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EphrinB–EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1

Abstract

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization1. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 — a phosphatidylinositol 5′-phosphatase that is involved in clathrin-mediated endocytosis2,3 — as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5′-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB–EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.

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Figure 1: Tyrosine phosphorylation of synaptojanin 1 by EphB receptor.
Figure 2: Inhibition of the interaction between synaptojanin 1 and endophilin by EphB2-mediated tyrosine phosphorylation.
Figure 3: Regulation of PtdIns(4,5)P2 metabolism by ephrinB–EphB signalling.
Figure 4: Effect of EphB receptor activation on transferrin uptake and transport.
Figure 5: Involvement of EphB receptors and synaptojanin 1 signalling in AMPA glutamate receptor endocytosis in hippocampal neurons.

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Acknowledgements

We thank F. Valencia for establishing the EphB2-B35 cell line; T. Williams for MALDI-TOF analysis; P. De Camilli and P. McPherson for their gift of antibodies; and W. Stallcup for critical reading of the manuscript. This work was supported by NIH grant P01 HD25938.

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Correspondence to Fumitoshi Irie.

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Supplementary figures S1, S2, S3 and supplementary table S1 (PDF 1371 kb)

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Irie, F., Okuno, M., Pasquale, E. et al. EphrinB–EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1. Nat Cell Biol 7, 501–509 (2005). https://doi.org/10.1038/ncb1252

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