Cytoplasmic Relaxation of Active Eph Controls Ephrin Shedding by ADAM10

Abstract

Release of cell surface-bound ligands by A-Disintegrin-And-Metalloprotease (ADAM) transmembrane metalloproteases is essential for signalling by cytokine, cell adhesion, and tyrosine kinase receptors. For Eph receptor ligands, it provides the switch between cell-cell adhesion and repulsion. Ligand shedding is tightly controlled by intrinsic tyrosine kinase activity, which for Eph receptors relies on the release of an inhibitory interaction of the cytoplasmic juxtamembrane segment with the kinase domain. However, a mechanism linking kinase and sheddase activities had remained elusive. We demonstrate that it is a membrane-proximal localisation of the latent kinase domain that prevents ephrin ligand shedding in trans. Fluorescence lifetime imaging microscopy and electron tomography reveal that activation extends the Eph receptor tyrosine kinase intracellular domain away from the cell membrane into a conformation that facilitates productive association with ADAM10. Accordingly, EphA3 mutants with constitutively-released kinase domains efficiently support shedding, even when their kinase is disabled. Our data suggest that this phosphorylation-activated conformational switch of EphA3 directly controls ADAM-mediated shedding. The Eph transmembrane receptors are part of the receptor tyrosine kinase family and play important roles in communication between neighbouring cells. An Eph receptor binds to its ligand, membrane-tethered ephrin, on a neighbouring cell so as to form a stable complex and activate downstream signalling events. One such event is regulation of ADAM10, a transmembrane protease of the ADAM metalloprotease family, which provides a feedback mechanism to Eph signalling. ADAM10 is located on Eph-expressing cells and cleaves ephrin from its membrane tether on the opposite cell (through its so-called sheddase activity), thereby separating the cell-cell connection and allowing the signalling complex to internalise. In other biological contexts, activity of the ADAM metalloprotease family underlies signalling mechanisms such as oncogenic EGF-receptor transactivation, adhesion molecule shedding and cytokine/chemokine release. In general, ADAM function is enhanced when receptor tyrosine signalling is active and repressed when tyrosine kinase signalling is inhibited. However, the mechanism through which receptor tyrosine kinase signalling regulates ADAM10, have remained elusive. By combining fluorescence lifetime imaging microscopy (FLIM) and electron microscopic tomography of EphA3, we have demonstrated in live cells at molecular resolution that tyrosine phosphorylation of activated EphA3 triggers a measurable movement of the kinase domain away from the plasma membrane. Only this conformation of the EphA3 kinase domain away from the plasma membrane permits ADAM10 to come close enough to EphA3 so that it can reach its tightly EphA3-bound substrate, ephrin-A5. Our findings delineate a new regulatory concept in cell-cell communication, whereby control over proteolytic sheddase activity is provided by an activation-induced switch in the conformation of the cytoplasmic domain of a receptor tyrosine kinase, rather than by a cytosolic signalling pathway.