The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alaninesubstituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.The mammalian discoidin domain receptors (DDRs), 5 DDR1, and DDR2, are receptor tyrosine kinases (RTKs) that function as collagen receptors (1, 2). Several collagen types, in particular fibrillar collagens, bind to and activate the DDRs, with the two receptors displaying different specificities toward certain collagen types (3, 4). DDR activation by collagen is strictly dependent on the native, triple-helical conformation of collagen (1, 2, 5). The DDRs are unique among RTKs in being activated by a component of the extracellular matrix; most RTKs are activated by small diffusible proteins such as growth factors. Like conventional RTKs, the DDRs regulate fundamental cellular processes including cell proliferation, adhesion, and migration, but the DDRs additionally control remodeling of the extracellular matrix (6 -9). Both receptors control developmental processes, such as mammary gland development (DDR1) (10) and the growth of long bones (DDR2) (11), and are associated with human diseases, including fibrotic diseases of the liver, kidney, and lung, atherosclerosis, osteoarthritis, and several types of cancer (reviewed in Ref. 12).The homologous DDRs are composed of an N-terminal discoidin homology (DS) domain followed by a stalk region unique to DDRs (ϳ220 amino acids), a transmembrane domain, a large cytosolic juxtamembrane domain, and a C-terminal tyrosine kinase domain (13). DDR activation, manifested by autophosphorylation, is a consequence of collagen binding to a specific site in the DS domain (5, 14). Collagen-induced DDR autophosphorylation is unusually slow and sustained (1, 2) when compared with the rapid response of canonical RTKs to their ligands.Collagens form a large protein family that is characterized by repeating glycine-X-XЈ sequences, where X and XЈ are often proline and 4-hydroxyproline, respectively (15). Three collagen chains form a right-handed triple-helical struct...

SummaryThe discoidin domain receptors, DDR1 and DDR2, are widely expressed receptor tyrosine kinases that are activated by triple-helical collagen. They control important aspects of cell behavior and are dysregulated in several human diseases. The major DDR2-binding site in collagens I–III is a GVMGFO motif (O is hydroxyproline) that also binds the matricellular protein SPARC. We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide. The GVMGFO motifs of two collagen chains are recognized by an amphiphilic pocket delimited by a functionally critical tryptophan residue and a buried salt bridge. Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation. A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. We have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha2beta1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where O is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

Background: Hedgehog acyltransferase (HHAT) palmitoylates hedgehog proteins and is a potential target in cancer.Results: HHAT has ten transmembrane domains, two reentrant loops, and four palmitoylation sites.Conclusion: HHAT topology is determined, and protein is multipalmitoylated, which modulates protein function.Significance: Elucidating HHAT topology and posttranslational modifications is crucial to understand its acyltransferase activity and to develop new strategies to treat cancer.