alpha 11beta 1 Integrin Recognizes the GFOGER Sequence in Interstitial Collagens*

Wan-Ming ZhangDagger , Jarmo Käpylä§, J. Santeri Puranen, C. Graham Knight||, Carl-Fredrik TigerDagger , Olli T. Pentikäinen, Mark S. Johnson, Richard W. Farndale||, Jyrki Heino§, and Donald GullbergDagger **

From the Dagger  Department of Medical Biochemistry and Microbiology, Biomedical Center, Box 582, Uppsala University, Uppsala S-751 23, Sweden, the § Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä FIN-40351, Finland, the  Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku FIN-20520, Finland, and the || Department of Biochemistry, University of Cambridge, Bldg. O, Downing Site, Tennis Court Rd., Cambridge CB2 1QW, United Kingdom

Received for publication, October 8, 2002, and in revised form, December 20, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The integrins alpha 1beta 1, alpha 2beta 1, alpha 10beta 1, and alpha 11beta 1 are referred to as a collagen receptor subgroup of the integrin family. Recently, both alpha 1beta 1 and alpha 2beta 1 integrins 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, alpha 11beta 1, recognizes collagens using C2C12 cells transfected with alpha 11 cDNA and the bacterially expressed recombinant alpha 11 I domain. The ligand binding properties of alpha 11beta 1 were compared with those of alpha 2beta 1. Mg2+-dependent alpha 11beta 1 binding to type I collagen required micromolar Ca2+ but was inhibited by 1 mM Ca2+, whereas alpha 2beta 1-mediated binding was refractory to millimolar concentrations of Ca2+. The bacterially expressed recombinant alpha 11 I domain preference for fibrillar collagens over collagens IV and VI was the same as the alpha 2 I domain. Despite the difference in Ca2+ sensitivity, alpha 11beta 1-expressing cells and the alpha 11 I domain bound to helical GFOGER sequences in a manner similar to alpha 2beta 1-expressing cells and the alpha 2 I domain. Modeling of the alpha  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 alpha 1beta 1, alpha 2beta 1, and also alpha 11beta 1 integrins. Although alpha 10 and alpha 11 chains show the highest sequence identity, alpha 2 and alpha 11 are more similar with regard to collagen specificity. Future studies will reveal whether alpha 2beta 1 and alpha 11beta 1 integrins also show overlapping biological functions.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The collagen family currently includes at least 24 members (1, 2), and four different collagen-binding integrins alpha 1beta 1, alpha 2beta 1, alpha 10beta 1 (3) and alpha 11beta 1 (4) are known. The alpha 3beta 1 integrin does not interact directly with collagen, but it does act as a laminin receptor (5) that can affect the activity of the collagen receptor alpha 2beta 1 through receptor cross-talk (6). alpha 1beta 1, alpha 2beta 1, alpha 10beta 1, and alpha 11beta 1 possess an inserted, or I domain1 closely related to the von Willebrand factor A domain, which mediates binding to native collagens. In different in vitro assays, alpha vbeta 3 also appears to be able to interact with type I collagen (7-9). However, this interaction most likely involves RGD motifs in denatured or partially unfolded collagen chains.

Studies of collagen-binding integrins in various in vitro assays show that they take part in cell adhesion, cell migration, control of collagen synthesis, matrix metalloproteinase synthesis, remodeling of collagen matrices, and influence such complex processes as cell proliferation, cell differentiation, angiogenesis, platelet adhesion/aggregation, and epithelial tubulogenesis (10, 11).

Using transfected cells and recombinant I domain, alpha 1beta 1 has been shown to bind collagens, with a preference for collagens IV and VI over collagen I and II. Collagen XIII in vitro is also a ligand for alpha 1beta 1 (12). Other identified ligands include laminin-1/-2 (13, 14), the cartilage protein matrilin-1 (15), and the C-propeptide of collagen I (16). The affinity of alpha 1beta 1 for laminin-1 has been reported to be about 10-fold lower than for collagen IV (17). In accordance with this, when the alpha 1 integrin chain is expressed in K562 cells, alpha 1beta 1 will bind type IV collagen, but it requires activation to bind laminin-1 (18). In Chinese hamster ovary cells, alpha 1beta 1 integrin does not mediate spreading on collagen II (12). It has been suggested that in these cells a coreceptor is needed for alpha 1beta 1-mediated spreading on collagen II.

Integrin alpha 2beta 1 and its alpha  I domain have been shown to bind a variety of collagens (19-21), the C-propeptide of collagen I (16, 22), laminin-1 (23), laminin-2 (14), decorin (24), and the cartilage protein chondroadherin (25). Unlike the other ligands for alpha 1beta 1 and alpha 2beta 1, chondroadherin does not support cell spreading. Early studies using antibodies showed that alpha 2beta 1 on some cells (melanoma LOX cells), but not others (fibroblasts, platelets), mediated the binding to laminin-1 (26).

The integrin subunit alpha 10 was originally identified by affinity purification of collagen type II-binding integrins from adult chondrocytes (3). The rather restricted expression of alpha 10beta 1 to cartilage indicates that the ligands are to be found in the cartilage extracellular matrix. Intriguingly, using recombinant protein, the collagen binding preference of the alpha 10 I domain is most similar to that of the alpha 1 I domain, so that the alpha 10 I domain prefers the basement membrane collagen IV and the beaded filament-forming collagen VI over the interstitial collagens I and II (27). In the same study, mutational analysis of the I domains showed that the amino acid residues Arg-218 in alpha 1 and alpha 10 and Asp-219 in alpha 2 are involved in determining this collagen preference.

alpha 11 was initially detected in differentiating human fetal muscle cells (28). alpha 11 protein and mRNA expression analysis in human embryos, however, revealed that expression is localized to mesenchymal non-muscle cells in areas of highly organized interstitial collagen networks. No expression was seen in muscle cells in vivo (29). In the developing skeletal system, alpha 10beta 1 and alpha 11beta 1 thus show nonoverlapping, complementary expression patterns (11). In accordance with the expression of alpha 11beta 1 in areas rich in interstitial collagens, alpha 11beta 1 binds more efficiently to collagen I than to collagen IV (29).

