Stimulated ErbB4 internalization is necessary for neuregulin signaling in neurons

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Abstract

Neuregulin-1 (NRG1) plays an important role in neural development, synapse formation, and synaptic plasticity by activating ErbB receptor tyrosine kinases. Although ligand-induced endocytosis has been shown to be important for many receptor tyrosine kinases, whether NRG1 signaling depends on ErbB endocytosis remains controversial. Here, we provide evidence that ErbB4, a prominent ErbB protein in the brain, becomes internalized in NRG1-stimulated neurons. The induced ErbB4 endocytosis requires its kinase activity. Remarkably, inhibition of ErbB endocytosis attenuates NRG1-induced activation of Erk and Akt in neurons. These observations indicate a role of ErbB endocytosis in NRG1 signaling in neurons.

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Materials and methods

Materials. Antibodies used were: ErbB2 (sc-284), ErbB3 (sc-285), and ErbB4 (sc-283) from Santa Cruz Biotechnology (Santa Cruz, CA); phospho-Erk (Thr202/Tyr204, #9101), phospho-Akt (Ser473, #4051), and Akt (#2966) from Cell Signaling Technology (Beverly, MA); HRP-conjugated secondary antibodies (GH9596614) were from Pierce (Rockford, IL). Streptavidin agarose-beads were from Molecular Probes (Eugene, OR). EZ-Link sulfo-NHS-biotin was from Pierce. Chemicals used were: monodansylcadavenrine (MDC,

ErbB2 and ErbB4 were internalized in NRG1-stimulated neurons

To determine whether ErbB proteins undergo endocytosis in neurons, hippocampal neurons were incubated with NHS-SS-biotin at 4 °C for 1 h to label surface proteins. Un-bound NHS-SS-biotin was washed off and neurons were incubated at 37 °C to initiate internalization. Biotinylated proteins remained on cell surface was debiotinylated by cleaving the NHS-SS-biotin disulfide bond in the cleavage buffer containing glutathione. Neurons were lysed, and internalized biotinylated proteins were purified by

Discussion

For many growth factors, intracellular signaling may be terminated by ligand-induced endocytosis of the receptor tyrosine kinases and their subsequent degradation [28]. On the other hand, receptor internalization may be necessary to mediate growth factor-stimulated signaling. For example, endocytosis of activated Trk receptors is necessary for some biological functions of neurotrophins in neurons [29], [30], [31]. Internalized Trk kinases could remain tyrosine phosphorylated and active, with

Acknowledgments

This work was supported in parts by grants from NIH (L. Mei and W.C. Xiong) and MDA (L. Mei). R.S.W. was supported in part by a Korea Research Foundation Grant (MOEHRD, KRF-2004-214-H00004).

References (33)

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