Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jan;30(1):16-26.
doi: 10.1016/j.matbio.2010.10.004. Epub 2010 Oct 28.

Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

Huifang Xu  1 , Nicolas Raynal, Stavros Stathopoulos, Johanna Myllyharju, Richard W Farndale, Birgit Leitinger
Affiliations

Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

Huifang Xu et al. Matrix Biol. 2011 Jan.

Abstract

The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Schematic representation of recombinant DDR-Fc constructs. The DDR extracellular domains contain an N-terminal DS domain that contains the collagen binding site, followed by a so-called stalk region. Fc-tagged proteins are fused to the Fc fragment of human IgG1 or IgG2, as indicated, with the Fc sequences (and linker amino acids) added after the last amino acid of the DDR ectodomains (T416 for DDR1, T398 for DDR2). The new constructs are shown on the right, and contain a three amino acid linker (Ala-Ala-Ala), symbolized by a triangle, between the DDR ectodomains and the IgG2 Fc sequence.
Fig. 2
Fig. 2
Identification of DDR1 and DDR2 binding sites on collagen II. Binding of recombinant DDR1-Fc2 or DDR2-Fc to immobilized collagen II Toolkit peptides in a solid phase binding assay. Recombinant DDR-Fc proteins were added for 3 h at room temperature to 96 wells coated with collagen or peptides at 10 μg/ml. Bound proteins were detected with anti-Fc antibodies and measured as absorbance at 492 nm. (A) Binding of DDR1-Fc2, added at 20 μg/ml (220 nM). (B) Binding of DDR2-Fc, added at 10 μg/ml (110 nM). Shown are the mean +/− SD of three to four independent experiments, each performed in triplicates. CI: rat tail collagen I; CII: bovine collagen II; Cas: casein; GPP and CRP: peptides without specific collagen sequences. GPP: (GPP)10; CRP: (GPO)10.
Fig. 3
Fig. 3
Identification of DDR1 and DDR2 binding sites on collagen III. Binding of recombinant DDR1-Fc2 or DDR2-Fc to immobilized collagen III Toolkit peptides in a solid phase binding assay. Recombinant DDR-Fc proteins were added for 3 h at room temperature to 96 wells coated with collagen or peptides at 10 μg/ml. Bound proteins were detected with anti-Fc antibodies and measured as absorbance at 492 nm. (A) Binding of DDR1-Fc2, added at 20 μg/ml (220 nM). (B) Binding of DDR2-Fc, added at 10 μg/ml (110 nM). Shown are the mean +/− SD of three to four independent experiments, each performed in triplicates. CI: rat tail collagen I; CIII: recombinant human collagen III; Cas: casein; GPP and CRP peptides as in Fig. 2.
Fig. 4
Fig. 4
Characterisation of collagen sequence required for DDR1 binding. Recombinant DDR1-Fc protein was added for 3 h at room temperature to 96 wells coated with collagen-derived peptides or collagen at 10 μg/ml. (A) Binding of DDR1-Fc to collagen III Toolkit peptide, III-23, and truncated peptides derived from this sequence (Table 1). (B) Binding of DDR1-Fc to alanine-substituted peptides derived from collagen III Toolkit peptide, III-23 (Table 1). Shown are the mean +/− SD of four independent experiments, each performed in triplicates. Black bars, DDR1-Fc added at 15 μg/ml (165 nM); grey bars, DDR1-Fc added at 3 μg/ml (33 nM).
Fig. 5
Fig. 5
DDR1 binding peptides mediate autophosphorylation of cell surface DDR1. Full length DDR1a was transiently expressed in HEK293 cells. After stimulation for 90 min with collagen peptides at 100 μg/ml, cell lysates were analysed by SDS-PAGE and Western blotting. Peptide names refer to Table 1. The blot was probed with anti-phosphotyrosine mAb 4G10 (upper panel), followed by stripping and reprobing with anti-DDR1 (lower panel). The positions of molecular weight markers (in kDa) are indicated. The experiment was carried out four times with very similar results.
Fig. 6
Fig. 6
DDR2 binding peptides that do not contain the GVMGFO motif do not mediate strong autophosphorylation of cell surface DDR2. Full length DDR2 was transiently expressed in HEK293 cells. After stimulation for 90 min with collagen peptides, cell lysates were analysed by SDS-PAGE and Western blotting. Peptide names refer to Table 1. Peptides were used at 500 μg/ml, except peptide GPR-GFO, which was used at 100 μg/ml. Cell lysates were resolved on two gels. The corresponding blots were probed with anti-phosphotyrosine mAb 4G10 (upper panel) or anti-DDR2 (lower panel). The positions of molecular weight markers (in kDa) are indicated.