Cyanogen bromide cleavage of collagen chains identified the non-RGD-containing helical CB3 fragment of collagen alpha 1 I as a cell-binding fragment that could be used to purify alpha 1beta 1 (30). The alpha 1 I and alpha 2 I integrin binding site located within triple-helical alpha 1 I CB3 has been identified as GFOGER (31, 32). Two related sequences, GLOGER2 and GASGER, were identified elsewhere in collagen I (33), and other GER-containing sequences in the collagen chains can also mediate cell adhesion through alpha 2beta 1.3 The GER motif thus appears to be a major cell adhesion motif used by collagen-binding integrins.

Examination of the crystal structure of an alpha 2 I domain-GFOGER complex suggested that other hydrophobic residues might replace phenylalanine, which together with the glutamate and arginine residues provided the main side chain interactions between the collagen-like peptide and the integrin (34). Interactions also occurred with the main chain carbonyl group of the hydroxyproline residue, suggesting that hydroxyproline itself may not be required specifically for collagen-integrin interaction.

Arginine interacts with negatively charged Asp-219 on the surface of the alpha 2 I domain, and although this appears a relatively nonspecific interaction, GEK will not substitute fully in human platelets. The ligand binding groove of the alpha 2 I domain is relatively deep compared with that of alpha 1. Thus, the coordination of Mg2+ in the metal ion dependent adhesion site (MIDAS) may only be achieved by glutamate residues from the GER motif, aspartate being too short for this purpose. This may not be the case for other integrins, and the abundance of GDR triplets within the collagens suggests the possibility that GDR motifs might serve as well. The present study was designed in part to test the possibility that alpha 11, which lacks an acidic residue equivalent to Asp-219, and whose structure is not yet defined, might recognize GEK and GDR triplets, both of which occur frequently in human collagens.

Other modes of collagen binding also exist, and alpha 1beta 1 binds to collagen type IV using the amino acids arginine and aspartate contributed from different collagen alpha  chains (35, 36). The residues recognized by alpha 1beta 1 in collagen XIII, lacking a GFOGER sequence, have not been identified. Studies with fragments of laminins have shown that the I domain integrin binding sites are present in the short arm of the alpha  chain, but the exact region(s) has not yet been mapped (13, 14).

The interaction between integrins and their physiological ligands displays several requirements for divalent cations. First, the GER glutamate residue binds directly to a Mg2+ ion coordinated within the I domain MIDAS. Other divalent cations will serve this purpose, notably Mn2+ and Co2+, but in nature such ions are unlikely to be sufficiently abundant to contribute significantly to the adhesion process. Integrins possess several other divalent cation binding sites, three or four within the blades of the alpha  subunit beta  propeller, and perhaps two within the beta  subunit I-like domain. Some of these sites likely bind Ca2+ and account for the biphasic role of Ca2+ in the competence of the integrins. Adhesion of collagen to alpha 2beta 1 in human platelets, in common with the binding of ligand by other integrins, has recently been shown to require micromolar Ca2+ (37) and to be inhibited by millimolar Ca2+. Conceivably the latter effect reflects competition for Ser-123 lying between Mg2+ in the MIDAS and Ca2+ in the ADMIDAS sites of the beta  subunit I-like domain, such that high levels of Ca2+ render the integrin beta  subunit incapable of regulating alpha  subunit function properly. The requirement for Ca2+ appears to differ even for the same integrin when expressed in different cells. Thus, although a biphasic effect of Ca2+ on alpha 2beta 1 competence in platelets can clearly be shown, the sensitivity of alpha 2beta 1 to either high or low levels of Ca2+ in HT1080 cells is much less obvious.4

In the present study we set out to study further the mechanism whereby alpha 2beta 1 and alpha 11beta 1 integrins bind collagens. Our data suggest that the approximated Kd of the alpha 11 I domain for type I collagen is higher than that of the alpha 2 I domain and that in C2C12 cells the alpha 11beta 1-mediated binding to collagen I, but not that of alpha 2beta 1, is sensitive to the presence of Ca2+. However, both integrins display a similar collagen specificity, and both recognize the helical GFOGER sequence. Modeling of alpha 2 I and alpha 11 I domain-ligand complexes could in part explain the observed differences in alpha 2 I and alpha 11 I domain binding to different collagen peptides. The results are potentially promising for future attempts to generate reagents effective in blocking multiple collagen-binding integrins simultaneously.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Production of Human Recombinant Integrin alpha 1 I, alpha 2 I, and alpha 11 I Domains as Fusion Proteins

