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• W1569377852 abstract "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. 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. metal ion-dependent adhesion site polymerase chain reaction maltose-binding protein glutathioneS-transferase phosphate-buffered saline bovine serum albumin Collagens are abundant structural proteins in the extracellular matrix. So far, 19 different triple helical protein trimers have been classified as a collagen subtype (1Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1388) Google Scholar). The collagens can be grouped into subclasses according to their structural details. Many collagen subtypes (namely types I, II, III, V, and XI) have long continuous triple helices, and they can form large fibrils. In other collagens the triple helix has interruptions. Some collagens form networks (types IV, VIII, and X) or beaded filaments (type VI). Other collagen subclasses include fibril-associated collagen with short interruptions in the triple helices (collagen types IX, XII, XIV, XVI, and XIX), anchoring fibril-forming collagen (type VII), and transmembrane collagen (types XIII and XVII). Collagen types XV and XVIII are found in association with basement membranes (the multiplexins; see Ref. 2Oh S.P. Warman M.L. Seldin M.F. Cheng S.D. Knoll J.H. Timmons S. Olsen B.R. Genomics. 1994; 19: 494-499Crossref PubMed Scopus (118) Google Scholar). The integrins form a large family of heterodimeric cell surface receptors involved in cell-extracellular matrix as well as in cell-cell adhesion and communication. Of the 24 different integrin receptors presently known, 5 function as a collagen receptor (3Heino J. Matrix Biol. 2000; 19: 319-323Crossref PubMed Scopus (242) Google Scholar). The collagen receptors are composed of the β1 subunit in complex with either an α1, α2, α3, α10, or an α11 subunit. Integrin α1β1 is the collagen receptor of many mesenchymal cells. α1 null mice are viable (4Gardner H.A. Kreidberg J. Koteliansky V. Jaenisch R. Dev. Biol. 1996; 175: 301-313Crossref PubMed Scopus (228) Google Scholar), but they seem to have defects in the feedback regulation of collagen synthesis (5Gardner H.A. Broberg A. Pozzi A. Laato M. Heino J. J. Cell Sci. 1999; 112: 263-272Crossref PubMed Google Scholar), in the regulation of matrix metalloproteinase expression (5Gardner H.A. Broberg A. Pozzi A. Laato M. Heino J. J. Cell Sci. 1999; 112: 263-272Crossref PubMed Google Scholar, 6Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (350) Google Scholar), in tumor-related angiogenesis (6Pozzi A. Moberg P.E. Miles L.A. Wagner S. Soloway P. Gardner H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2202-2207Crossref PubMed Scopus (350) Google Scholar), and in lymphocyte function (7Meharra E.J. Schön M. Hassett D. Parker C. Havran W. Gardner H. Cell. Immunol. 2000; 201: 1-5Crossref PubMed Scopus (46) Google Scholar). Integrin α2β1 is an important collagen receptor on platelets and epithelial cells (8Zutter M.M. Santoro S.A. Am. J. Pathol. 1990; 137: 113-120PubMed Google Scholar). Little is known about the biology of the recently identified α10β1 and α11β1integrins. α10β1 is one of the collagen receptors on chondrocytes (9Camper L. Hellman U. Lundgren-Åkerlund E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), and α11β1was originally found in fetal muscle (10Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Integrin α3β1 can bind some collagens, but it may function as an assisting collagen receptor rather than as a primary receptor (11DiPersio C.M. Shah S. Hynes R.O. J. Cell Sci. 1995; 108: 2321-2336Crossref PubMed Google Scholar). One notable feature common to the primary collagen receptors is the existence of an I (“inserted”) domain at the N terminus of the α subunit. No other extracellular matrix receptor contains this independently folding domain, but five integrin-type cell-cell adhesion receptors do have it. The I domain is built up of β sheets surrounded by amphipathic α helices (12Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (806) Google Scholar). I domains are homologous to the A domains found in von Willebrand factor and in cartilage matrix protein, in some collagen subtypes, and in components of the complement system. The A and I domains are commonly involved in molecular interactions, and they are responsible for the collagen binding activity of von Willebrand factor and collagen receptor integrins (13Cruz M.A. Yuan H. Lee J.R. Wise R.J. Handin R.I. J. Biol. Chem. 1995; 270: 10822-10827Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 14Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 15Kern A. Briesewitz R. Bank I. Marcantonio E.E. J. Biol. Chem. 1994; 269: 22811-22816Abstract Full Text PDF PubMed Google Scholar). At the “top” of the I domain, where ligands bind, resides a metal ion in a