Eph receptors and ephrins: therapeutic opportunities

Abstract

The erythropoietin-producing hepatocellular carcinoma (Eph) receptor tyrosine kinase family plays important roles in developmental processes, adult tissue homeostasis, and various diseases. Interaction with Eph receptor-interacting protein (ephrin) ligands on the surface of neighboring cells triggers Eph receptor kinase-dependent signaling. The ephrins can also transmit signals, leading to bidirectional cell contact-dependent communication. Moreover, Eph receptors and ephrins can function independently of each other through interplay with other signaling systems. Given their involvement in many pathological conditions ranging from neurological disorders to cancer and viral infections, Eph receptors and ephrins are increasingly recognized as attractive therapeutic targets, and various strategies are being explored to modulate their expression and function. Eph receptor/ephrin upregulation in cancer cells, the angiogenic vasculature, and injured or diseased tissues also offer opportunities for Eph/ephrin-based targeted drug delivery and imaging. Thus, despite the challenges presented by the complex biology of the Eph receptor/ephrin system, exciting possibilities exist for therapies exploiting these molecules.

Keywords: angiogenesis; cancer; neurological disease; tyrosine kinase; viral infection.

Figure 1

Eph receptor/ephrin domain structure and signal transduction. (a) Bidirectional signaling at cell-cell contact sites. Red circles indicate tyrosine phosphorylation sites; purple circles indicate serine phosphorylation sites; SH2 indicates signaling proteins that interact with phosphorylated motifs through their SH2 domain; PDZ indicates binding partners containing a PDZ domain; other binding partners/signaling effectors (Eff.) are also shown schematically. Ephrin-As mediate reverse signals through association with a transmembrane signaling partner such as p75NTR or the TrkB and Ret receptor tyrosine kinases. LBD, ligand-binding domain; FNIII, fibronectin type III domain. (b) Examples of Eph receptor/ephrin non-canonical signaling modalities occurring through interplay with other signaling systems and independently of Eph receptor-ephrin interaction. For example, EphA2 can be phosphorylated by the serine/threonine kinase AKT activated downstream of receptor tyrosine kinases (RTKs), such as members of the EGF receptor family, or as a consequence of cancer mutations. EphA2 phosphorylated on serine 897 has unique signaling activities. Ephrin-B1 can control the interaction of cardiomocytes with the extracellular matrix by interacting with the claudin 5/ZO1 complex.

Figure 2

Modulation of Eph receptor/ephrin signaling by proteases. Proteases can cleave Eph receptors and ephrins in their extracellular, transmembrane and intracellular regions in a manner that can be dependent or independent of Eph receptor-ephrin interaction (although for simplicity only unbound Eph receptors and ephrins are shown). These cleavages are important for biological effects that involve separation of Eph receptor and ephrin expressing cells, including neuronal growth cone collapse, cell-cell repulsion and cell segregation. The Eph/ephrin proteolytic fragments generated can also have distinctive signaling functions in the extracellular space, cytoplasm and nucleus. Ephrin-As are cleaved near their C terminus by matrix metalloproteases (MMPs) such as MMP1, MMP2, MMP9 and MMP13, which release soluble monomeric ephrin-As that can activate EphA receptors in a paracrine manner (15). Proteases of the ADAM (A disintegrin and metalloproteinase) family, such as ADAM10 and ADAM12, can associate with EphA receptors and cleave in trans ephrin-As expressed in neighboring cells, allowing EphA/ephrin-A endocytosis and cell separation or weakening intercellular junctions (8; 170). Ephrin-Bs can also be cleaved extracellularly by ADAM8, ADAM10 and ADAM13 to regulate angiogenesis, neural tube morphogenesis, and induction of cranial neural crest (8; 171; 172). Interaction with EphB receptors can enhance ephrin-B extracellular cleavage by MMPs, such as MMP8 in the case of ephrin-B1 (171), followed by intramembrane cleavage by γ-secretase (8). The ephrin-B cytoplasmic fragment generated can enhance phosphorylation of uncleaved ephrin-Bs by SRC (8) and translocate to the nucleus to regulate transcription (8; 171). RHBDL2, a rhomboid transmembrane serine protease, cleaves the transmembrane segment of ephrin-Bs, with a preference for ephrin-B3 (171). EphA receptors such as EphA4 can be cleaved in the second fibronectin domain by MMPs activated by calcium influx independently of ephrin binding, followed by intramembrane cleavage by γ-secretase (8). The EphA4 cytoplasmic fragment generated promotes dendritic spine formation in neurons by activating the RHO family GTPase RAC1. EphA4 can also be cleaved in the kinase domain by caspases such as caspase-3 to promote apoptosis, an effect that can be reversed by ephrin-B3 binding (4). EphA2 is cleaved in the first fibronectin domain by the transmembrane metalloprotease MMP14 (MT1-MMP), which enables receptor internalization, RHOA activation and cell-cell separation (7). EphB receptors such as EphB4 can be cleaved near the transmembrane segment by ADAM8, ADAM9 and ADAM17 (171). EphB2 can be cleaved near the transmembrane segment by a metalloprotease activated by calcium influx, such as ADAM10, or by a distinct yet to be identified metalloprotease activated by ephrin binding (8). Ephrin-B binding also induces MMP7/MMP9-dependent EphB2 cleavage at two sites in the first fibronectin domain (one of which is conserved within the Eph family), which prolongs receptor activation and promotes RHOA signaling and cell-cell repulsion (171). KLK8 (Kallikrein 8 or Neuropsin) cleaves EphB2 in the brain in a stress-dependent manner leading to anxiety (8) and other kallikreins (KLKs), cleave the EphB4 extracellular domain at least in vitro (171). These extracellular EphB cleavages are typically followed by intramembrane cleavage by γ-secretase, which generates an EphB2 cytoplasmic fragment that phosphorylates the NMDA receptor and promotes its cell surface localization, thus modulating synaptic function (8). Caspases can cleave the kinase domain of EphB3 not bound to ephrins, leading to neuronal apoptosis after adult brain injury (24).