cDNAs encoding alpha 1 I and alpha 2 I domains were generated by PCR as described earlier (27) using human integrin alpha 1 and alpha 2 cDNAs as templates. Vectors pGEX-4T-3 and pGEX-2T (Amersham Biosciences) were used to generate recombinant glutathione S-transferase (GST) fusion proteins of human alpha 1 I and alpha 2 I domains, respectively. Human integrin alpha 11 cDNA (4) was used as a template when the alpha 11 I domain was generated by PCR. The PCR product having BamHI and EcoRI sites was cloned to pGEX-KT, and the DNA sequence was checked by sequencing the whole insert. The same vector was used for expression of recombinant GST fusion proteins of the human alpha 11 I domain. Competent Escherichia coli BL21 cells were transformed with the plasmids for protein production. 500 ml of LB medium (Biokar) containing 100 µg/ml ampicillin was innoculated with a 50-ml overnight culture of BL21/palpha 1 I, BL21/palpha 2 I, or BL21/palpha 11 I, and the cultures were grown at 37 °C until the A600 of the suspension reached 0.6-1.0. Cells were induced with isopropyl-1-thio-beta -D-galactopyranoside and allowed to grow for an additional 4-6 h before harvesting by centrifugation. Pelleted cells were resuspended in PBS (pH 7.4) and then lysed by sonication followed by the addition of Triton X-100 to a final concentration of 2%. After incubation for 30 min on ice, suspensions were centrifuged, and supernatants were pooled. Glutathione-Sepharose (Amersham Biosciences) was added to the lysate, which was incubated at room temperature for 30 min with gentle agitation. The lysate was then centrifuged, the supernatant was removed, and glutathione-Sepharose with bound fusion protein was transferred into disposable chromatography columns (Bio-Rad). The columns were washed with PBS, and fusion proteins were eluted using 30 mm glutathione. Purified recombinant and glutathione-tagged alpha 1 I, alpha 2 I, and alpha 11 I domains were analyzed by SDS and native PAGE. The recombinant alpha 1 I domain produced was 227 amino acids in length, corresponding to amino acids 123-338 of the whole alpha 1 integrin, whereas the alpha 2 I domain was 223 amino acids long, which corresponded to amino acids 124-339 of the whole alpha 2 integrin. The carboxyl termini of the alpha 1 I and alpha 2 I domains contained 10 and 6 non-integrin amino acids, respectively. Recombinant alpha 11 I domain contains a total of 204 amino acids: at the amino terminus there are 2 extra residues (GS) before the alpha 11 I domain, which starts from CQTY and ends with SLEG (residues 159-354); at the carboxyl terminus there are 6 extra amino acids (EFIVTD). The recombinant alpha 11 I domain contains some GST as an impurity caused by endogenous protease activity during expression and purification. Recombinant I domains were used as GST fusion proteins for collagen binding experiments.

Synthesis of Peptides

Peptides were synthesized as carboxyl-terminal amides on TentaGel R RAM resin in a PerSeptive Biosystems 9050 Plus PepSynthesizer exactly as described in our earlier studies (30, 31). Peptides were purified by reverse phase high performance liquid chromatography (HPLC) on a column of Vydac 219TP101522 using a linear gradient of 5-45% acetonitrile in water containing 0.1% trifluoroacetic acid. Fractions containing homogeneous product were identified by analytical HPLC on a column of Vydac 219TP54, pooled, and freeze dried. All peptides were found to be of the correct theoretical mass by mass spectrometry. The triple-helical stability of each peptide was assessed by polarimetry as described previously.

Solid Phase Binding Assay for alpha 1 I, alpha 2 I, and alpha 11 I Domains

The coating of a 96-well high binding microtiter plate (Nunc) was done by exposure to 0.1 ml of PBS containing 5 µg/cm2 (15 µg/ml) collagens or 20 µg/ml synthetic triple-helical collagen peptides overnight at 4 °C. Type I rat (rat tail) collagen, type IV mouse (basement membrane of Engelbreth-Holm-Swarm mouse sarcoma) collagen, and type IV human "cut" (human placenta) collagen were purchased from Sigma. Type IV human collagen and type II bovine collagen were purchased from Biodesign International and Chemicon, respectively. Type I bovine (bovine dermal) collagen was from Cellon S. A. Blank wells were coated with a 1:1 solution of 0.1 ml Delfia® Diluent II (Wallac) and PBS. Residual protein absorption sites on all wells were blocked with a 1:1 solution of 0.1 ml of Delfia® Diluent II and PBS. Recombinant proteins alpha 1 I-GST, alpha 2 I-GST, and alpha 11 I-GST were added to the coated wells at the desired concentration in Delfia® assay buffer and incubated for 1 h at room temperature. Europium-labeled anti-GST antibody (Wallac) was then added (typically 1:1,000), and the mixtures were incubated for 1 h at room temperature. All incubations mentioned above were done in the presence of 2 mM MgCl2. Delfia® enhancement solution (Wallac) was added to each well, and the europium signal was measured by time-resolved fluorometry (Victor2 multilabel counter, Wallac). In every case, at least three parallel wells were analyzed.

Cells

Murine C2C12 myoblast cells from the American Type Culture Collection were provided by A. Starzinski-Powitz. The generation of C2C12 cells stably transfected with integrin alpha 2 cDNA or integrin alpha 11 cDNA has been described previously (29). Cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Statens veterinärmedicinska anstalt, Uppsala). The cells were grown to subconfluence and passaged every 2-3 days.

Antibodies

Rabbit antibodies to the cytoplasmic tail of alpha 11 integrin have been described previously (4). To immunoprecipitate beta 1 integrins, a polyclonal antibody to rat integrin beta 1 chain was used (38).

