Eph Receptor Signal Transduction in Cancer Biology and Neurobiology

We identified several Eph receptors and ephrins, and research in our laboratory has been dedicated to the characterization of their biological functions and signal transduction pathways using biochemical, cell biology and molecular biology approaches in conjunction with animal models.

Signal Transduction by Eph Receptors

Receptor tyrosine kinases transmit signals across the plasma membrane, from the cell exterior to the cytoplasm. The interaction of the external domain of a receptor tyrosine kinase with the ligand up-regulates the enzymatic activity of the intracellular catalytic domain, which causes tyrosine phosphorylation of cytoplasmic signaling molecules modifying their activities. To understand how Eph receptors influence cell behavior, we investigated their signal transduction mechanisms. Initial work identified tyrosine phosphorylation sites of Eph receptors and ephrin-B ligands in cultured cells and in vivo in retinal tissue using mass spectrometry approaches. We further determined that two conserved Eph receptor phosphorylation sites not only serve as binding sites for SH2 domain containing molecules but, surprisingly, also regulate receptor kinase activity. We also found that the Src and Abl cytoplasmic tyrosine kinases and a novel SH2 domain containing protein (Shep1) are binding partners of the Eph receptors and identified signaling connections between Eph receptors and integrins. Further work has elucidated signaling pathways that mediate the activities of the Eph receptors in cancer cells, as discussed below.

Tumor Suppressor and Tumor Promoting Activities of Eph Receptors in Cancer

Many Eph receptors are highly expressed in tumor tissue, but we are only beginning to understand their role in cancer. Certain Eph receptors and ephrins promote tumor angiogenesis. We demonstrated that the EphB4 receptor expressed on the surface of tumor cells promotes tumor growth by enhancing tumor angiogenesis through interactions with its preferred ligand, ephrin-B2, expressed in tumor endothelial cells. In addition, multiple intriguing roles for the Eph receptors in cancer progression are beginning to emerge. We found that the EphB4 receptor expressed in tumor cells that are not near blood vessels is typically not engaged with ephrin-B2 and, therefore, not activated. Interestingly, we found that inducing widespread EphB4 activation with a soluble dimeric form of the ephrin-B2 ligand inhibits cancer cell proliferation/survival and migration/invasion in culture and tumor growth in vivo. These tumor suppressor effects of EphB4 rely on the cytoplasmic tyrosine kinase Abl, which phosphorylates and inactivates the proto-oncogene Crk. These findings suggest that anti-cancer strategies to inhibit EphB4 interaction with ephrin-B2 while at the same time stimulating EphB4 signaling should be particularly effective. There is also evidence that the Eph receptors can influence the properties of cancer cells even when there is no ephrin ligand to activate them. We are currently investigating ephrin-dependent as well as –independent pathways in cancer cells.

Activities of Eph Receptors in Cancer Cells. (Left) Eph receptor-ephrin binding at cell-cell contact sites results in the lateral aggregation of Eph-ephrin complexes, and initiation of “forward” signals through the receptor cytoplasmic domain (“reverse” signals through the ephrins can also be generated). Tyrosine phosphorylation sites (yellow circles) promote Eph kinase activity and also provide binding sites for signaling proteins containing SH2 domains. Other proteins that mediate Eph signals are PDZ domain-containing proteins (PDZ) and guanine nucleotide exchange factors for Rho GTPases (GEF). (Right) Eph receptors may potentiate the oncogenic effects of other receptors, an activity that is likely independent of ephrin binding and whose mechanisms are not well understood.

Neuron Communication Through EphA4 and Ephrin-A3

Eph receptors and ephrins play an important role in the proper development of the nervous system by influencing cell position, axon guidance, and the formation of synaptic structures. In the adult brain, Eph receptors and ephrins have been implicated in multiple aspects of synaptic function, including modulating neurotransmitter receptors, modifying the shape of post-synaptic terminals, and influencing long-term synaptic plasticity and memory. We have discovered a new form of neuron-glia communication mediated by the EphA4 receptor expressed in dendritic spines of adult hippocampal pyramidal neurons and the ephrin-A3 ligand expressed in perisynaptic astrocyte processes. Dendritic spines are small protrusions on dendrites that are juxtaposed to pre-synaptic terminals and contain the postsynaptic portion of excitatory synapses. The shape of dendritic spines mainly depends on the actin cytoskeleton and influences synaptic transmission and plasticity. EphA4-ephrin-A3 bidirectional communication regulates dendritic spine shape and length as well as the molecular properties of astrocyte processes surrounding synapses, ultimately affecting synaptic plasticity. We have identified EphA4-dependent signal transduction mechanisms that regulate dendritic spines, which involve inactivation of beta1 integrins and their downstream signaling pathways in neurons. Integrin inactivation likely occurs as a consequence of ephrin-dependent EphA4 association with the SPAR GTPase-activating protein, which leads to inactivation of the Ras family protein Rap1, a well-known regulator of integrin activity. We also found that ephrin-A3 is concentrated in the astrocyte processes that surround excitatory synapses and colocalizes with the glial transporters that remove the neurotransmitter glutamate from the extracellular space. Interestingly, ephrin-A3 reverse signals decrease the glutamate uptake activity of the glial glutamate transporters, thus affecting synaptic function. We are currently investigating the signaling mechanisms involved in regulation of glutamate transporters by ephrin-A3.

Neuron-glia Bidirectional Communication Through EphA4 and Ephrin-A3. (Left) The EphA4 receptor is expressed in the dendritic spines of pyramidal neurons and the ephrin-A3 ligand is on astrocyte processes that surround synapses. EphA4-ephrin-A3 interaction triggers bidirectional signals that regulate the length and shape of dendritic spines and the uptake of the neurotransmitter glutamate in perisynaptic astrocyte processes. (Right) Confocal image showing a mouse hippocampal dendrite and dendritic spine expressing yellow flurescent protein (appearing green) juxtaposed to a presynaptic terminal labeled in blue with anti-synaptophysin antibodies and surrounded by astrocyte processes labeled in red with anti-ephrin-A3 antibodies. Scale bar = 0.5 μm. (Confocal image by Keith Murai)

Inhibiting Eph Receptor Interactions with Peptides and Small Molecules

We have identified by using phage display a number of peptides that target Eph receptors and inhibit ephrin binding. Unlike the natural ephrin ligands, each of which binds to many Eph receptors, some of these peptides are highly selective and target only one Eph receptor. Furthermore, we have recently developed high-throughput assays to screen for chemical inhibitors of Eph receptor ligand binding. Initial screen have identified small molecule inhibitors of ligand binding to Eph receptors. Collaborating groups have characterized the structural features of the antagonistic peptides and small molecules identified in our laboratory in complex with the ephrin-binding domain of Eph receptors. The Eph receptor-targeting peptides and small molecules represent valuable tools to study Eph receptor function in the cancer and the nervous system. Furthermore, their improved derivatives could be used as leads to develop therapies against cancer and neurological diseases, and to promote regeneration after nervous system injury. Current work focuses on improving Eph receptor-targeting agents in collaboration with medicinal chemists and structural biologists, and testing them in cell culture and in vivo animal models.