conserved coordination site called MIDAS1 (metal ion-dependent adhesion site; see Ref. 16Mischishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar). Five amino acid side chains bind the magnesium ion, directly or through water molecules, and a water molecule or glutamate from a collagenous ligand may complete the coordination to the metal ion. The MIDAS site is centered on a groove restricted on one side by a helix called the αC helix. This αC helix can be found only in collagen binding integrin αI domains. Ligand binding induces a conformational change in the α2I domain resulting in the unwinding of the αC helix and opening of the binding site (17Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (845) Google Scholar). Recombinant α2I domain missing the αC helix has been shown to have altered kinetics in binding type I collagen (18Käpylä J. Ivaska J. Riikonen R. Nykvist P. Pentikäinen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). A prominent feature in the αI domain binding surface is the presence of several charged amino acids surrounding the MIDAS site. The binding surface of α2I is more negatively charged than the binding surfaces of α1I and α10I (19Pentikäinen O. Hoffren A.-M. Ivaska J. Käpylä J. Nyrönen T. Heino J. Johnson M.S. J. Biol. Chem. 1999; 274: 31493-31505Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Despite the structural similarity of the integrins α1β1 and α2β1, their binding specificity differs from each other. Integrin α1β1 prefers network-forming collagen type IV over fibrillar collagen type I, whereas α2β1 binds type I collagen more strongly than collagen type IV (20Kern A. Eble J. Golbik R. Kuhn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar). Moreover, α1β1is a much better receptor for type XIII collagen than α2β1 (21Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The same binding pattern can also be seen with the corresponding recombinant I domains. Here we have tested the binding of α1I and α2I domains to various collagen subtypes. We have also unveiled the ligands recognized by the α10I domain. The similarities and differences in the ligand binding patterns were correlated with the atomic structures, and based on experiments with mutated αI domains, we suggest that Asp-219 in the α2I domain, and the corresponding amino acid Arg-218 in the α1I and α10I domains, is a critical residue in the determination of collagen binding specificity. α10I domain cDNA was generated by reverse transcription-PCR from RNA isolated from KHOS-240 cells (human Caucasian osteosarcoma). Total cellular RNA was isolated by using an RNeasy Mini Kit (Qiagen). Reverse transcription-PCR was done using the Gene Amp PCR kit (PerkinElmer Life Sciences). The α10I forward primer (5′-CAG GGA TCC CCA ACA TAC ATG GAT GTT GTC-3′) contained a Bam HI restriction site at the 5′-end. The reverse primer (5′-GGC TGA ATT CCC CTT CAA GGC CAA AAA TCC G-3′) also contained an Eco RI restriction site at the 5′-end. The primers were delivered by CyberGene, Sweden. Forty cycles of PCR amplification were done in 2 mm MgCl2 using the following protocol: denaturation for 1 min at 94 °C, annealing for 1 min at 67 °C, and extension for 2 min at 72 °C. The amplified α10I domain cDNA was digested along with the pMAL-c expression vector (New England Biolabs) or pGEX-2T expression vector (Amersham Pharmacia Biotech) using the Bam HI and Eco RI restriction enzymes (Promega). The cDNA was ligated to the pMAL-c vector with T4 DNA ligase (Promega). To the pGEX-2T vector the α10I domain cDNA was ligated with the SureClone ligation kit (Amersham Pharmacia Biotech). The constructs were transformed into the Escherichia coli BL21 strain for production. The DNA sequences of the constructs were checked with DNA sequencing and compared with the published α10 DNA sequence (10Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) (data not shown). Site-directed mutation of the α10I domain cDNA in a pGEX-2T vector was made using PCR according to Stratagene’s QuikChange mutagenesis kit instructions. Briefly, mutagenesis was based on primers that contained the planned mutations (primers were delivered by CyberGene). At 68 °C the proofreading Pfu DNA polymerase (Promega) makes only one copy of the plasmid without replacing the primers. The mutated strands were then selected from parental ones by digesting the template withDpn I endonuclease (Promega), which digests only methylated and hemimethylated DNA. By that means only mutated strands remained. The presence of mutations was checked by DNA sequencing. Mutant constructs were then transformed into E. coli strain BL21 for production of recombinant protein. The α1I domain was cloned into the pGEX-4T vector (Amersham Pharmacia Biotech) as described earlier by Nykvistet al. (21Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The full-length integrin α1cDNA was a gift from Dr. E. Marcantonio (Columbia University, New York). The α1I domain cDNA sequence differed from the originally published sequence (23Briesewitz R. Epstein M.R. Marcantonio E.E. J. Biol. Chem. 1993; 268: 2989-2996Abstract Full Text PDF PubMed Google Scholar) at one position, and as a result the amino acid lysine was replaced with glutamate at position 170 (numbered from the beginning of the mature peptide). Site-directed mutations to α1I domain cDNA were made as stated above for the α10I domain. Human integrin α2I domain was generated as described earlier by Ivaska et al. (24Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.