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The Mechanism of Ligand‐Induced Activation of the Tie Family of Receptor Tyrosine Kinases
Author(s) -
MADUKWE JERRY,
FERGUSON KATHRYN
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.809.10
Subject(s) - receptor tyrosine kinase , chemistry , microbiology and biotechnology , angiogenesis , fibronectin , tyrosine kinase , ligand (biochemistry) , proto oncogene tyrosine protein kinase src , angiopoietin , angiopoietin receptor , receptor , sh2 domain , receptor protein tyrosine kinases , ectodomain , grb2 , biochemistry , biology , cancer research , extracellular matrix , vascular endothelial growth factor , vegf receptors
Tie1 and Tie2, members of the tyrosine kinase family with immunoglobulin and EGF homology domains, are receptor tyrosine kinases found primarily in endothelial cells with key roles in development and maintenance of the vasculature and in angiogenesis. They are attractive targets for therapeutic intervention in tumor angiogenesis, inflammation, and sepsis. Tie2 is regulated directly by the multimeric angiopoietin (Ang) ligands, with Ang1 being its primary activator. We have previously shown that the Tie2 extracellular region (ECR) forms strong dimers even in the absence of bound ligand and that dimerization is mediated by membrane‐proximal fibronectin type III (FNIII) domains that were omitted in previous structural studies as revealed in the 2.5‐Å resolution X‐ray crystal structure of the membrane‐proximal three Tie2 FNIII domains. However, it is not known whether the membrane proximal FNIII domains of Tie receptors mediate homo‐ and heteromeric interactions that are key in determining the signaling outcome of ligand binding to Tie2. We propose to elucidate the role of the membrane proximal FNIII domains of Tie2 receptors on ligand binding using purified proteins in biophysical and cell‐based experiments. We have successfully purified significant yields of Tie2 and Tie1 ECR lacking or containing the proximal FNIII domains. We are currently optimizing our yields of purified angiopoietin ligands of defined oligomeric states. The oligomeric states of the Ang ligands will be confirmed by size exclusion chromatography and small angle x‐ray scattering analysis. We will use surface plasmon resonance to assess binding of the Ang ligands to the Tie2 ECR variants. We will examine the size of the complex formed between the Ang ligands and Tie2 variants using size exclusion chromatography/multi angle laser light scattering analysis. In addition, we will determine a high‐resolution structure of the complete ECR of Tie2 with or without Ang ligands bound. Taken together, these studies will reveal the effect of the Tie2 extracellular proximal FNIII domain on ligand binding and complex formation. Finally, we will assess the importance of our biophysical experiments findings on cellular signaling using cell‐based assays. We will test the ability of Ang ligands with defined oligomeric states to activate Tie2. Phosphorylation status of Tie2 and downstream signaling molecules such as Akt will serve as a measure of Tie2 activation. Support or Funding Information NIH grant R01 CA214704 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .