Abstract
Brain-derived neurotrophic factor (BDNF) signaling through its receptor, TrkB, modulates survival, differentiation, and synaptic activity of neurons. Both full-length TrkB (TrkB-FL) and its isoform T1 (TrkB.T1) receptors are expressed in neurons; however, whether they follow the same endocytic pathway after BDNF treatment is not known. In this study we report that TrkB-FL and TrkB.T1 receptors traverse divergent endocytic pathways after binding to BDNF. We provide evidence that in neurons TrkB.T1 receptors predominantly recycle back to the cell surface by a "default" mechanism. However, endocytosed TrkB-FL receptors recycle to a lesser extent in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner which relies on its tyrosine kinase activity. The distinct role of Hrs in promoting recycling of internalized TrkB-FL receptors is independent of its ubiquitin-interacting motif. Moreover, Hrs-sensitive TrkB-FL recycling plays a role in BDNF-induced prolonged mitogen-activated protein kinase (MAPK) activation. These observations provide evidence for differential postendocytic sorting of TrkB-FL and TrkB.T1 receptors to alternate intracellular pathways.
Highlights
Brain-derived neurotrophic factor (BDNF)3 has been shown to play critical roles in vertebrate nervous system development and function [1,2,3]
Cling receptor, as determined by confocal microscopy (Fig. 3C). These results suggest that TrkB.T1 receptor is predominantly sorted to the recycling pathway, which is consistent with its minimal BDNF-dependent degradation (Fig. 1A), whereas TrkB-FL receptor recycles to a lesser extent after advantage of a novel feature of the amino-terminal FLAG Kinase Domain Is the Key Motif Responsible for the Differenepitope-tagged versions of Trk receptors, in which calcium- tial TrkB-FL and TrkB.T1 Endocytic Recycling—To define the sensitive fluorescent anti-FLAG antibodies can be rapidly potential region in TrkB-FL receptor that is responsible for the dissociated with phosphate-buffered saline (PBS)/EDTA [12]
Overexpression of full-length hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), which both inhibited TrkB-FL degradation and recycling, enhanced BDNF-induced sustained mitogen-activated protein kinase (MAPK) activation. These results suggest that the Hrs-sensitive TrkB-FL recycling may play a role in BDNF-induced prolonged MAPK activation
Summary
Reagents and Antibodies—Human recombinant BDNF was obtained from PeproTech (Rocky Hill, NJ). Two parallel control coverslips were included, one in which cells were fixed after a 300-min incubation in the absence of ligand and without EDTA stripping step (100% surface receptor control), and one in which cells were stripped immediately after the feeding step (0% Internalization control). Cells transiently transfected with TrkB receptors were grown on glass coverslips and incubated with Alexa-488-conjugated M1 anti-FLAG antibody after serum starvation. Cells were examined by epifluorescence microscopy by using appropriate filter sets to selectively detect Alexa-488 or Alexa-594, and staining intensities of each fluor in individual cells were integrated using MetaMorph software (Molecular Devices) This analysis indicated that the efficiency of the EDTA strip (reduction of Alexa-594 staining intensity in the 0% recycled control relative to the 100% surface receptor control) was Ͼ95%.
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