Ligand binding and calcium influx induce distinct ectodomain/gamma-secretase-processing pathways of EphB2 receptor.

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 gamma-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.

Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling.

Bidirectional signaling triggered by interacting ephrinB receptors (EphB) and ephrinB ligands is crucial for development and function of the vascular and nervous systems. A signaling cascade triggered by this interaction involves activation of Src kinase and phosphorylation of ephrinB. The mechanism, however, by which EphB activates Src in the ephrinB-expressing cells is unknown. Here we show that EphB stimulates a metalloproteinase cleavage of ephrinB2, producing a carboxy-terminal fragment that is further processed by PS1/gamma-secretase to produce intracellular peptide ephrinB2/CTF2. This peptide binds Src and inhibits its association with inhibitory kinase Csk, allowing autophosphorylation of Src at residue tyr418. EphrinB2/CTF2-activated Src phosphorylates ephrinB2 and inhibits its processing by gamma-secretase. These data show that the PS1/gamma-secretase system controls Src activation and ephrinB phosphorylation by regulating production of Src activator ephrinB2/CTF2. Accordingly, gamma-secretase inhibitors prevented the EphB-induced sprouting of endothelial cells and the recruitment of Grb4 to ephrinB. PS1 FAD and gamma-secretase dominant-negative mutants inhibited the EphB-induced cleavage of ephrinB2 and Src autophosphorylation, raising the possibility that FAD mutants interfere with the functions of Src and ephrinB2 in the CNS.

Amyloid-beta protein precursor (AbetaPP) intracellular domain-associated protein-1 proteins bind to AbetaPP and modulate its processing in an isoform-specific manner.

The amyloid-beta protein precursor (AbetaPP) is a type I transmembrane molecule that undergoes several finely regulated cleavage events. The physiopathological relevance of AbetaPP derives from the fact that its aberrant processing strongly correlates with the onset of Alzheimer's disease (AD). AD is a neurodegenerative disorder characterized by neuronal cell death, loss of synapses, and deposition of misfolded protein plaques in the brain; the main constituent of these plaques is the amyloid-beta peptide, a 40-42 amino-acid-long protein fragment derived by AbetaPP upon two sequential processing events. Mutations in the genes encoding for AbetaPP and some of the enzymes responsible for its processing are strongly associated with familial forms of early onset AD. Therefore, the elucidation of the mechanisms underlying AbetaPP metabolism appears crucial to understanding the basis for the onset of AD. Apart from Abeta, upon processing of AbetaPP other fragments are generated. The long extracellular domain is released in the extracellular space, whereas the short cytoplasmic tail, named AbetaPP intracellular domain (AID) is released intracellularly. AID appears be involved in several cellular processes, apoptosis, calcium homeostasis, and transcriptional regulation. We have recently reported the cloning and characterization of different isoforms of AID associated protein-1 (AIDA-1), a novel AID-binding protein. Here we further analyzed the interaction between several AIDA-1 isoforms and the cytoplasmic tail of AbetaPP. Our data demonstrated that the interaction between the two molecules is regulated by alternative splicing of the AIDA-1 proteins. Furthermore, we provide data supporting a possible function for AIDA-1a as a modulator of AbetaPP processing.

The intracellular localization of amyloid beta protein precursor (AbetaPP) intracellular domain associated protein-1 (AIDA-1) is regulated by AbetaPP and alternative splicing.

The Amyloid-beta Protein Precursor (AbetaPP) is a widely expressed transmembrane protein that is extensively processed in intracellular vesicular compartments and on the cell membrane. As a result of two sequential proteolytic cleavages, AbetaPP releases the Amyloid-beta (Abeta) peptide, which accumulates in insoluble plaques in the brain of patients affected by Alzheimer's Disease (AD). Another peptide, a C-terminal fragment named AbetaPP Intracellular Domain (AID), is generated by AbetaPP processing and is released intracellularly. Several functions for AID have been proposed: pro-apoptotic peptide, regulator of calcium homeostasis, molecule involved in transcriptional regulation. Many intracellular proteins, such as Fe65, Jip-1, Shc, Numb and X11alpha, interact with AID and modulate its function by different mechanisms. Here we report the cloning and initial characterization of two isoforms of a novel protein that we named AID Associated protein-1a (AIDA-1a), AIDA-1b and AIDA-1bDeltaAnk. We show that AbetaPP and the AIDA-1 proteins interact in vitro, in living cells and, endogenously, in leukemia cell lines. Transfected AIDA-1a, AIDA-1b and AIDA-1bDeltaAnk localize in different compartments and the intracellular distribution of AIDA-1a can be modified by over-expression of AbetaPP. AIDA-1 proteins are expressed at high levels in the brain; thus, studying their involvement in AbetaPP processing and AID function might give new insights regarding a possible role for these molecules in normal brain development and in the pathogenesis of AD.