Immunoprecipitation and Electrophoresis

Cell cultures were washed three times in Dulbecco's modified Eagle's medium devoid of cysteine and methionine and metabolically labeled overnight in the presence of 25 µCi/ml [35S]methionine/cysteine (pro-Mix 35S cell labeling mix; Amersham Biosciences). Proteins were extracted from the tissue culture dishes by the addition of 1 ml of solubilization buffer (1% Triton X-100, 0.15 M NaCl, 20 mM Tris-HCl pH 7.4, 1 mM MgCl2, 1 mM CaCl2) containing protease inhibitors (1 mM Pefabloc SC (Roche Molecular Diagnostics), 1% aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin). Solubilized proteins were centrifuged for 10 min at 15,000 × g. The centrifuged supernatant was precleared by incubating with 100 µg/ml preimmune IgG and protein A-Sepharose CL4B (Amersham Biosciences) for 2 h. After centrifugation, immune IgG was incubated with the extract for 2 h. Specifically bound proteins were recovered with protein A-Sepharose. The precipitate was washed three times with buffer A (1% Triton X-100, 0.5 M NaCl, 20 mM Tris-HCl pH 7.4, 1 mM MgCl2, 1 mM CaCl2) and three times with buffer B (0.1% Triton X-100, 0.15 M NaCl, 20 mM Tris-HCl pH 7.4, 1 mM MgCl2, 1 mM CaCl2) prior to solubilization in electrophoresis sample buffer. Proteins were separated on 6% SDS-polyacrylamide gels and processed for fluorography.

Cell Attachment Assay

General Setup-- Ligands and metal ions are described separately for the two experiments. 24-well cell culture plates (Nunc) were coated with ligands (500 µl to a 2-cm2 well) diluted in PBS overnight at 4 °C, followed by blocking with 2% BSA in PBS for 2 h at room temperature and then washed in Puck's saline (137 mM NaCl, 5 mM KCl, 4 mM Na2CO3, 5.5 mM D-glucose, pH 7.0). Transfected cells were trypsinized, washed four times in Puck's saline, deeded into the wells at a concentration of 250,000 cells/well, and were allowed to attach for 45 min at 37 °C and 5% CO2. Wells were washed three times in Puck's saline, and plates were rapidly frozen at -20 °C for later assay using the hexoseaminidase test as described previously (29). For each cell line used, a cell number standard was made. Each experiment was performed in triplicate. To minimize errors from unequal trypsinization stress between cell lines and handling of plates, for example, data were normalized as follows. For each plate the adhesion to 10 µg/ml fibronectin (provided by S. Johansson, Uppsala University) was used as the 100% reference level, and the background found on BSA-only coated wells was used as the base-line (0%) reference level.

Ca2+ Inhibition Setup-- Wells were coated with 10 µg/ml bovine collagen type I (Vitrogen®100, Cohesion) or 10 µg/ml human plasma fibronectin. Wells were filled with Puck's saline, and MgCl2 + EGTA was added to obtain a final concentration (after addition of cells) of 2 mM MgCl2 and 0.01 mM EGTA. CaCl2 was added according to Fig. 1.

Cell Attachment to Synthetic Peptides-- 24-well plates were coated with 10 µg/ml synthetic triple-helical collagen peptides at 4 °C overnight according to Fig. 4, or 10 µg/ml bovine collagen type I (Vitrogen®100), or 10 µg/ml fibronectin. MgCl2 and CaCl2 were added to a final concentration of 2 mM MgCl2 and 0.01 mM CaCl2.

Homology Modeling

Sequences of integrin alpha 1 (accession code P56199 (39)) and alpha 11 (Q9UKX5 (40)) were obtained from SWISS-PROT (41). The crystal structure of the alpha 2 I domain in complex with the triple-helical collagen mimetic peptide (PDB code 1dzi (34)) was obtained from the Protein Data Bank (42).

The sequence alignment was made using the program MALIGN (43) in the BODIL modeling environment5 (www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html) using a structure-based sequence comparison matrix (44) with a gap penalty of 40.

Homology models were built with HOMODGE in BODIL. The amino acid side chain rotamer library incorporated into BODIL was used to evaluate alternative possibilities for side chain conformations for sequence differences in the alignment of alpha 1 and alpha 11 I domain sequences with the template structure.

In the alpha 2 I domain-peptide complex structure, 1dzi, and the three peptide chains (identical in sequence but having different interactions with the alpha 2 I domain) of the collagen mimetic tripeptide are labeled B, C, and D. The corresponding chain labels are used for the tripeptides docked to the model structures built for the alpha 1 I and alpha 11 I domains.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Influence of Ca2+ on Cell Attachment to Collagen-- To compare the mechanism whereby alpha 11beta 1 and alpha 2beta 1 recognize collagens, we used the satellite cell line C2C12 transfected with alpha 2 or alpha 11 cDNAs (C2C12 alpha 2+ and C2C12 alpha 11+, respectively). The parental cell line C2C12 expresses members of the beta 1 subfamily, such as alpha 5beta 1 and alpha 7beta 1 (45), but does not adhere to collagen (29). C2C12 cells transfected with either alpha 2 or alpha 11 acquire the ability to interact with collagens I and IV, with a preference for collagen I (29). The alpha 2beta 1-mediated binding of platelets to collagen has been reported to require micromolar Ca2+ but to be inhibited by millimolar Ca2+ in the presence of Mg2+ (37). To test the effect of Ca2+ on cell adhesion to collagen I, the transfected C2C12 cells were plated on collagen I in the presence of Mg2+ and EGTA with increasing concentrations of Ca2+ added. In the absence of Ca2+, adhesion of cells expressing alpha 11beta 1 was virtually absent, as reported for human platelets (45), and a biphasic response to added Ca2+ was observed with peak adhesion occurring at a free Ca2+ of around 30 µM and adhesion being substantially abolished at 4 mM, with an IC50 of about 1 mM. In marked contrast, C2C12 alpha 2+ cell adhesion to collagen I was largely refractory to both the removal of Ca2+ using EGTA or the subsequent addition of millimolar Ca2+ (Fig. 1).


