Ligand Binding and Calcium Influx Induce Distinct Ectodomain/γ-Secretase-processing Pathways of EphB2 Receptor

Abstract

Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance, and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface, but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin-dependent ␥-secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by presenilin 1 familial Alzheimer disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis, and the ectodomain cleavage is sensitive to peptide inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx-and N-methyl-D-aspartic acid-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by N-benzyloxycarbonyl-Val-Leu-leucinal. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2, indicating that it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor.The Ephrin (Eph) 2 receptors are the largest family of receptor tyrosine kinase proteins. They bind membrane ligand proteins, called ephrins, on adjacent cells forming multimeric clusters that bridge juxtaposed cells. These binding interactions trigger signaling cascades in both the receptor-expressing cells (forward signaling) and the ligand-expressing cells (reverse signaling) stimulating functions that modulate cell morphogenesis, tissue patterning, and angiogenesis (1-4). In the developing central nervous system, binding of ephrin ligands to Eph receptors regulates axon guidance and synapse formation (5, 6). Paradoxically, depending on specific conditions such as the expression levels of Eph receptors and their ligands, signaling events initiated by the Eph-ephrin interactions can lead either to increased cell-cell adhesion or to repulsion and separation of the involved cells (4). In the adult brain, the Eph-ephrin systems regulate memory-related functions, including synaptic structure and long term potentiation (5,7,8). There are two subclasses of Eph receptors, EphA and EphB, which are selectively activated by ephrinA and ephrinB ligands, respectively, although exceptions to this rule have been observed (4).The EphB-ephrinB system regulates the development of many tissues, including the vasculature and the c...

Proteolytic processing of protocadherin proteins requires endocytosis

“…This proposal is consistent with a report that regulated vesicular trafficking controls the formation of Pcdh-Pcdh contacts between neurons in a manner that requires the Pcdh ICDs (29). For the Notch receptor, endocytosis and γ-secretase cleavage are dependent on monoubiquitination, and sorting of Notch to the lysosome is dependent on the Nedd4 family of E3 ligases (31,40,(41)(42)(43). Processing of Pcdh may be regulated in a manner similar to that of Notch.…”

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

“…Despite the transiency of Ca 2þ microdomains and despite the buffering of Ca 2þ by Ca 2þ -binding proteins such as calmodulin, 107 increased concentrations of Ca 2þ inside microdomains suffice for a local activation of calpains. While the cleavages of proteins by calpains are the chief proteolytic consequence of Ca 2þ transients, these fluxes are also capable of activating some secretases, [108][109][110] and caspases as well, in part through activation of the apoptosome and inflammasome. 111,112 Many, possibly most neurons contain activated caspases such as caspase-3 (an effector caspase) under conditions in which caspase activation and caspase-mediated protein cuts are relatively constrained and do not cause cell death.…”

Interleukin-1 Receptor Type 1 Is a Substrate for γ-Secretase-dependent Regulated Intramembrane Proteolysis

“…7C). Considering that it has been recently reported that RIP of the p75 neurotrophin receptor (18), EphB2 receptor (123), and Notch (124) occurs subsequent to endocytosis on the endosome, determining whether ␥-secretase-dependent proteolysis of IL-1R1 CTF occurs at the plasma membrane or endosome requires further experimentation. Highlighting the physiological importance of this model, we show that pharmacological inhibitors of ␥-secretase activity inhibit generation of IL-1R1-ICD and antagonize IL-1␤-induced JNK activation.…”