Four integrins, namely α₁β₁, α₂β₁, α₁₀β₁, and α₁₁β₁, form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their α subunit. We have produced the human integrin α₁₀I domain as a recombinant protein to reveal its ligand binding specificity. In general, α₁₀I did recognize collagen types I–VI and laminin-1 in a Mg²⁺-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When α₁₀I was tested together with the α₁I and α₂I domains, all three I domains seemed to have their own collagen binding preferences. The integrin α₂I domain bound much better to fibrillar collagens (I–III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin α₁I had the opposite binding pattern. The integrin α₁₀I domain was similar to the α₁I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the α₁I and α₂I domains, we modeled the structure of the α₁₀I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the αI domains, and their binding to types I, IV, and VI collagen was tested. In the α₂I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the α₁I and α₁₀I domains is oppositely charged (Arg-218). The mutation D219R in the α₂I domain changed the ligand binding pattern to resemble that of the α₁I and α₁₀I domains and, vice versa, the R218D mutation in the α₁I and α₁₀I domains created an α₂I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.

The integrins α₁β₁, α₂β₁, α₁₀β₁, and α₁₁β₁ are referred to as a collagen receptor subgroup of the integrin family. Recently, both α₁β₁ and α₂β₁integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, α₁₁β₁, recognizes collagens using C2C12 cells transfected with α₁₁ cDNA and the bacterially expressed recombinant α₁₁ I domain. The ligand binding properties of α₁₁β₁ were compared with those of α₂β₁. Mg²⁺-dependent α₁₁β₁ binding to type I collagen required micromolar Ca²⁺ but was inhibited by 1 mmCa²⁺, whereas α₂β₁-mediated binding was refractory to millimolar concentrations of Ca²⁺. The bacterially expressed recombinant α₁₁ I domain preference for fibrillar collagens over collagens IV and VI was the same as the α₂ I domain. Despite the difference in Ca²⁺ sensitivity, α₁₁β₁-expressing cells and the α₁₁ I domain bound to helical GFOGER sequences in a manner similar to α₂β₁-expressing cells and the α₂ I domain. Modeling of the α I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by α₁β₁, α₂β₁, and also α₁₁β₁ integrins. Although α₁₀and α₁₁ chains show the highest sequence identity, α₂ and α₁₁ are more similar with regard to collagen specificity. Future studies will reveal whether α₂β₁ and α₁₁β₁integrins also show overlapping biological functions.

Human filamins are large actin‐crosslinking proteins composed of an N‐terminal actin‐binding domain followed by 24 Ig‐like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 Å resolution structure of a three‐domain fragment of human filamin A (IgFLNa19–21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N‐terminus of IgFLNa20 forms a β‐strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20–IgFLNa21 interaction enhances filamin binding to integrin β‐tails. Structural and functional analysis of other IgFLN domains suggests that auto‐inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin–ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.

Receiver operating characteristics (ROC) curve with the calculation of area under curve (AUC) is a useful tool to evaluate the performance of biomedical and chemoinformatics data. For example, in virtual drug screening ROC curves are very often used to visualize the efficiency of the used application to separate active ligands from inactive molecules. Unfortunately, most of the available tools for ROC analysis are implemented into commercially available software packages, or are plugins in statistical software, which are not always the easiest to use. Here, we present Rocker, a simple ROC curve visualization tool that can be used for the generation of publication quality images. Rocker also includes an automatic calculation of the AUC for the ROC curve and Boltzmann-enhanced discrimination of ROC (BEDROC). Furthermore, in virtual screening campaigns it is often important to understand the early enrichment of active ligand identification, for this Rocker offers automated calculation routine. To enable further development of Rocker, it is freely available (MIT-GPL license) for use and modifications from our web-site ( http://www.jyu.fi/rocker ).