scholarly journals Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Victoria Juskaite ◽  
David S Corcoran ◽  
Birgit Leitinger
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Drug Target ◽  
Kinase Activity ◽  
Transmembrane Domain ◽  
Activation Mechanism ◽  
Collagen Binding ◽  
Wide Range ◽  
Different Types ◽  
In Trans ◽  
Discoidin Domain Receptor 1

The collagen-binding receptor tyrosine kinase DDR1 (discoidin domain receptor 1) is a drug target for a wide range of human diseases, but the molecular mechanism of DDR1 activation is poorly defined. Here we co-expressed different types of signalling-incompetent DDR1 mutants (‘receiver’) with functional DDR1 (‘donor’) and demonstrate phosphorylation of receiver DDR1 by donor DDR1 in response to collagen. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor function, we show that receiver dimers are phosphorylated in trans by the donor; this process requires the kinase activity of the donor but not that of the receiver. The receiver ectodomain is not required, but phosphorylation in trans is abolished by mutation of the transmembrane domain. Finally, we show that mutant DDR1 that cannot bind collagen is recruited into DDR1 signalling clusters. Our results support an activation mechanism whereby collagen induces lateral association of DDR1 dimers and phosphorylation between dimers.

scholarly journals DDR1 autophosphorylation is a result of aggregation into dense clusters

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
David S. Corcoran ◽  
Victoria Juskaite ◽  
Yuewei Xu ◽  
Frederik Görlitz ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Super Resolution ◽  
Activation Kinetics ◽  
Molecular Events ◽  
Wide Range ◽  
Human Disorders ◽  
Resolution Imaging ◽  
Collagen Receptor

AbstractThe collagen receptor DDR1 is a receptor tyrosine kinase that promotes progression of a wide range of human disorders. Little is known about how ligand binding triggers DDR1 kinase activity. We previously reported that collagen induces DDR1 activation through lateral dimer association and phosphorylation between dimers, a process that requires specific transmembrane association. Here we demonstrate ligand-induced DDR1 clustering by widefield and super-resolution imaging and provide evidence for a mechanism whereby DDR1 kinase activity is determined by its molecular density. Ligand binding resulted in initial DDR1 reorganisation into morphologically distinct clusters with unphosphorylated DDR1. Further compaction over time led to clusters with highly aggregated and phosphorylated DDR1. Ligand-induced DDR1 clustering was abolished by transmembrane mutations but did not require kinase activity. Our results significantly advance our understanding of the molecular events underpinning ligand-induced DDR1 kinase activity and provide an explanation for the unusually slow DDR1 activation kinetics.

scholarly journals Two-step release of kinase autoinhibition in discoidin domain receptor 1

2020 ◽  
Vol 117 (36) ◽  
pp. 22051-22060
Author(s):  
Douglas Sammon ◽  
Erhard Hohenester ◽  
Birgit Leitinger
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Active Site ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Crystal Structure Analysis ◽  
Activation Loop ◽  
Discoidin Domain Receptor ◽  
Important Regulator ◽  
Discoidin Domain Receptor 1

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase with important functions in organogenesis and tissue homeostasis. Aberrant DDR1 activity contributes to the progression of human diseases, including fibrosis and cancer. How DDR1 activity is regulated is poorly understood. We investigated the function of the long intracellular juxtamembrane (JM) region of human DDR1 and found that the kinase-proximal segment, JM4, is an important regulator of kinase activity. Crystal structure analysis revealed that JM4 forms a hairpin that penetrates the kinase active site, reinforcing autoinhibition by the activation loop. Using in vitro enzymology with soluble kinase constructs, we established that release from autoinhibition occurs in two distinct steps: rapid autophosphorylation of the JM4 tyrosines, Tyr569 and Tyr586, followed by slower autophosphorylation of activation loop tyrosines. Mutation of JM4 tyrosines abolished collagen-induced DDR1 activation in cells. The insights may be used to develop allosteric, DDR1-specific, kinase inhibitors.