-M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The integrin α2 cDNA was a gift from Dr. M. Hemler, Dana Farber, Boston. The α2I domain was cloned into the pGEX-2T vector (Amersham Pharmacia Biotech). The site-directed mutations were made as stated above for the α10I domain. To produce the wild type α10I domain as a fusion with the maltose-binding protein (MBP), 500 ml of LB medium containing 50 μg/ml ampicillin was inoculated with an overnight culture of 10 ml ofE. coli BL21 cells transformed with the pMAL-cα10I vector. Cells were grown at 37 °C for about 4 h. Protein production was induced with isopropyl-β-d-thiogalactoside (Amersham Pharmacia Biotech) and allowed to continue for 2 h. The cells were pelleted by centrifugation (4,000 × g, 20 min, 4 °C). Cell pellets were resuspended in column buffer (20 mm Tris-HCl, pH 7.4, 200 mm NaCl, 1 mm EDTA, 10 mm β-mercaptoethanol) and stored at −20 °C until purification. The cell suspension was thawed on ice and sonicated in 15-s bursts for 3 min. Cell debris was removed by centrifugation (9,000 × g, 30 min, 4 °C). The supernatant was incubated with amylose resin (New England BioLabs) in an end-over-end rotor at 4 °C overnight. Amylose resin with bound MBP-α10I fusion was transferred to 10-ml columns (Bio-Rad) and washed three times with 10 ml of column buffer. The bound fusion protein was eluted with 5 mm maltose in column buffer after a 2–3-h incubation at room temperature. Maltose was removed using hydroxyapatite, first washed with column buffer. One ml of washed hydroxyapatite was mixed with eluted protein and incubated in an end-over-end rotor for 1 h at 4 °C, after which the fusion protein was attached to the hydroxyapatite. Soluble maltose was washed away with column buffer, repeated three times. The fusion protein was eluted from the hydroxyapatite with sodium phosphate buffer after a 30-min incubation at 4 °C. The protein concentrations were measured using Bradford’s method (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). The purity and folding of the proteins were checked by SDS- and native polyacrylamide gel electrophoresis (PhastSystem, Amersham Pharmacia Biotech; data not shown). The wild type and mutated α1I, α2I, and α10I domains were produced as glutathioneS-transferase (GST)-αI domain fusion proteins. 500 ml of LB medium (with 50 μg/ml ampicillin) was inoculated with an overnight culture of E. coli BL21 cells transformed with either wild type or mutant plasmid. The cultures were grown at 37 °C until theA 600 was in the range of 0.5–1. Next, an inducer of the tac promoter, isopropyl β-d-thiogalactoside, was added to a final concentration of 0.4 mm, and the temperature was lowered to room temperature. The bacteria were allowed to produce the recombinant protein for 3–4 h after which the cultures were centrifuged (5,550 rpm, 10 min, 4 °C) to collect the cells. Cells were stored until purification at −20 °C overnight and at −70 °C for longer times. The bacterial cells were resuspended in PBS for purification. The cell suspension was sonicated and detergent (Triton X-100) was added to a final concentration of 1%. To dissolve the recombinant protein, the disrupted cells were incubated with agitation and Triton X-100 for 1 h. Cell debris was removed by centrifugation (17,000 rpm, 10 min, 4 °C). Glutathione-Sepharose 4B (Amersham Pharmacia Biotech) was added to the supernatant, and the mixture was incubated for 1–1.5 h in gentle agitation. The glutathione-Sepharose 4B was then transferred to columns (Bio-Rad), and unbound proteins were washed away with PBS. The bound fusion protein was eluted with reduced glutathione (Sigma). Protein concentrations were measured with Bradford’s method (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). The purity and folding of the fusion proteins were checked with SDS- and native polyacrylamide gel electrophoresis (data not shown). The binding of wild type or mutated I domains to collagen was studied using solid phase binding assays. Ninety-six-well microtiter plates (Wallac) were coated with each collagen type: I (rat tail, Sigma), II (bovine, Chemicon), III (human, Chemicon), IV (Engelbreth-Holm-Swarm mouse sarcoma basement membrane, Sigma), V (human, Chemicon), or