EphrinA1 Is Released in Three Forms from Cancer Cells by Matrix Metalloproteases

Abstract

EphrinA1 is a glycosylphosphatidylinositol (GPI)-linked ligand for the EphA2 receptor, which is overexpressed in glioblastoma (GBM), among other cancers. Activation of the receptor by ephrinA1 leads to a suppression of oncogenic properties of GBM cells. We documented that a monomeric functional form of ephrinA1 is released from cancer cells and thus explored the mechanism of ephrinA1 release and the primary protein sequence. We demonstrate here that multiple metalloproteases (MMPs) are able to cleave ephrinA1, most notably MMP-1, -2, -9, and -13. The proteolytic cleavage that releases ephrinA1 occurs at three positions near the C terminus, producing three forms ending in valine-175, histidine-177, or serine-178. Moreover, deletion of amino acids 174 to 181 or 175 to 181 yields ephrinA1 that is still GPI linked but not released by proteolysis, underlining the necessity of amino acids 175 to 181 for release from the membrane. Furthermore, recombinant ephrinA1 ending at residue 175 retains activity toward the EphA2 receptor. These findings suggest a mechanism of release and provide evidence for the existence of several forms of monomeric ephrinA1. Moreover, ephrinA1 should be truncated at a minimum at amino acid 175 in fusions or conjugates with other molecules in order to prevent likely proteolysis within physiological and pathobiological environments.

EFN-4/Ephrin functions in LAD-2/L1CAM-mediated axon guidance in Caenorhabditis elegans

“…In these studies, soluble forms of ephrin A1 were released from these cells in a matrix metalloprotease-dependent manner and are independent of cellcell contact. Moreover, when cultured cells, including embryonic neurons, were exposed to soluble ephrin A1-conditioned medium, diverse physiological changes were observed, including growth cone collapse and attenuated cell migration (Beauchamp et al, 2012;Wykosky et al, 2008). These studies, combined with our results, reveal that conserved ephrin-mediated processes probably include the use of endogenous soluble ephrins acting as diffusible factors.…”

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

“…Deg-eA1 tested on GBM, breast, and prostate cancer cells overexpressing EphA2 did not induce receptor downregulation or the typical cell rounding observed with active eA1-WT. In addition, neither migration nor anchorage-independent growth alterations were observed in the presence of Deg-eA1 indicating lack of activation of downstream signaling pathways (12,17,18).…”

“…However, as our laboratory recently demonstrated, eA1 wild-type (eA1-WT) full-length (aa. 1-205) is a GPI-anchored cell membrane protein that, on cell surface, is cleaved by several metalloproteinases like MMP-1,-2,-9, and-13 (17), and released as a soluble fully active monomeric protein (12,18).…”

Eph receptor and ephrin function in breast, gut, and skin epithelia

“…However, this is not an absolute requirement for Eph receptor signaling as ligand-independent functions for these RTKs have been described and there is evidence that EphA receptors can be activated by soluble ephrin-A ligands present in the microenvironment. 4,5 Ligand binding to Eph receptors at cell-cell contacts rapidly leads to the formation of higher order signaling clusters required for robust kinase activation, effector molecule recruitment, and transmission of a variety of downstream signaling cascades. 6,7 Contact-dependent signaling is evident where there is complementary expression of Eph receptor and ephrin in distinct cell types but there also appears to be a major role for Eph receptor signaling complexes in cell types where there is overlapping expression of receptor and ligand, particularly in epithelial tissues.…”

“…This large family of RTKs is subdivided into 2 subclasses, EphA (1)(2)(3)(4)(5)(6)(7)(8)10) and EphB (1)(2)(3)(4)6) receptors based on amino acid sequence homology and relative binding affinities to glycosylphosphatidylinositol (GPI)-linked ephrin-A (1-5) or transmembrane ephrin-B (1-3) ligands. There is extensive promiscuity for ligand binding within receptor subclasses and more limited interaction between receptors and ligands in different subclasses.…”