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Fig. 1.   Effects of Ca2+ on alpha 11beta 1-mediated cell adhesion to collagen I. C2C12 alpha 2+ and C2C12 alpha 11+ cells were allowed to adhere to collagen I in the presence of 1 mM Mg2+, 10 µM EGTA, and varying concentrations of Ca2+. Adhesion was measured as described under "Materials and Methods."

alpha 11 and alpha 2 I Domains Differ in Their Affinity for Collagen I-- To estimate the Kd of alpha 11 for collagen I, we produced an alpha 11 I domain in E. coli. Initial attempts to express the alpha 11 I domain as a bacterial GST fusion protein yielded low amounts of protein. We therefore expressed the alpha 11 I domain as a His-tagged fusion protein in Pichia pastoris. After purification on nickel-Sepharose only low binding to collagen I was noted, and background binding was high. Gel filtration revealed that a majority of protein appeared in a high molecular weight fraction, indicating aggregation (data not shown). Production of alpha 11 I-GST in E. coli was subsequently optimized. Large scale expression of the alpha 11 I domain yielded enough protein to perform binding studies. Approximated Kd values based on solid phase binding assays (26) and the use of the Michaelis-Menten equation suggested a relatively low alpha 11 I domain avidity to collagen I (750 ± 50 nM) when compared with the alpha 2 I domain binding to collagen I (20 ± 5 nM (27)) (Fig. 2).


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Fig. 2.   Binding of the alpha 11 I domain to collagen as a function of alpha 11 I domain concentration. Microtiter plates were coated with 15 µg/ml collagen types I (rat col-I), II (human col-II), and IV (mouse col-IV native) overnight. Diluent containing BSA was used as a background control and to block the wells. The GST fusion alpha 1 I domain (4-408 nM) was allowed to bind for 1 h in the presence of 2 mM MgCl2. The wells were washed three times. Bound alpha  I domain was detected with europium-labeled anti-GST antibody. Time-resolved fluorescence measurements were used. The data are the means of three parallel determinations (±S.D.).

Collagen Preference of alpha 11 I Domain-- Previous studies from several laboratories have shown that alpha 2beta 1 integrin prefers fibril-forming collagens over network-forming type IV and beaded filament-forming type VI collagen. The same pattern can be seen in the binding of the alpha 2 I domain. Here alpha 2- and alpha 11-mediated binding to different collagens was compared. The alpha 11 I domain was shown to prefer the fibril-forming collagen types I and II (Fig. 3), whereas its binding was weaker to type III (data not shown), a member of the same collagen subgroup. Collagens IV and VI were poor ligands for the alpha 11 I domain. Thus, in the terms of its binding pattern the alpha 11 I domain was closer to the alpha 2 I domain than to either the alpha 1 or alpha 10 I domain.


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Fig. 3.   Binding of the alpha 11 I domain to ECM ligands. Microtiter plates were coated with 15 µg/ml collagen types I (rat col-I, bovine col-I), II (bovine col-II), IV (human col-IV cut, mouse col-IV native), and VI (human col-VI) overnight. Diluent containing BSA was used as a background control and to block the wells. GST fusion alpha 11 I domain (408 nM) was allowed to bind for 1 h in the presence of 2 mM MgCl2. Wells were washed three times. Bound alpha  I domain was detected with europium-labeled anti-GST antibody. Time-resolved fluorescence measurements were used. The data are the means of three parallel determinations (±S.D.).

Binding to GER-containing Peptides-- Helical GFOGER and GFOGER-like peptides have recently been shown to represent high affinity integrin recognition motifs in collagens (32, 33). To determine whether alpha 11beta 1 also differed from alpha 2beta 1 with regard to its recognition sequences, C2C12 alpha 2+ and C2C12 alpha 11+ cells were tested for their ability to attach to different collagen-like peptides. C2C12 alpha 2+ cells adhered to GFOGER and GFOGEK peptides as reported previously. C2C12 alpha 11+ cells also bound these peptides in a similar pattern, whereas untransfected cells failed to do so (Fig. 4).To confirm that the observed binding occurred via the alpha  I domain, I domains from alpha 1, alpha 2, and alpha 11 were compared with regard to binding to the different collagen-derived peptides (Fig. 5). In these studies we also included the GLOGER peptide (33) which bound all I domains. The alpha 11 I domain preferred the GFOGER peptide followed by the GFOGEK and GLOGER peptides, much like the alpha 2 I domain. The alpha 1 I domain bound the GLOGER peptide as efficiently as the GFOGER peptide. The peptide containing the sequence GASGER, reported as a weak binding site for alpha 2beta 1 and alpha 1beta 1 showed only low capacity to bind any of the I domains, and substitution of aspartate for glutamate within the GER triplet similarly abolished I domain binding. The sequence GPOGES, from the collagen I alpha 2 chain where it corresponds to GFOGER in the alpha 1 I chain, was similarly without significant binding activity. Comparing the overall peptide binding pattern, alpha 2 I and alpha 11 I domains appear most similar in their peptide binding preferences.


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Fig. 4.   alpha 11beta 1 binds the helical GFOGER sequence. C2C12, C2C12 alpha 2+, and C2C12 alpha 11+ cells were allowed to adhere to synthetic collagen peptides in the presence of 1 mM Mg2+ and 10 µM Ca2+, and cell adhesion (triplicate wells) was evaluated (±S.D.).