scholarly journals ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1)

2015 ◽  
Vol 26 (4) ◽  
pp. 659-673 ◽  
Author(s):  
Yasuyuki Shitomi ◽  
Ida B. Thøgersen ◽  
Noriko Ito ◽  
Birgit Leitinger ◽  
Jan J. Enghild ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Receptor Tyrosine Kinase ◽  
Ectodomain Shedding ◽  
Efficient Manner ◽  
Collagen Binding ◽  
Collagen Matrices ◽  
Discoidin Domain Receptor ◽  
Discoidin Domain Receptor 1

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and transmits signals from various collagens in epithelial cells. However, how DDR1–dependent signaling is regulated has not been understood. Here we report that collagen binding induces ADAM10-dependent ectodomain shedding of DDR1. DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner. DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1. Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor. Our data also revealed that ADAM10 plays an important role in regulating DDR1-mediated cell adhesion to achieve efficient cell migration on collagen matrices.

c-kit associated with the transmembrane 4 superfamily proteins constitutes a functionally distinct subunit in human hematopoietic progenitors

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4413-4421 ◽  
Author(s):  
Naoyuki Anzai ◽  
Younghee Lee ◽  
Byung-S. Youn ◽  
Seiji Fukuda ◽  
Young-June Kim ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Myeloid Cell ◽  
Hematopoietic Progenitors ◽  
Modulate Function ◽  
Kit Receptor ◽  
Wide Range ◽  
Transmembrane 4 Superfamily

The transmembrane 4 superfamily (TM4SF) has come into prominence for its association with a wide range of cell surface molecules, especially integrins. We report that TM4SF molecules CD9, CD63, and CD81 are physically associated with c-kit receptor tyrosine kinase in the human factor–dependent myeloid cell line, MO7e. We characterized this complex using coimmunoprecipitation and colocalization methods. The c-kit coimmunoprecipitated with anti-TM4SF antibodies showed several distinct phenotypes compared to the total c-kit immunoprecipitated with anti–c-kit antibody. These included: (1) higher basal level of tyrosine phosphorylation without elevated kinase activity in the absence of Steel factor (SLF), (2) deficient enhancement of tyrosine phosphorylation and kinase activity in response to SLF, (3) elevated binding rate of SLF shown in chemical cross-linking studies, and (4) little internalization and degradation after SLF treatment. Cocapping studies in living cells showed that c-kit colocalized with TM4SF molecules after SLF stimulation, suggesting confirmation of the biochemical data obtained by the coimmunoprecipitation studies. Colocalization of c-kit with CD81 by SLF was also observed in cord blood CD34+ cells, suggesting the existence of functional units of c-kit in TM4SF complexes in primary hematopoietic cells. This suggests that some TM4SF members may negatively modulate function of c-kit receptor tyrosine kinase and thus regulate receptor sensitivity to SLF in hematopoietic progenitors.

scholarly journals A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Daiane S Alves ◽  
Justin M Westerfield ◽  
Xiaojun Shi ◽  
Vanessa P Nguyen ◽  
Katherine M Stefanski ◽  
...  
Keyword(s):  
Cell Migration ◽  
Receptor Tyrosine Kinase ◽  
Lipid Membranes ◽  
Transmembrane Domain ◽  
Activation Mechanism ◽  
Extracellular Medium ◽  
Akt Phosphorylation ◽  
Signaling Axis ◽  
Membrane Peptide ◽  
Ph Dependent

Misregulation of the signaling axis formed by the receptor tyrosine kinase (RTK) EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts that contribute to metastasis. Solid tumors are characterized by an acidic extracellular medium. We intend to take advantage of this tumor feature to design new molecules that specifically target tumors. We created a novel pH-dependent transmembrane peptide, TYPE7, by altering the sequence of the transmembrane domain of EphA2. TYPE7 is highly soluble and interacts with the surface of lipid membranes at neutral pH, while acidity triggers transmembrane insertion. TYPE7 binds to endogenous EphA2 and reduces Akt phosphorylation and cell migration as effectively as ephrinA1. Interestingly, we found large differences in juxtamembrane tyrosine phosphorylation and the extent of EphA2 clustering when comparing TYPE7 with activation by ephrinA1. This work shows that it is possible to design new pH-triggered membrane peptides to activate RTK and gain insights on its activation mechanism.

