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Abstract
The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.
Highlights
The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen
Soluble DDR extracellular domains (ECDs) Bind to Immobilized Collagen with High Affinity—Human DDR1 and DDR2 are classified as collagen receptors [3, 4], but a direct protein-protein binding assay that allows an estimation of binding strength has been lacking
Recombinant proteins corresponding to the ECDs of the DDRs, N-terminally tagged with a His tag and a Myc epitope were produced in stably transfected human 293-EBNA cells and purified from serum-free medium. his-DDR2 exhibited dosedependent, saturable binding to rat tail collagen I, whereas his-DDR1 did not display any binding above background levels (Fig. 2A). his-DDR2 binding to collagen I was of high affinity, with half-maximal binding at ϳ10 –20 nM his-DDR2
Summary
The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. The two closely related receptors of the discoidin domain receptor (DDR) RTK subfamily, DDR1 and DDR2, are unusual in that they are activated by an extracellular matrix protein, triple-helical collagen [3, 4]. This activation is independent of the major cellular collagen receptors, 1 integrins, as shown for DDR1 [5]. To gain insight into the molecular basis of DDR-collagen signaling, I have studied an array of recombinant DDR proteins, obtained by eukaryotic expression, in collagen binding
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