CDB15:0000519 EFNB2 — EPHB2

ABSTRACT: EphB receptors provide crucial adhesive and repulsive signals during cell migration and axon guidance, but it is unclear how they switch between these opposing responses. Here we provide evidence of an important role for matrix metalloproteinases (MMPs) in repulsive EphB2 signaling. We found that EphB2 is cleaved by MMPs both in vitro and in vivo, and that this cleavage is induced by interaction with its ligand ephrin-B2. Our findings demonstrate that MMP-2/MMP-9-specific inhibition or cleavage-resistant mutations in the ectodomain of EphB2 can prevent EphB2-mediated cell-cell repulsion in HEK293 cells, and block ephrin-B1-induced growth cone withdrawal in cultured hippocampal neurons. Transient expression of wtEphB2, but not noncleavable EphB2-4/5 mutant, restored ephrin-B1-induced growth cone collapse and withdrawal in EphB-deficient neurons. The inhibition of EphB2 cleavage also had potent regulatory effects on EphB2 activity. This study provides the first evidence that MMP-mediated cleavage of EphB2 is induced by receptor-ligand interactions at the cell surface and that this event triggers cell-repulsive responses.

ABSTRACT: The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.