title: Normal activation of discoidin domain receptor 1 mutants with disulfide cross-links, insertions, or deletions in the extracellular juxtamembrane region: mechanistic implications.
abstract: The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.
authors.affiliation: From the National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom and.
authors.forename: Huifang
authors.initials: H
authors.lastname: Xu
authors.forename: Takemoto
authors.initials: T
authors.lastname: Abe
authors.forename: Justin K H
authors.initials: JK
authors.lastname: Liu
authors.forename: Irina
authors.initials: I
authors.lastname: Zalivina
authors.forename: Erhard
authors.initials: E
authors.lastname: Hohenester
authors.forename: Birgit
authors.initials: B
authors.lastname: Leitinger
mesh_terms: D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000070818:Discoidin Domain Receptors; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D004220:Disulfides; D011975:Receptors, Mitogen; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D003545:Cysteine
keywords: Activation Mechanism; Collagen; Cysteine Scanning; Mutagenesis Site-specific; Receptor Regulation; Receptor Structure-Function; Receptor Tyrosine Kinase; Signal Transduction