Eph receptor signaling complexes in the plasma membrane

Abstract

Eph receptor tyrosine kinases, together with their cell surface-anchored ephrin ligands, constitute an important cell-cell communication system that regulates physiological and pathological processes in most cell types. This review focuses on the multiple mechanisms by which Eph receptors initiate signaling via the formation of protein complexes in the plasma membrane. Upon ephrin binding, Eph receptors assemble into oligomers that can further aggregate into large complexes. Eph receptors also mediate ephrin-independent signaling through interplay with intracellular kinases or other cell-surface receptors. The distinct characteristics of Eph receptor family members, as well as their conserved domain structure, provide a framework for understanding their functional differences and redundancies. Possible areas of interest for future investigations of Eph receptor signaling complexes are also highlighted.

Keywords: Eph receptor; ephrin; phosphorylation; receptor tyrosine kinase; signaling platform.

Figure 1.. The Eph receptors and ephrin ligands.

(A) The Eph receptor family in mammals comprises 9 EphA and 5 EphB receptors. Their activating ligands include the 5 glycosylphosphatidylinositol (GPI)-linked ephrinAs, which preferentially bind to EphA receptors (although EFNA5 can also bind to EPHB2), and the 3 transmembrane ephrinBs, which preferentially bind to EphB receptors and EPHA4. Gene names are indicated next to the cartoons illustrating the domain structures. Arrows point to the ephrin-binding and ATP-binding pockets of Eph receptors. Asterisks indicate the kinase inactive EPHA10 and EPHB6. LBD, ligand-binding domain; EGF-like, epidermal growth factor-like domain; FN1 and FN2, first and second fibronectin type III domains; JM, juxtamembrane segment; kinase, kinase domain; SAM, sterile alpha motif domain; PDZ binding, PDZ domain-binding motif at the end of the C-terminal tail. (B) Eph receptors forward signaling and ephrin reverse signaling. Forward signaling by both EphA and EphB receptors depends on ephrin-induced oligomerization, which leads to cross-phosphorylation on tyrosine residues. Effector signaling proteins typically contain domains that mediate binding to the Eph receptor (yellow) as well as domains that mediate binding to each other or have other functions (gray). Both ephrinAs and ephrinBs can mediate reverse signaling when bound to Eph receptors, affecting the cells in which they are expressed. EphrinA signaling is likely mediated by a transmembrane signaling partner. EphrinB reverse signaling involves phosphorylation of the ephrinB intracellular region by tyrosine and serine/threonine kinases and binding of SH2 and PDZ domain-containing effector proteins. DH-PH, Dbl homology-pleckstrin homology; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; PTB, phosphotyrosine binding; SAM, sterile-alpha motif.

Figure 2.. Multiple Eph receptor signaling modalities in the plasma membrane.

(A) Tyrosine phosphorylation-dependent signaling. (a) An Eph receptor phosphorylated by another tyrosine kinase can participate in signaling as a scaffolding protein and through increased kinase activity. (b) Eph receptors not bound to ephrins, particularly if highly expressed, can form dimers that interact through a Sushi-Sushi domain interface (Sushi interf., dark purple) leading to tyrosine phosphorylation (red circle) and downstream signaling. (c,d) Top (c) and side (d) view of a signaling complex including two Eph receptor and two ephrin molecules interacting via two low and two high affinity receptor ligand-binding domain (LBD)-ephrin interfaces and via a receptor LBD-LBD interface (light purple). (e,f) Soluble (e) and cell surface-associated (f) ephrins can induce Eph receptor oligomers that are stabilized by LBD-LBD and Sushi-Sushi interfaces leading to forward signaling. (g) Ephrins can induce the formation of large Eph receptor signaling platforms, which may have properties of biomolecular condensates leading to stronger and more persistent forward signaling and activation of some distinctive downstream signaling pathways. Whether reverse signaling is also potentiated in large Eph receptor/ephrin clusters remains to be determined. (B) Ligand-independent non-canonical signaling. (a) Non-canonical signaling depends on a cluster of serine/threonine phosphorylation sites in the kinase-SAM linker region. The best studied of these sites is S897 in EPHA2 (black circle). (b) LBD-FN2 head-to-tail interaction may enhance non-canonical signaling by increasing the spacing between EPHA2 intracellular regions, thus facilitating access of the kinases that phosphorylate the linker.

Figure 3.. Eph receptor domains and their signaling functions.

Percentage (%) amino acid (aa) identities are graphically represented using violin plots, which are constructed from MUSCLE sequence alignments (www.ebi.ac.uk/jdispatcher/msa/muscle). The median is indicated in bold and by a thicker line; the 25^th and 75^th percentiles are also indicated, not in bold and by thinner lines. Colors indicate the degree of conservation of the indicated domain or region, from lowest (white) to highest (red). The functions outlined have been demonstrated for some or all Eph receptors. Abbreviations: EGF, epidermal growth factor; FN, fibronectin type III; TM, transmembrane; SAM, sterile alpha motif.

Figure I for Box 1.. Conserved and divergent Eph receptor glycosylation sites.

Experimentally identified N-glycosylation sites are shown above the Eph receptor schematic and O-glycosylation sites are shown below. The thicker outline marks the 3 most conserved N-glycosylation sites, which are located in the FN1 domain. The experimentally identified sites are shown and the number of additional Eph receptors with a predicted N-glycosylation site at that position is also indicated (phosphosite.org; uniprot.org). EGF, epidermal growth factor-like; FN, fibronectin type III; JM, juxtamembrane segment; LBD, ligand-binding domain; S, signal peptide; SAM, sterile alpha motif; T, transmembrane helix. The names of the Eph receptors are indicated in abbreviated form.

Figure II for Box 2.. Conserved and divergent Eph receptor phosphorylation sites.

Tyrosine phosphosites are shown above the Eph receptor schematic and serine/threonine phosphosites are shown below (phosphosite.org). Phosphosites identified in ≥20 studies are indicated in bold black font and in ≥5 studies in black font. Only few phosphosites identified in <5 studies that have demonstrated or likely functional significance are shown in gray font. The red outline indicates the conserved regulatory tyrosine phosphosites in the juxtamembrane segment and in the activation loop. The blue outline indicates the phosphosites in the kinase-SAM linker region. The green outline indicates extracellular phosphosites. Clusters of phosphosites, or phosphosites with neighboring positions in different Eph receptors, are indicated together with a single thicker connecting line. EGF, epidermal growth factor-like; FN, fibronectin type III; JM, juxtamembrane segment; LBD, ligand-binding domain; S, signal peptide; SAM, sterile alpha motif; T, transmembrane helix.

Figure III for Box 3.. Common and distinct Eph receptor proximal proteins.

(A) Percentage of prey proteins identified in proximity of the indicated number of bait Eph receptors (out of a total of 674 preys identified with a stringent Bayesian false discovery rate (FDR) ≤0.01%, including 400 preys for EPHA2, 307 for EPHA4, 125 for EPHB2, 236 for EPHB3, 194 for EPHB4 and 430 for EPHB6) [23,27,28]. (B) Dot plots showing selected transmembrane proteins identified in proximity of the indicated Eph receptors. RTK, receptor tyrosine kinase. Plots were generated with the prohits-viz web resource (prohits-viz.lunenfeld.ca) using data from references [23,27,28].