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Fig. 5.   Binding of alpha 1 I, alpha 2 I, and alpha 11 I domains to synthetic triple-helical collagen peptides. Microtiter plates were coated with 20 µg/ml collagen peptides. Diluent II containing BSA was used as a background control and to block the wells. GST-fusion alpha 1 I (A), alpha 2 I (B), and alpha 11 I domains (C) were allowed to bind for 1 h in the presence of 2 mM MgCl2. The wells were washed three times. Bound alpha  I domains were detected with europium-labeled anti-GST antibody. Time-resolved fluorescence measurements were used. The data are the means of three parallel determinations (±S.D.).

Modeling of Collagen Peptide Binding to the alpha 11 I Domain-- The basic assumption in modeling was that the collagen mimetic tripeptide GFOGER and its mutants bind to all of the integrin alpha  I domains in a way similar to that seen in the crystal structure of the complex between alpha 2 I domain and the GFOGER triple-helical peptide (34). The sequence identities of the alpha 1 and alpha 11 I domains to the alpha 2 I domain are 51 and 45% respectively, thus experience dictates that high quality models will be produced. Only one region of the alpha 11 I domain model is uncertain, where Pro-310 (threonine in the alpha 1 and alpha 2 I domains) is located within a region that corresponds to helix 6 of the open fold of the alpha 1 and alpha 2 I domains. Proline generally does not promote helix stability, so it is very likely that the helix begins at or after position 310 in the alpha 11 I domain. In addition, the local alignment of residues 179 and 180 seems peculiar because the charged residue Glu-180 would be buried, and the hydrophobic residue Val-179 would be exposed toward the solvent. If Glu-180 is buried, then the conserved residue Tyr-157 may change its conformation in the alpha 11 I domain and affect the binding of the collagen mimetic tripeptide. Thus, it is possible that the binding conformation seen in alpha 2 I domain-tripeptide complex structure may be different in the case of alpha 11.

Glutamate in the Collagen Mimetic Tripeptide-- In the crystal structure of the integrin alpha 2 I domain in complex with the collagen mimetic peptide (3 × GFOGER), the side chain of only one of the glutamate residues in the collagen mimetic tripeptide, that of the middle strand, chain C, interacts with the I domain. This glutamate is coordinated to the metal ion of the MIDAS motif and thus, represents a key interaction in tripeptide binding (Fig. 6). Moreover, even a conservative change, mutation to aspartate, lowers the binding dramatically for the alpha 1 I, alpha 2 I, and alpha 11 I domains (GFOGDR; Fig. 4). Aspartate is one methylene group shorter than glutamate and would not reach the metal ion of the MIDAS motif as easily as glutamate can.


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Fig. 6.   Stereo view of the interactions between the arginine from chain C of the collagen mimetic tripeptide and integrin alpha 2 (1dzi) (A), alpha 1 (B), and alpha 11 (C) I domains. Detailed interactions of Arg right-arrow Lys mutation of the collagen mimetic tripeptide with the integrin alpha 2 I domain in the same region are shown in D. The backbone of the collagen mimetic tripeptide is shown as yellow cords, and the I domain backbone is shown in blue. Strong electrostatic interactions are shown as black dotted lines, and weak electrostatic interactions are shown as yellow dotted lines.

In the alpha 2 I domain there is a leucine residue at position 286, which forms an interaction with phenylalanine of the collagen mimetic tripeptide from chain B, whereas both the alpha 1 and alpha 11 I domains have tyrosine at that position. Tyrosine could either form a hydrophobic interaction with a planar face of the Arg-148 side chain, or tyrosine could form a hydrogen bond with glutamate from the collagen mimetic tripeptide chain B. If the latter case is true then the hydrogen bond would not be possible in the collagen mimetic tripeptide containing the Glu right-arrow Asp mutation.

In the alpha 1 I domain model, Arg-218 (aspartate in alpha 2 and threonine in alpha 11) may form an additional interaction with the glutamate of chain D of the collagen mimetic tripeptide. This interaction would still be possible in the tripeptide containing the aspartate mutation.

Arginine in the Collagen Mimetic Tripeptide-- In the crystal structure of the alpha 2 I domain (1dzi), arginine in chain C of the collagen mimetic tripeptide is bound to the area where Asp-219, Asn-189, and Leu-220 are located. The Nepsilon and Neta 2 atoms of arginine would interact with side chain carboxylate of Asp-219 but only weakly because the angle is not optimal for forming a strong hydrogen-bond/salt bridge. In addition, the side chain of Leu-220 forms an optimal site for hydrophobic interactions with a planar face of the arginine side chain (Fig. 6A). In both alpha 1 and alpha 11, the arginine of the tripeptide could form a salt bridge with glutamate at the position equivalent to Asn-189 in alpha 2 (Fig. 6, B and C). Furthermore, in alpha 11 there is a threonine equivalent to Asp-219 in alpha 2 whose side chain hydroxyl group can accept a hydrogen bond from the Nepsilon of arginine from the tripeptide (Fig. 6C). For alpha 2, the Arg right-arrow Lys mutation in the collagen mimetic tripeptide (GFOGEK) does not affect binding as dramatically as seen for alpha 1 and alpha 11 (Fig. 5). In alpha 2, the repulsion resulting from the charged amino group of lysine positioned near the Leu-220 side chain would be offset by the formation of a somewhat more optimal hydrogen bond/salt bridge between lysine and Asp-219 (Fig. 6D). The mutation of arginine to lysine reduces the binding affinity of collagen mimetic tripeptide to the alpha 1 and alpha 11 I domains because the salt bridge to glutamate, at the position equivalent to Asn-189 in alpha 2, cannot be maintained. When arginine of the collagen mimetic tripeptide is mutated to lysine, lysine cannot reach glutamate because a lysine residue is shorter than an arginine residue. Moreover, in alpha 11 the lysine residue can form a hydrogen bond with the threonine equivalent to Asp-219 in alpha 2, and thus, the effect of the mutation is not as dramatic as for alpha 1.