Fig. 7
Fig. 7
Sequence conservation of the DDR collagen binding sites. (A) Sequence alignment of the DS domains of human DDR1 and DDR2. Secondary structure elements are given above the alignment, sequence numbering to the right of the alignment. Conserved residues are highlighted in grey. The central residues of the GVMGFO binding interface (Trp52, Thr56, Asp69, Arg105, Glu113, and Cys73-Cys177 disulfide bridge (see Carafoli et al., 2009)), are indicated by two asterisks. Additional residues that lose ≥ 5 Å2 of their solvent-accessible surface upon collagen binding (see Carafoli et al., 2009) are indicated by one asterisk. Non-conserved residues that lose ≥ 5 Å2 of their solvent-accessible surface upon collagen binding are highlighted in white on black background. (B) Amino acid replacements in DDR2–DDR1 hybrid constructs.
Fig. 8
Fig. 8
Replacing five amino acids in the collagen binding site of DDR2 with DDR1 amino acids leads to collagen IV recognition. Solid phase binding assay with recombinant DDR2 wild-type and DDR2–DDR1 hybrid constructs. DDR2-Fc proteins were added for 3 h at room temperature to 96 wells coated with collagen at 10 μg/ml. Bound proteins were detected with anti-Fc antibodies and measured as absorbance at 492 nm. (A) DDR2 wild-type, (B) DDR2-H1, (C) DDR2-H2, (D) DDR2-H3 binding to different immobilized proteins: BSA (▲); human placental collagen IV (□); rat tail collagen I (●). Shown is a representative of three independent experiments, each performed in duplicates.
Fig. 9
Fig. 9
Replacing five amino acids in the collagen binding site of DDR2 with DDR1 amino acids leads to collagen IV-induced signalling of cell surface DDR2-H3. Full length DDR1a, DDR2 wild-type or DDR2-DDR1 hybrid constructs were transiently expressed in HEK293 cells. After stimulation for 90 min with collagen at 10 μg/ml (collagen I, CI) or 50 μg/ml (collagen IV, CIV), cell lysates were analysed by SDS-PAGE and Western blotting. (A) DDR1 expression. The blot was probed with anti-phosphotyrosine mAb 4G10 (upper panel), followed by stripping and reprobing with anti-DDR1 (lower panel). (B) DDR2 wild-type and DDR2-DDR1 hybrid receptor expression. Cell lysates were resolved on two gels. The corresponding blots were probed with anti-phosphotyrosine mAb 4G10 (upper panel) or anti-DDR2 (lower panel). The positions of molecular weight markers (in kDa) are indicated. The experiment was carried out three times with very similar results.
Fig. 10
Fig. 10
Characterisation of binding sites for the DDR2–DDR1 hybrid protein DDR2-H3 on collagens II and III. Binding of recombinant DDR2-H3-Fc to immobilized collagen II Toolkit peptides (A) or collagen III Toolkit peptides (B) in a solid phase binding assay. Recombinant DDR2-H3-Fc protein was added at 10 μg/ml (110 nM) for 3 h at room temperature to 96 wells coated with collagen or peptides at 10 μg/ml. Bound protein was detected with anti-Fc antibodies and measured as absorbance at 492 nm. Shown are the mean +/− SD of four independent experiments, each performed in triplicates. CI: rat tail collagen I; CII: bovine collagen II; CIII: recombinant human collagen III; CIV: placental collagen IV; GPP and CRP peptides as in Fig. 2.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abdulhussein R., McFadden C., Fuentes-Prior P., Vogel W.F. Exploring the collagen-binding site of the DDR1 tyrosine kinase receptor. J. Biol. Chem. 2004;279:31462–31470. - PubMed
    1. Abdulhussein R., Koo D.H., Vogel W.F. Identification of disulfide-linked dimers of the receptor tyrosine kinase DDR1. J. Biol. Chem. 2008;283:12026–12033. - PMC - PubMed
    1. Ali B.R., Xu H., Akawi N.A., John A., Karuvantevida N.S., Langer R., Al-Gazali L., Leitinger B. Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients. Hum. Mol. Genet. 2010;19:2239–2250. - PMC - PubMed
    1. Alves F., Vogel W., Mossie K., Millauer B., Hofler H., Ullrich A. Distinct structural characteristics of discoidin I subfamily receptor tyrosine kinases and complementary expression in human cancer. Oncogene. 1995;10:609–618. - PubMed
    1. Bargal R., Cormier-Daire V., Ben-Neriah Z., Le Merrer M., Sosna J., Melki J., Zangen D.H., Smithson S.F., Borochowitz Z., Belostotsky R., Raas-Rothschild A. Mutations in DDR2 gene cause SMED with short limbs and abnormal calcifications. Am. J. Hum. Genet. 2009;84:80–84. - PMC - PubMed

Publication types

MeSH terms