VI (human, Biodesign International) or with laminin-1 (Engelbreth-Holm-Swarm mouse sarcoma basement membrane, Sigma), tenascin (chicken, Chemicon) 5 μg/cm2 in PBS and control wells with Diluent II (containing BSA, Wallac) 1:2 in PBS at 4 °C overnight. Before carrying out the binding assays, wells were blocked with Diluent II for 1 h at 37 °C. Dilutions of the I domains were made with assay buffer (Wallac) and added to the wells. A concentration series of 1–500 nm was used for the I domains. The I domains were allowed to attach to the wells for 1 h at 37 °C in the presence of 2 mm MgCl2, unless stated otherwise. The unattached I domain was washed away with PBS containing 2 mm MgCl2, repeated three times. Detection of the I domain was based on the existence of the GST or MBP fusion partner. Goat anti-GST antibody (Amersham Pharmacia Biotech) or anti-MBP (New England Biolabs) was added to the wells in a 1:8,000 or 1:1,000 dilution, respectively, in assay buffer and incubated for 1 h at 37 °C. The wells were then washed three times with PBS containing 2 mm MgCl2. Europium3+-labeled protein G (Wallac) (1:100 in assay buffer) was then added to the wells and incubated for 1 h at 37 °C. Wells were washed three times with PBS containing MgCl2. Enhancement solution (Wallac) was used to dissociate the highly fluorescent Europium3+ label. The fluorescence was measured with a time-resolved fluorometer (Victor2, Wallac). All assays were performed at least in triplicate. Three-dimensional structures of the integrin α1I domain in the apo form (1qcy, see Ref. 25Salminen T.A. Nymalm Y. Kankare J. Käpylä J. Heino J. Johnson M.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1365-1367Crossref PubMed Scopus (13) Google Scholar; Mg2+ present but no bound ligand) and the apo and holo (bound hexapeptide collagen fragment) forms (1aox, Ref. 26Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar; 1dzi, Ref. 17Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (845) Google Scholar) were obtained from the Protein Data Bank (27Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27938) Google Scholar). The sequence alignment of human α1I, α2I, and α10I domains was made using MALIGN (28Johnson M.S. Overington J.P. J. Mol. Biol. 1993; 233: 716-738Crossref PubMed Scopus (266) Google Scholar) in the BODIL modeling package 2J. Lehtonen, V.-V. Rantanen, D.-J. Still, M. Gyllenberg, and M. S. Johnson, unpublished results. using a structure-based sequence comparison matrix (29Johnson M.S. May A.C.W. Rodionov M.A. Overington J.P. Methods Enzymol. 1996; 266: 575-598Crossref PubMed Google Scholar). MALIGN constructs a multiple sequence alignment from pairwise alignments according to a tree relating the sequences being matched. The alignment between the three I domains contains no gaps and for α1I and α2I matches their structural alignment. The sequence identity between the α2I domain and the α1I domain is 52% and between the α2I domain and the α10I domain is 46%, thus leading to high quality reliable models. The program MODELLER 4.0 (30Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar) was used to construct three-dimensional model structures of the holo form of the α1I domain and both the apo and holo forms of the α10I domain. α10I domain was produced in E. coli as a recombinant protein fused to MBP or GST. The recombinant proteins were purified with affinity chromatography and characterized with SDS- and native polyacrylamide gel electrophoresis (data not shown). The proteins appeared as one band in native polyacrylamide gel electrophoresis, suggesting the presence of a single molecular form. The binding of purified recombinant α10I domain to collagen was studied using a solid phase binding assay. Wells were coated with different collagens, laminin-1, or tenascin (5 μg/cm2). α10I fusion protein (300 nm) was allowed to bind to the coated wells. The α10I domain was found to bind to cartilage-derived type II collagen in a magnesium-dependent manner (Fig.1 A) because 5 mmEDTA inhibited binding completely. Thus, the basic binding mechanism of α10I is cation-dependent as are the other integrin αI domains (16Mischishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 17Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (845) Google Scholar). To investigate whether other fibrillar collagens, namely subtypes I, II, III, and V, also can function as ligands for α10I, a binding assay was carried out (Fig.1 B). The α10I domain bound to the collagen subtypes I–III nearly equally, but in repeated experiments the binding to type V collagen was weaker. Collagen receptors α1β1 and α2β1can both bind to laminin-1 (31Tomaselli K.J. Hall D.E. Flier L.A. Gehlsen K.R. Turner D.C. Carbonetto S. Reichardt L.F. Neuron. 