Profiling Eph receptor expression in cells and tissues

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.

Eph Receptor Signaling and Ephrins

“…The peculiar characteristics of the Eph RTKs include the membrane-bound nature of the ephrins, the bidirectional mode of Eph receptor-ephrin signaling, the ability of the ephrins to stimulate but also attenuate Eph receptor signaling, and the ability of the Eph receptors to signal without ephrin involvement and even independently of kinase activity. Given the emerging view that different co-expressed Eph receptors signal cooperatively (Janes et al 2011), to correctly interpret the results of signaling studies it will be important to profile the entire repertoire of Eph receptors present in a biological system (Noberini et al 2012b) as well as survey their post-translational modifications, including tyrosine and serine/threonine phosphorylation and ubiquitination. Systems biology approaches are also essential for a comprehensive understanding of the complexities of Eph receptor signaling networks and feedback loops, and the ability of these receptors to produce widely different biological outcomes (Jorgensen et al 2009; Bush and Soriano 2012).…”

Ephrin-Eph signaling in embryonic tissue separation

“…The affinity values published so far were obtained using soluble recombinant proteins, either as monomers or as preclustered complexes. [62][63][64] Despite the problematic physiological validity of these values, and a relatively large variability between different studies, it can be concluded with good confidence that interactions are far less promiscuous than generally assumed ( Table 2) 62,65 In many but not all cases, pairs showing a high affinity in vitro were also identified as interaction partners based on biological functions. 62,66 However, the biological relevance of several high affinity ephrin-Eph pairs has yet to be determined.…”

Δ5-Cholenoyl-amino acids as selective and orally available antagonists of the Eph–ephrin system

“…Relevant to this point, tumor progression is often a highly redundant process in which multiple receptors and signaling pathways contribute to an uncontrolled proliferation of cancer cells. As several ephrins and Eph receptor subtypes are co-expressed and biologically active in different solid tumors [38,39], the ability of 10 to block different Eph receptors might be exploited for the design and development of multi-target Eph receptor antagonists featured by a significant anti-tumor efficacy in vivo.…”