In the alpha 2 I domain, the arginine from chain B of the collagen mimetic tripeptide interacts mainly with other parts of the tripeptide and not with the I domain. Nepsilon is hydrogen-bonded to the main chain oxygen of arginine in chain C, and the planar end of the arginine side chain has a hydrophobic interaction with proline in peptide chain C. In addition, hydrophobic interactions with the hydrophobic part of the Glu-256 side chain and weak electrostatic interactions with the main chain oxygen atom of Ser-257 can be seen. These interactions should be present and identical in each of the I domains in this study. Thus, the effect of the mutation of arginine to lysine, caused by chain C, should be same for all I domains.

In the alpha 2 structure, the arginine from chain D of the collagen mimetic tripeptide is exposed to the solvent, and thus, the mutation can only have an indirect influence on I domain binding.

Phenylalanine in the Collagen Mimetic Tripeptide-- In the alpha 2 I domain structure, the phenyl ring of phenylalanine in chain B of the collagen mimetic tripeptide is stacked with the phenol ring of the conserved tyrosine (position 157 in alpha 2). This phenylalanine also has hydrophobic interactions with Leu-286 in alpha 2. In addition, the phenylalanine of chain B forms an unfavorable interaction with the main chain oxygen atom of Tyr-285 (Fig. 7A ). A corresponding view of phenylalanine interactions with the alpha 1 I domain is shown in Fig. 7B.


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Fig. 7.   Stereo view of the interactions between the phenylalanine from chain B of the collagen mimetic peptide and integrin I domains alpha 2 (1dzi) (A) and alpha 1 (B). The collagen mimetic peptide backbone is shown in yellow, and the I domain backbone is shown in blue.

When phenylalanine of the collagen mimetic tripeptide is mutated to leucine, some favorable interactions would be lost, but this loss is offset by the removal of unfavorable interactions with the main chain oxygen atom of the residue at position 285 (tyrosine in alpha 2 and alpha 11; serine in alpha 1). Thus, there is a small change in the binding affinity of alpha 1 and alpha 2 when the Phe right-arrow Leu mutant is compared with the "wild type" tripeptide (Fig. 5). The effects seen for alpha 11 are difficult to predict because the model is inaccurate in this region. The binding affinity is lowered dramatically when phenylalanine is replaced with alanine, resulting in the loss of all favorable interactions (Fig. 5).

In the alpha 2 I domain structure, the phenylalanine in chain C of the collagen mimetic tripeptide leans against the side chain of Asn-154, which is conserved in the alpha 1, alpha 2, and alpha 11 I domains. This interaction is not very critical, and thus the mutation of phenylalanine to leucine or alanine would not affect the binding affinity by much. The phenylalanine in chain D is exposed to the solvent, and thus it has practically no role in binding.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In recent years an increasing effort has been spent trying to understand the mechanism whereby cells bind collagen. In vertebrates more than 24 different collagens exist, and the role of some of these is yet unclear. Integrins are major receptors for collagens. A common feature of the collagen-binding integrins is the presence of an alpha  I domain that is directly involved in ligand binding.

The I domain is not found in integrin alpha  chains from the invertebrate Drosophila melanogaster but it is present in 9 of the 18 currently known vertebrate integrin alpha  chains (11) including alpha L, alpha M, alpha X, alpha D, and alpha E, which are all involved in different aspects of leukocyte functions and pair exclusively with the beta 2 subunit (10). The overall importance of integrin-mediated cell-collagen interactions involving the alpha 1, alpha 2, alpha 10, and alpha 11 integrin chains is largely unknown because of the limited information available for alpha 10beta 1 and alpha 11beta 1. Based on the appearance of I domain integrin alpha  chains during vertebrate evolution it is possible that these integrin chains play important roles in vertebrate-specific structures of the musculoskeletal system.

Gene knockout experiments and recombinant expression of the alpha  I domains have yielded considerable information about the characteristics and functions of collagen-binding integrins alpha 1beta 1 and alpha 2beta 1 (11, 46, 47). Phylogenetically, alpha 1 and alpha 2 form a subfamily distinct from alpha 10 and alpha 11, which most likely have formed through two distinct gene duplication events.

Both alpha 1 and alpha 2 integrin chains are fairly widely expressed throughout the body. Gene knockout experiments of alpha 1 and alpha 2 chains have shown that inactivation of the individual collagen-binding integrins does not seem to impair embryonic development (46, 47). Rather, mild phenotypes are observed where either fibroblast and leukocyte interactions with collagens or platelet interactions with collagens are affected. Recent analysis of alpha 10 and alpha 11 expression (3, 29) reveals a restricted embryonic expression pattern, which is not overlapping but complementary. In the near future it will be important to determine to what extent the collagen-binding integrins show overlapping functions and to what extent different collagen-binding integrins can functionally compensate for each other's absence. Crossing different mice strains lacking certain collagen receptors will shed light on these issues.

As a part of understanding the biological function of collagen-binding integrins it is important to characterize all of the different collagen-binding integrins with regard to collagen affinity, collagen specificity, divalent ion requirements, and ligand recognition motifs. Studies of alpha 1 I, alpha 2 I, and alpha 10 I domains have shown that they bind collagens with different specificity (27). This specificity seems in part to be determined by residues located outside the MIDAS motif in the alpha  I domain. Data from several groups have convincingly shown that alpha 1 prefers collagens IV and VI over collagen I and that the preferences of alpha 2 are opposite. More recently the alpha 10 I domain was shown to display a collagen binding specificity similar to that of alpha 1 (27).