1990; 5: 651-662Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 32Languino L.R. Gehlsen K.R. Wayner E. Carter W.G. Engvall E. Ruoslahti E. J. Cell Biol. 1989; 109: 2455-2462Crossref PubMed Scopus (259) Google Scholar). α2β1is also able to bind tenascin (33Sriramarao P. Mendler M. Bourdon M.A. J. Cell Sci. 1993; 105: 1001-1012Crossref PubMed Google Scholar). The binding of α10I to these extracellular matrix proteins was also studied. The α10I domain was found to bind to laminin-1, but binding to tenascin was only slightly better than to BSA (Fig.1 C). The ligand recognition patterns of α10I-GST and α10I-MBP were identical. In some experiments the α10I-MBP showed higher unspecific binding to BSA than α10I-GST (data not shown). Integrins α1β1 and α2β1and their corresponding αI domains differ in collagen binding specificity even though they are structurally highly similar. Overall about half of their αI domain sequence is identical, and the αI domain binding surfaces resemble each other. The α1I domain prefers type IV collagen over type I collagen, whereas the preference of α2I is reversed (20Kern A. Eble J. Golbik R. Kuhn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar). To study the binding of these αI domains to collagen type VI in addition to collagen types I and IV, binding assays with an αI domain concentration series of 1–500 nm were carried out (Fig.2). The results were fit to the Michaelis-Menten equation to obtain estimates of theKd of binding. In accordance with previous studies, the integrin α1I domain bound better to type IV collagen (Kd ≈ 65 ± 20 nm;panel A) than type I collagen (Kd ≈ 160 ± 40 nm). The integrin α2I domain, in contrast, associated considerably better with collagen type I (Kd ≈ 20 ± 5 nm) than with collagen type IV (Kd ≈ 140 ± 30 nm; panel B). Type VI collagen forms beaded filaments, and it has been reported to be a ligand of both α1β1 and α2β1 integrins (34Loeser R.F. Arthritis Rheum. 1997; 40: 270-276Crossref PubMed Scopus (114) Google Scholar). However, in chondrocytes α1β1 may be the preferred receptor for collagen type VI (35Loeser R.F. Sadiev S. Tan L. Goldring M.B. Osteoarthr. Cartil. 2000; 8: 96-105Abstract Full Text PDF PubMed Scopus (160) Google Scholar). Here, the binding of the α1I and α2I domains to type VI collagen was tested (Fig. 2). Integrin α1I domain showed the strongest binding (Kd ≈ 200 ± 80 nm; Fig. 2 C), whereas the binding of the α2I domain was so weak that no reasonable estimate of the half-maximal value for binding could be obtained (Fig. 2 D). To test whether the α10I domain can also bind collagens other than the fibrillar subtypes and to get quantitative information, a binding assay with collagen types I, IV, and VI was carried out. In the solid phase assay the range of α10I domain concentrations spanned 1–500 nm. The α10I domain was found to bind type IV collagen slightly better (Kd ≈ 300 ± 100 nm) than collagen types I and VI (Fig.3). The estimatedKd for binding to fibrillar collagen type I was 350 ± 150 nm, similar to the value for collagen type VI binding (Kd ≈ 350 ± 200 nm). In terms of binding properties, these results place the α10I domain closer to the α1I domain, which prefers collagen type IV over collagen type I or VI. Moreover, the fact that the α10I domain binds well to collagen type VI, in contrast to the α2I domain, suggests that the binding preferences of the α1I and α10I domains are similar. Sequence differences in the vicinity of the binding site were identified by using the sequence alignment of the α1I, α2I, and α10I domains, and the x-ray structures of the apo form of the α1I domain (25Salminen T.A. Nymalm Y. Kankare J. Käpylä J. Heino J. Johnson M.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1365-1367Crossref PubMed Scopus (13) Google Scholar) and both the apo and holo forms (1aox, Ref. 26Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar; 1dzi, Ref.17Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (845) Google Scholar) of the α2I domain. To make it easier to visualize the differences among the surface regions surrounding the MIDAS site, both the α1I (holo form) and α10I (apo and holo forms) domain structures were modeled using MODELLER 4.0. The electrostatic potentials were mapped to the solvent accessible surfaces of the apo and holo forms (Fig. 4) for each of the αI domains, and the sequence differences are listed in Table I.Table ISequence differences in the vicinity of the MIDAS site in the α1I, α2I, and α10I domainsAmino acid numberingThree-letter code for an amino acidα1,10α2α1α2α10218219ArgAspArg219220QlnLeuGlu259260AsnGlyGly260261HisSerGlu285286TyrLeuTyr288289GlyAsnArg290291LeuLeuArg291292SerAspAsp294295LysAsnSer297298GluLysArg301302SerAlaThrAmino acids that are conservatively replaced among the integrins or are less likely to be involved in ligand bindingα1,10α2α1α2α10284285SerTyrHis286287AsnAsnLeu289290AsnAlaQln292293ThrThrPro293294" @default.
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