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.