Prior to this study no binding studies had been performed with the alpha 11 I domain. It thus appears that although alpha 1 is, in terms of evolution, more similar to alpha 2, and alpha 10 is more closely related to alpha 11, another grouping can be made based on their collagen specificity. The finding that alpha 11 I prefers interstitial collagens over nonfibrillar collagens supports our previous cell binding data (29), but the difference is even more pronounced at the I domain level. A candidate amino acid that might play a role in determining this preference is Thr-238 found in a position corresponding to Arg-218 in alpha 1.

The relatively low avidity for collagens estimated for both alpha 10 I and alpha 11 I domains is intriguing. Our experience is that as recombinant GST fusion proteins, these I domains are less soluble than alpha 1 I and alpha 2 I domains, and they might have a tendency to form aggregates. This may affect the Kd estimates. Furthermore, we have shown that in the length of the produced protein a difference of one amino acid residue might lead to changes in the avidity of collagen binding (48). Thus, the approximated Kd values should be used for comparing the binding of a recombinant alpha  I domain with different collagens rather than for comparing the alpha  I domains with each other. Low avidity may indicate that the major function for alpha 10 and alpha 11 is not that of firm adhesion but that these integrins engage in dynamic interactions with collagen during events such as cell migration. It is also possible that the true ligands have not yet been identified. For example, for alpha 10, a cartilage ligand, possibly a collagen other than collagen II, might be the preferred ligand. In the case of alpha 11, a perichondrium ligand other than collagen I might bind this integrin with higher affinity.

The alpha 2beta 1-mediated binding of platelets to collagen I is inhibited by mM concentrations of Ca2+ (37). The finding that alpha 2beta 1 is not inhibited by Ca2+ when expressed in C2C12 cells is intriguing. In the case of platelets and C2C12 cells this difference might be related to the activation status of the integrin. Whereas platelets and leukocytes have a more elaborate system for regulating integrin activation status, integrins in C2C12 cells are expected to be mainly in the activated state, displaying a higher affinity. alpha 11beta 1 is not expressed on platelets, so a direct comparison with alpha 2beta 1 is not possible.6 However, when expressed in C2C12 cells, alpha 11beta 1 binding to collagen I requires µM Ca2+ and is sensitive to mM concentrations of Ca2+, as alpha 2beta 1 when expressed on the platelet surface. The recent crystallization of soluble alpha vbeta 3 supports a role of Ca2+ ions in allosteric modulation of integrin conformation (49). It is possible that a high affinity interaction with collagen I, such as that mediated by alpha 2beta 1, is less affected by Ca2+-induced allosteric conformational changes. Conversely, a lower affinity interaction with collagen I, such as that mediated via alpha 11beta 1 might be more sensitive to allosteric changes in other regions of the receptor. This differential sensitivity to Ca2+ might be physiologically important in the formation and turnover of the musculoskeletal system, where the local concentration of Ca2+ varies.

Despite the differences in collagen specificity, helical GFOGER-like sequences are recognized by alpha 1beta 1, alpha 2beta 1, and as shown in this study, also by alpha 11beta 1. Careful analysis of the occurrence of GFOGER-like peptides has shown that in addition to the CB3-derived sequences GFOGER and GFOGEK, which are present in the central part of the collagen chain, an amino-terminal alpha 2 I and alpha 1 I domain binding site overlaps with the peptide GLOGER (33). As shown in this study, the binding of the different I domains to different collagen peptides varied somewhat. The glutamate in GFOGER was central for the binding of all I domains, whereas the phenylalanine seemed to be more important for alpha 11 binding, and the arginine was especially important for alpha 1 binding. It is possible that in vivo collagen-binding integrins prefer certain sites on the collagen molecules. In a particular cell expressing multiple collagen receptors, a number of factors might determine which region in collagen is bound by a particular integrin. Some of the factors that might affect ligand binding include local Ca2+ concentrations, expression levels of the different receptors, and subcellular localization within the cell. Collagen receptors have been shown to affect collagen and matrix metalloproteinase synthesis. Already now it is possible to envisage how GFOGER peptides have the potential to become universal reagents blocking cell-collagen interactions. It will be important to determine whether alpha 10beta 1 also binds GFOGER peptides. Triple-helical collagen peptides might be of use in conditions characterized by excessive collagen production such as various fibrotic conditions.

    FOOTNOTES

* This work was supported by grants from the Swedish Research Council (to D. G.), the Medical Research Council (to R. W. F.), the British Heart Foundation (to R. W. F.), the Wenner-Gren Foundation (to W.-M. Z.), and the Kung Gustaf V:s 80-års Fond (to D. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 46-18-471-4175; Fax: 46-18-471-4673; E-mail: donald.gullberg@imbim.uu.se.

Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M210313200

3 R. W. Farndale, P. R.-M. Siljander, and C. G. Knight, in preparation.

4 R. W. Farndale, P. R.-M. Siljander, and C. G. Knight, unpublished observation.

5 J. V. Lehtonen, V. V. Rantanen, D. J. Still, M. Gyllenberg, and M. S. Johnson, unpublished observation.

6 R. W. Farndale, unpublished observation.

2 Where single letter amino acid nomenclature is used, O = hydroxyproline.

    ABBREVIATIONS

The abbreviations used are: I domain, inserted domain; BSA, bovine serum albumin; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline.

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