scholarly journals Her2 activation mechanism reflects evolutionary preservation of asymmetric ectodomain dimers in the human EGFR family

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Anton Arkhipov ◽  
Yibing Shan ◽  
Eric T Kim ◽  
Ron O Dror ◽  
David E Shaw
Keyword(s):  
Molecular Dynamics ◽  
Tyrosine Kinase ◽  
Molecular Dynamics Simulations ◽  
Receptor Tyrosine Kinase ◽  
Drug Target ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Egfr Family ◽  
Dynamics Simulations ◽  
Necessary And Sufficient

The receptor tyrosine kinase Her2, an intensely pursued drug target, differs from other members of the EGFR family in that it does not bind EGF-like ligands, relying instead on heterodimerization with other (ligand-bound) EGFR-family receptors for activation. The structural basis for Her2 heterodimerization, however, remains poorly understood. The unexpected recent finding of asymmetric ectodomain dimer structures of Drosophila EGFR (dEGFR) suggests a possible structural basis for Her2 heterodimerization, but all available structures for dimers of human EGFR family ectodomains are symmetric. Here, we report results from long-timescale molecular dynamics simulations indicating that a single ligand is necessary and sufficient to stabilize the ectodomain interface of Her2 heterodimers, which assume an asymmetric conformation similar to that of dEGFR dimers. This structural parallelism suggests a dimerization mechanism that has been conserved in the evolution of the EGFR family from Drosophila to human.

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

2017 ◽  
Author(s):  
Jana Shen ◽  
Zhi Yue ◽  
Helen Zgurskaya ◽  
Wei Chen
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Proton Release ◽  
Intrinsic Resistance ◽  
Constant Ph ◽  
Wide Range ◽  
Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood. <p><br></p>

Nanoparticles Functionalized with Venom-Derived Peptides and Toxins for Pharmaceutical Applications

2020 ◽  
Vol 21 (2) ◽  
pp. 97-109 ◽  
Author(s):  
Ana P. dos Santos ◽  
Tamara G. de Araújo ◽  
Gandhi Rádis-Baptista
Keyword(s):  
Oral Absorption ◽  
Current Data ◽  
Pharmacological Activities ◽  
Venom Peptides ◽  
Essential Components ◽  
Wide Range ◽  
Different Types ◽  
Direct Use ◽  
Animal Venoms ◽  
Pharmaceutical Biotechnology

Venom-derived peptides display diverse biological and pharmacological activities, making them useful in drug discovery platforms and for a wide range of applications in medicine and pharmaceutical biotechnology. Due to their target specificities, venom peptides have the potential to be developed into biopharmaceuticals to treat various health conditions such as diabetes mellitus, hypertension, and chronic pain. Despite the high potential for drug development, several limitations preclude the direct use of peptides as therapeutics and hamper the process of converting venom peptides into pharmaceuticals. These limitations include, for instance, chemical instability, poor oral absorption, short halflife, and off-target cytotoxicity. One strategy to overcome these disadvantages relies on the formulation of bioactive peptides with nanocarriers. A range of biocompatible materials are now available that can serve as nanocarriers and can improve the bioavailability of therapeutic and venom-derived peptides for clinical and diagnostic application. Examples of isolated venom peptides and crude animal venoms that have been encapsulated and formulated with different types of nanomaterials with promising results are increasingly reported. Based on the current data, a wealth of information can be collected regarding the utilization of nanocarriers to encapsulate venom peptides and render them bioavailable for pharmaceutical use. Overall, nanomaterials arise as essential components in the preparation of biopharmaceuticals that are based on biological and pharmacological active venom-derived peptides.

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