6 research outputs found

    Tyrosine Phosphorylation of Mig6 Reduces Its Inhibition of the Epidermal Growth Factor Receptor

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    Under physiological conditions, epidermal growth factor receptor (EGFR) tyrosine kinase activity is tightly controlled through the coordinated action of both positive and negative regulators. Aberrant EGFR activation occurs frequently in many cancer types, and the endogenous EGFR feedback inhibitor, Mig6/RALT, is more efficiently phosphorylated by oncogenic EGFR variants. We have utilized expressed protein ligation to generate semisynthetic Tyr394 phosphorylated and unphosphorylated forms of the Mig6 protein and shown that phosphorylation of Mig6 reduces its ability to inhibit purified, near full-length EGFR (tEGFR). We also demonstrate that the kinetic parameters of tEGFR are similar whether solubilized in detergent or reconstitutued in nanodisc bilayers. These findings suggest a mechanism by which EGFR and its family members evade negative regulation by Mig6 under pathological conditions

    Mapping of the mAb 1479 epitope.

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    <p>(A) A schematic illustration of different His-tagged extracellular domain constructs generated to map mAb 1479 binding to ErbB4. (B) Western analysis of conditioned media from COS-7 cell transfectants expressing the different His-tagged subdomains of ErbB4 extracellular domain using mAb 1479 or anti-pentaHis as the primary antibody. (C, D) Crystal structure of the sErbB4:Fab 1479 complex. (C) A ribbon diagram of the sErbB4:1479 Fab complex is shown. sErbB4 is colored blue (subdomain I), green (subdomain II), yellow (subdomain III), and red (subdomain IV). The 1479 heavy chain is colored cyan and the light chain palecyan. The side chains of sErbB4 residues containing atoms within 4 Å of 1479 atoms are shown as stick models. (D) An expanded view of the sErbB4:Fab 1479 interface with sErbB4 residues containing atoms within 4 Å of Fab 1479 shown as stick models. The orientation is similar to that shown in panel C.</p

    Immunohistochemical and ELISA analysis of ErbB4 expression in breast cancer tissues and matched serum samples.

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    <p>(A, B) Representative examples of immunohistochemical analysis of breast cancer sections with an antibody (HFR-1) recognizing the intracellular domain of ErbB4 demonstrate strong cytoplasmic immunoreactivity in the absence of nuclear staining (A), or strong cytoplasmic and strong nuclear immunoreactivity (B). (C) ELISA analysis of ErbB4 ectodomain levels in serum samples from 30 healthy individuals (white dots) and 238 breast cancer patients (black dots). (D) ROC analysis of the value of serum ErbB4 ectodomain concentration in differentiating between normal and cancer. AUC for ErbB4 concentration in serum was 0.76. At a cut-off of 40 ng/mL of ErbB4 ectodomain, the specificity of the assay was 100% and the sensitivity 43%. All samples with ErbB4 level below the detection limit of the assay (51% of cancer samples, 47% of control samples) were excluded from the analysis.</p

    Effect of estrogen on ErbB4 shedding.

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    <p>(A) Western analysis of MCF-7 cells treated with or without 10 nM estradiol or 100 ng/mL PMA for 24 hours. The amount of shed ErbB4 ectodomain (100 kD) was detected with mAb 1479 under non-reducing conditions from culture medium and full-length ErbB4 (180 kD) with anti-ErbB4 (E-200) from cell lysates. Anti-actin was used as a loading control. The experiment was repeated four times with similar results. (B) TACE activity assay of MCF-7 cells treated for 30 min with or without 10 nM estradiol or 100 ng/mL PMA. After the treatments, TACE substrate peptide that becomes fluorescent upon cleavage was applied to the cells at time point 0. *, <i>P</i><0.05 compared to control at the time point.</p

    An Fc Domain Protein–Small Molecule Conjugate as an Enhanced Immunomodulator

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    Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A<sub>2A</sub>R) has been identified as a promising target for immunotherapy, small molecule A<sub>2A</sub>R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A<sub>2A</sub>R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A<sub>2A</sub>R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics
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