5T4 Regulates CXCR4 Chemotaxis Figure 5. Disruption of cytoskeleton and CXCL12 dependent signaling in 5T4KO MEF. WT MEF exhibited an increase in ERK phosphorylation in response to CXCL12 stimulation that was prevented by the MEK1 inhibitor PD98059 (M, 50 mM) and the CXCR4 inhibitor AMD3100 (C, 10 mM) but not by the PI3K inhibitor LY294002 (P, 50 mM). 5T4KO MEF did not respond to CXCL12 stimulation, and this lack of response was specifically related to CXCR4 function and not due to a generalized disruption of MAPK/ERK signaling as both WT and KO MEF exhibited an increase in ERK1/2 phosphorylation in response to PMA stimulation, which was blocked by MEK1 inhibition but independent of both CXCR4 and PI3K activity. Total ERK was used as a loading control. doi:10.1371/journal.pone.0009982.g005 and 5T4 molecules appear to show a stable association. There was some indication that cytochalasin increased CXCR4/5T4 expres- sion at the cell surface. The observation that following actin depolarization 5T4 and CXCR4 remain at the plasma membrane is consistent with several reports implicating actin binding proteins in CXCR4 internalization and degradation[38]. For example, actin binding cortactin and plectrin have been shown to be involved in CXCL12 induced CXCR4 internalization and recycling in HEK293 cells overexpressing CXCR4 [39,40]. Also, myosin IIA, a molecular motor involved in vesicle and protein trafficking along actin filaments, requires its actin binding domain for efficient endocytosis of CXCR4 [41]. Therefore the disruption of the cytoskeleton may have diminished the ability of 5T4 and CXCR4 to be endocytosed and subsequently degraded in the lysosomes thereby increasing their stability and half-life at the cell surface. The 5T4 gene is highly conserved across different vertebrate species and the TM region is completely conserved (Figure 12A). This explains why the human 5T4 and mouse 5T4 genes could equally restore CXCR4 cell surface expression in 5T4KO cells. Chemokine receptors, G-protein-coupled seven TM spanning proteins, are also highly conserved in evolution [42], with the hydrophobic amino acids of TM domains forming a-helical structures which anchor the receptors in the membrane [43]. Current understanding of the mechanisms of CXCL12-CXCR4 interaction support a two step mechanism where by the initial interaction occurs between the ligand b-sheet and its 50S and N- loop and the CXCR4 extracellular region which facilitates rapid binding and efficient anchoring on the extracellular side of the receptor [44]. The ligand N-terminus remains highly dynamic and searches for the binding cavities buried with the receptor TM helices. This second step interaction between the ligand N- terminus and the receptor TM region triggers conformational changes in the CXCR4 TM to induce G-protein signaling [44]. Importantly, the chemotactic response of both differentiated ES cells and MEF can be blocked by some but not all antibodies recognizing distinct parts and epitopes of m5T4 molecules. These data suggest that 5T4 contributes to functional integrity of the CXCR4 receptor expression at the cell surface. 5T4 antibody induced modulation of 5T4 molecules from the cell surface or prevention of CXCR4/5T4 co-localization do not appear to account for observed inhibition of some m5T4 mAbs. It seems more likely that the inhibition results from allosteric effects on CXCR4 altering the nature of ligand binding or its consequences. Since chimeric CD44/5T4TM molecules are functional in facilitating CXCR4 membrane expression and chemotactic response one might conclude that the 5T4 specific antibodies are unlikely to influence the initial direct binding of CXCL12. The most efficient inhibitory antibody (B1C3) showed differential activity in ELISA and western blotting of m5T4-Fc compared to the other mAbs, consistent with an epitope influenced partly by the presence of the 5T4 TM domain. It is possible that the 5T4 transmembrane region specifically recognizes intramembrane residues of CXCR4 and contributes not only to the stability of the CXCR4 expression in the plasma membrane but possibly also to conformational changes in the receptor which govern responsiveness to ligand. The latter may be influenced by binding PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e9982