting in the so called hybrid models. In this work, we present a novel application—3DIANA—specially targeted to those cases in which the EM map resolution is medium or low and additional experimental structural information is scarce or even lacking. In this way, 3DIANA statistically evaluates proposed/potential contacts between protein domains, presents a complete catalog of both structurally resolved and predicted interacting regions involving these domains and, finally, suggests structural templates to model the interaction between them. The evaluation of the proposed interactions is computed with DIMERO, a new method that scores physical binding sites based on the topology of protein interaction networks, which has recently shown the capability to increase by 200% the number of domain-domain interactions predicted in interactomes as compared to previous approaches. The new application displays the information at a sequence and structural level and is accessible through a web browser or as a Chimera plugin at http://3diana.cnb.csic.es.     

Phosphorylation of the Kinase Homology Domain Is Essential for Activation of the A-Type Natriuretic Peptide Receptor

Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (~20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.     

A GxxxG-like Motif within HIV-1 Fusion Peptide Is Critical to Its Immunosuppressant Activity,Structure, and Interaction with the Transmembrane Domain of the T-cell Receptor

To thrive in the human body, HIV fuses to its target cell and evades the immune response via several mechanisms. The fusion cascade is initiated by the fusion peptide (FP), which is located at the N-terminal of gp41, the transmembrane protein of HIV. Recently, it has been shown that the HIV-1 FP, particularly its 5–13 amino acid region (FP_5–13), suppresses T-cell activation and interacts with the transmembrane domain (TMD) of the T-cell receptor (TCR) complex. Specific amino acid motifs often contribute to such interactions in TMDs of membrane proteins. Using bioinformatics and experimental studies, we report on a GxxxG-like motif (AxxxG), which is conserved in the FP throughout different clades and strains of HIV-1. Biological activity studies and FTIR spectroscopy revealed that HIV FP_5–13-derived peptides, in which the motif was altered either by randomization or by a single amino acid shift, lost their immunosuppressive activity concomitant with a loss of the β-sheet structure in a membranous environment. Furthermore, fluorescence studies revealed that the inactive mutants lost their ability to interact with their target site, namely, the TMD of TCRα, designated CP. Importantly, lipotechoic acid activated macrophages (lacking TCR) were not affected by FP, further demonstrating the specificity of the immunosuppressant activity of CP. Finally, although the AxxxG WT and the GxxxG analog both associated with the CP and immunosuppressed T-cells, the AxxxG WT but not the GxxxG analog induced lipid mixing. Overall, the data support an important role for the AxxxG motif in the function of FP and might explain the natural selection of the AxxxG motif rather than the classical GxxxG motif in FP.     

The I Domain is Essential for Echovirus 1 Interaction with VLA-2

VLA-2, the α2β1 integrin, mediates cell adhesion to collagen and laminin, and is the receptor for the human pathogen echovirus 1. Because of its similarity to domains present in other proteins that interact with collagen, a 191 amino acid region within the α2 subunit (the I domain) has been proposed as a potential site for ligand interactions. Although the α2 subunits of human and murine VLA-2 are 84% identical, human α2 promotes virus binding whereas murine α2 does not. We used murine/human chimeric α2 molecules to identify regions of the human molecule essential for virus binding. Virus bound efficiently to a chimeric protein in which the human I domain was inserted into murine α2, indicating that the human I domain is responsible for specific virus interactions. Monoclonal antibodies that inhibited virus attachment all recognized epitopes within the human I do-main, further suggesting that virus interacts with this portion of the molecule. Similarly, antibodies that prevented VLA-2-mediated cell adhesion to collagen also mapped to the I domain. These results indicate that the I domain plays a role in VLA-2 interactions both with virus and with extracellular matrix ligands.