Eph/ephrin signaling: genetic, phosphoproteomic, and transcriptomic approaches

Abstract

The Eph receptor tyrosine kinases and their ephrin partners compose a large and complex family of signaling molecules involved in a wide variety of processes in development, homeostasis, and disease. The complexity inherent to Eph/ephrin signaling derives from several characteristics of the family. First, the large size and functional redundancy/compensation by family members presents a challenge in defining their in vivo roles. Second, the capacity for bidirectional signaling doubles the potential complexity, since every member has the ability to act both as a ligand and a receptor. Third, Ephs and ephrins can utilize a wide array of signal transduction pathways with a tremendous diversity of cell biological effect. The daunting complexity of Eph/ephrin signaling has increasingly prompted investigators to resort to multiple technological approaches to gain mechanistic insight. Here we review recent progress in the use of advanced mouse genetics in combination with proteomic and transcriptomic approaches to gain a more complete understanding of signaling mechanism in vivo. Integrating insights from such disparate approaches provides advantages in continuing to advance our understanding of how this multifarious group of signaling molecules functions in a diverse array of biological contexts.

Figure 1. Phospho-proteomic approaches to Eph/ephrin signaling networks

(A–C) The workflow utilized in recent phosphoproteomic studies identifying targets of forward signaling. Each of these studies employed anti-phosphotyrosine antibodies to purify phosphorylated peptides. Key advantages to (A)[35, 36] and (B)[43], compared with (C)[45], involved the use of incorporation of SILAC labeling for quantitation; (B)[43] utilized cellular ephrin-B1 instead of ephrin-B1-Fc; and (C)[45] utilized primary cells with known developmental relevance. (D–G) Venn diagrams of overlap between phosphorylated protein targets of ephrin-B1 induced forward signaling identified by the three studies. Yellow (Jorgensen et al., 2009); (Zhang et al., 2006, 2008); Blue (Bush et al., 2009). (D, E) Proteins with increased tyrosine phosphorylation compared with cellular ephrin-B1 induction (Jorgensen et al., 2009) (D) or pre-clustered ephrin-B1-Fc induction (E). (F, G) Proteins with decreased tyrosine phosphorylation compared with cellular ephrin-B1 induction (yellow) (D) or pre-clustered ephrin-B1-Fc induction (yellow) (E). In all cases, blue and red were induced by pre-clustered ephrin-B1-Fc.

Figure 2. Transcriptional regulation by EphB/ephrin-B signaling

Forward and reverse signaling through EphB/ephrin-B signaling can result in transcriptional regulation by a number of proposed mechanisms. Signaling pathways leading to transcriptional responses are connected by arrows; transcriptional activation or repression is indicated. The biological consequences of this regulation are indicated in red.