The endosomal sorting adaptor HD-PTP is required for ephrin-B:EphB signalling in cellular collapse and spinal motor axon guidance

Sylvie Lahaie,Charles-Etienne Castonguay,Daniel Morales,Jean-François Côté,Jalal M Kazan,Artur Kania,Noumeira Hamoud,Avihu Klar,Anne-Claude Gingras,Guillaume Desrochers,Arnim Pause,Halil Bagci

doi:10.1038/s41598-019-48421-9

Abstract

The signalling output of many transmembrane receptors that mediate cell-cell communication is restricted by the endosomal sorting complex required for transport (ESCRT), but the impact of this machinery on Eph tyrosine kinase receptor function is unknown. We identified the ESCRT-associated adaptor protein HD-PTP as part of an EphB2 proximity-dependent biotin identification (BioID) interactome, and confirmed this association using co-immunoprecipitation. HD-PTP loss attenuates the ephrin-B2:EphB2 signalling-induced collapse of cultured cells and axonal growth cones, and results in aberrant guidance of chick spinal motor neuron axons in vivo. HD-PTP depletion abrogates ephrin-B2-induced EphB2 clustering, and EphB2 and Src family kinase activation. HD-PTP loss also accelerates ligand-induced EphB2 degradation, contrasting the effects of HD-PTP loss on the relay of signals from other cell surface receptors. Our results link Eph function to the ESCRT machinery and demonstrate a role for HD-PTP in the earliest steps of ephrin-B:EphB signalling, as well as in obstructing premature receptor depletion.

Highlights

Eph receptor A and B subfamilies are defined by their ephrin ligands’ linkage to the cell membrane via a GPI anchor or a transmembrane domain, respectively
Our proteomics experiments identify a number of potential novel effectors of Eph signalling, and demonstrate that one such protein is the endosomal sorting complex required for transport (ESCRT) adaptor HD-PTP
Its association with EphB2 is induced by ephrin-B2 binding, and its function is required for repulsive responses to ephrin-B2 in cultured cells and motor neuron growth cones, as well as the normal guidance of ephrin-B-responsive spinal motor axons in vivo

Summary

Introduction

Eph receptor A and B subfamilies are defined by their ephrin ligands’ linkage to the cell membrane via a GPI anchor or a transmembrane domain, respectively. The endosomal internalisation of ephrin:Eph complexes is required for normal receptor signalling[15,16,17], and eventually leads to dephosphorylation of juxtamembrane tyrosines[18], ubiquitylation of the Eph cytoplasmic tail[19], and Eph recycling or degradation[20] It is unknown whether the fate of internalised Eph receptors depends on the ESCRT machinery, which detects ubiquitylated receptors and transfers them between specialised vesicles, where they are sorted back to the membrane or to the lysosome[2,21]. HD-PTP acts at a later step in the Eph signalling pathway where, in contrast to its described function for other receptors processed by ESCRTs24, it acts as a negative regulator of EphB2 degradation by the lysosome These results are the first to establish a functional link between Eph signalling and ESCRT accessory proteins, revealing their novel role in promoting cell surface receptor signalling

Methods

Results

Conclusion