Amyloid beta protein precursor is phosphorylated by JNK-1 independent of, yet facilitated by, JNK-interacting protein (JIP)-1.

Alzheimer's disease (AD) is genetically linked to the processing of amyloid beta protein precursor (AbetaPP). Aside from being the precursor of the amyloid beta (Abeta) found in plaques in the brains of patients with AD, little is known regarding the functional role of AbetaPP. We have recently reported biochemical evidence linking AbetaPP to the JNK signaling cascade by finding that JNK-interacting protein-1 (JIP-1) binds AbetaPP. In order to study the functional implications of this interaction we assayed the carboxyl-terminal of AbetaPP for phosphorylation. We found that the threonine 668 within the AbetaPP intracellular domain (AID or elsewhere AICD) is indeed phosphorylated by JNK1. We surprisingly found that although JIP-1 can facilitate this phosphorylation, it is not required for this process. We also found that JIP-1 only facilitated phosphorylation of AbetaPP but not of the two other family members APLP1 (amyloid precursor-like protein 1) and APLP2. Understanding the connection between AbetaPP phosphorylation and the JNK signaling pathway, which mediates cell response to stress may have important implications in understanding the pathogenesis of Alzheimer's disease.

Processing of beta-amyloid precursor-like protein-1 and -2 by gamma-secretase regulates transcription.

The familial Alzheimer's disease gene product beta-amyloid (Abeta) precursor protein (APP) is processed by the beta- and gamma-secretases to produce Abeta as well as AID (APP Intracellular Domain) which is derived from the extreme carboxyl terminus of APP. AID was originally shown to lower the cellular threshold to apoptosis and more recently has been shown to modulate gene expression such that it represses Notch-dependent gene expression while in combination with Fe65 it enhances gene activation. Here we report that the two other members of the APP family, beta-amyloid precursor-like protein-1 and -2 (APLP1 and APLP2), are also processed by the gamma-secretase in a Presenilin 1-dependent manner. Furthermore, the extreme carboxyl-terminal fragments produced by this processing (here termed APP-like Intracellular Domain or ALID1 and ALID2) are able to enhance Fe65-dependent gene activation, similar to what has been reported for AID. Considering that only APP and not the APLPs have been linked to familial Alzheimer's disease (AD), this data should help in understanding the physiologic roles of the APP family members and in differentiating these functions from the pathologic role of APP in Alzheimer's disease.

Evidence for a role of the nerve growth factor receptor TrkA in tyrosine phosphorylation and processing of beta-APP.

The cytoplasmic tail of the beta-amyloid precursor protein (APP) contains a Y(682)ENPTY(687) sequence through which APP associates with phosphotyrosine binding (PTB) domain containing proteins in a tyrosine phosphorylation-independent manner. We have recently found that tyrosine phosphorylation of APP-Y(682) promotes docking of Shc proteins that modulate growth factor signaling to the ERK and PI3K/Akt pathways. We have also shown that APP is phosphorylated on Y(682) in cells that overexpress a constitutively active form of the tyrosine kinase abl. Here we present evidence that the nerve growth factor receptor TrkA may also promote phosphorylation of APP. Overexpression of TrkA, but not of mutated, kinase inactive TrkA resulted in tyrosine phosphorylation of APP. Site-directed mutagenesis studies showed that TrkA overexpression was associated with phosphorylation of APP-Y(682). Moreover, overexpression of TrkA also affected APP processing reducing the generation of the APP intracellular domain (AID). Thus, tyrosine phosphorylation of APP may functionally link APP processing and neurotrophic signaling to intracellular pathways associated with cellular differentiation and survival.