Research ArticleBiochemistry

The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane

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Science Signaling  08 May 2018:
Vol. 11, Issue 529, eaao4006
DOI: 10.1126/scisignal.aao4006

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  • RE: RE: The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane

    June 3, 2018

    In order to allow proper separation of individual receptor particles on the cell surface for step-bleaching analysis, using the single molecule TIRF imaging experiments, we specifically imaged and analyzed only cells with sparse labeling at the cell surface. Furthermore, to ensure its physiological relevance, and working at physiological receptor dosage, only cells that were confirmed to express TrkB-ACP levels similar to endogenous TrkB ones (fig. S2D), were adequate for analysis. In HEK293T cells this sparse labeling was carried out by titering down the DNA concentration for transient expression of ACP-TrkB and performing the labeling 12–18 hours after transfection. In neurons, lentivirus was used in concentrations that showed expression in a small fraction of cultured cells, thus ensuring the expression was based on infection with a single viral unit. These measures, as well as the filtering out from analysis of cells with excessive labeling density, enabled a reliable analysis that supports our conclusion that the receptor tyrosine kinase TrkB can signal without dimerization on the plasma membrane.

    Indeed, TrkB is phosphorylated to a certain extent even in the absence of BDNF, as evident by the colocalization with phosphotyrosine labeling at the plasma membrane in figure 2E and more directly by the specific Trk-phospho extracted from the cell surface in figure S5A. Regardless of whether a basal level of ligand-independent TrkB autopho...

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    Competing Interests: None declared.
  • RE: The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane
    • Ichiro N. Maruyama, Educator/Full Professor, Okinawa Institute of Science and Technology Graduate University
    • Other Contributors:
      • Endang Rinawarti Purba, Postdoctoral Schalor, Okinawa Institute of Science and Technology Graduate University
      • Ei-ichiro Saita, Staff Scientist, Okinawa Institute of Science and Technology Graduate University

    May 26, 2018

    The paper claims that TrkB acts as a monomeric receptor at the plasma membrane regardless of whether it has bound BDNF. Based on cell imaging and biochemical assays, dimerization occurs only after the internalization and accumulation of TrkB monomers within BDNF-containing endosomes.

    To reach these conclusions, however, there are a number of points that must be clarified. From the photobleaching step counting of ACP-tagged TrkB expressed on HEK or SCN cells (Fig. 1B), the authors concluded that only 5–8% of TrkB molecules were in dimeric form. Although experimental conditions for fluorescent labeling of ACP-TrkB are not precisely described in the paper, it is extremely difficult to express cell surface receptors at <104 molecules per cell in order to observe them at the resolution of single molecules by TIRFM. Therefore, it is highly recommended to confirm that all cell-surface TrkB molecules were fluorescently labeled. This can be accomplished by changing substrate concentrations, incubation temperatures, and incubation periods.

    Partial labeling of cell-surface TrkB molecules with fluorophores is evident from results described in the paper. First, Qdot-BDNF bound to non-labeled TrkB (Figs. 2A and B), demonstrating that there are many non-labeled TrkB on the cell surface. Second, from images in Fig. 2C, BDNF-bound TrkB and ACP-tagged TrkB did not overlap, although the authors claimed that three out of eight BDNF-boun...

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    Competing Interests: None declared.

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