5T4 Regulates CXCR4 Chemotaxis Figure 3. Cellular location of CXCR4, 5T4 in undifferentiated and differentiating WT and 5T4KO-ES cells. (A), Shows lipid rafts in the membrane of all cells (i, vi, xi, xvi); CXCR4 is intracellular in undifferentiated WT-ES and all 5T4KO-ES cells (ii, vii) and cell surface 5T4 is only expressed on differentiation of WT-ES cells (xiv). The composite images (lipid = green, CXCR4 = red and 5T4 = blue) show co-localization of 5T4 and CXCR4 (purple) including in lipid rafts (white) in differentiating WT-ES cells (xiv) but no other cells (iv, ix, xix) (co-localized areas are shown in separate channel (v, x, xv, xx)). (B), RAd-m5T4 infection of 5T4KO-ES cells leads to cell surface expression of both 5T4 and CXCR4 only in differentiating cells but not in undifferentiated cells which are seen to co-localize (CXCR4 = green, 5T4 = red) in the composite (yellow)(co-localized areas are shown in separate channel). RAd-GFP showed no effect on CXCR4 expression (not shown). (C), Upper panels, Double labeling of WT or 5T4KO-ES cells with either NBD C6-Ceramide (Golgi) or Endotracker (ER) (both red) shows that in the absence of 5T4, CXCR4 (green) accumulates predominately in the Golgi and to a lesser extent the smooth ER (yellow) whereas cell surface labeling is apparent only in the differentiating WT-ES cells)(lower panels: co-localized areas are shown in separate channel). doi:10.1371/journal.pone.0009982.g003 The possibility that the inhibitory antibodies might differentially modulate cell surface 5T4 expression was tested. Flow cytometry analysis showed no significant differences in the modulation of cell surface expression of 5T4 molecules after 2 or 24 hours by P1C9 and B1C3 mAbs as assessed in B16m5T4 cells (data not shown). To assess any differential influence on recycling of CXCR4 molecules at the cell surface, MEF were treated with nocodazole to remove CXCR4 membrane expression and allowed to recover in the presence of either P1C9 or B1C3 mAbs. There was no significant difference in recovery of CXCR4 membrane expression in the presence of non-inhibitory P1C9 or inhibitory B1C3 mAb after 3 or 6 hours (data not shown). These data suggest that the influence of inhibitory 5T4 antibodies is not mediated through preventing colocalization of 5T4 and CXCR4 at the cell surface. 5T4 facilitates CXCR6 but not CXCR3 or CXCR7 cell surface expression and response to specific ligands While the sequence variation across the extracellular chemokine receptor domains may provide for ligand specificity, mechanisms of transmembrane domain interaction with 5T4 molecules may be shared with other molecules in the CXC receptor family. Since, in addition to CXCL12 and CD26, the microarray data identified significant upregulation of transcript levels in ES differentiation of the chemokine CXCL10, and because CD26 is able to regulate multiple chemokines, a role for 5T4 in the expression and function of other chemokine receptors, including the CXCL10 receptor CXCR3 [34] and the other CXCL12 receptor CXCR7, was investigated. The expression and cellular localization of 5T4 and CXCR6 molecules before and after differentiation was determined by immunofluorescence of fixed ES cells grown on glass plates. Undifferentiated WT-ES cells are 5T4-negative with CXCR6 expression low and intracellular. Following differentiation both molecules can be detected at the cell surface with some areas of co- localization (Figure 10A). To examine whether cell surface expression of CXCR6 was biologically functional, undifferentiated and differentiating WT and 5T4KO-ES cells were placed on a chemotactic gradient towards the chemokine CXCL16, which is a known ligand for CXCR6. Undifferentiated ES cells of either genotype exhibited no chemotaxis towards CXCL16. In contrast, differentiating WT-ES cells exhibited a significant increase in chemotaxis towards CXCL16 whilst differentiating 5T4KO-ES cells showed no functional chemotaxis towards CXCL16 (Figure 10B). This phenomenon was also evident in WT MEF with co-localization of 5T4 and CXCR6 on the cell surface. By contrast, 5T4KO MEF showed no cell surface expression of CXCR6. Analysis of cell surface expression of CXCR6 by flow cytometry confirmed results obtained by immunocytochemistry, showing CXCR6 cell surface expression on WT but not 5T4KO MEF (data not shown). Thus CXCR6 expression and its response to the chemokine CXCL16 does require 5T4 expression in embryonic mouse cells. In contrast to CXCR4 and CXCR6, CXCR3 expression and its response to its ligand CXCL10 does not require 5T4 expression. Expression of CXCR3 is found at the cell surface of both WT and 5T4KO undifferentiated (not shown) and differentiating ES cells (Figure 10C). However, a chemotactic response to CXCL10 is only seen after differentiation in both WT and 5T4KO-ES cells (Figure 10D). Further analysis of undifferentiated ES cells treated with the inhibitor Diprotin A (10 mM) suggests that lack of response in undifferentiated ES cells is partly due to CD26 activity which can destroy CXCL10 [35] (data not shown). Treatment of both WT and 5T4KO differentiating ES cells with the Gi protein inhibitor pertussis toxin (10 ng/ml) showed that Gi protein- chemokine receptor interaction occurs irrespective of 5T4 expression at the cell surface allowing CXCL10 chemotaxis (data not shown). Similarly cell surface expression of CXCR7 is also independent of 5T4 cell surface expression. Undifferentiated ES cells (either WT or 5T4KO) demonstrated both cytoplasmic and cell surface expression of CXCR7 (Figure 11). In contrast following 3 days under differentiating condition in WT-ES cells, CXCR7 is relatively down-regulated from the cell surface whereas in differentiating 5T4KO-ES surface CXCR7 is retained (Figure 11). Discussion In this study, we have demonstrated that in mouse differenti- ating ES cells and embryonic fibroblasts, 5T4 glycoprotein plays a critical role in the membrane expression of CXCR4 and the chemotactic response to CXCL12. We first showed that during ES cell differentiation, increased surface expression of 5T4 and CXCL12 production was accompanied by decreased membrane expression of the CXCL12 regulatory protease CD26 whereas total cell levels of CXCR4 were unchanged. Studies with 5T4KO and WT-ES cells and MEF established that 5T4 molecules are required for cell surface expression and intracellular signaling of the CXCL12 receptor CXCR4. Importantly, chemotaxis in response to CXCL12 is disrupted in the absence of 5T4. The 5T4 dependency for CXCR4 plasma membrane expression and co-localization studies support the direct molecular interaction of CXCR4 and 5T4 molecules. The transmembrane region of 5T4 is critical for this purpose as it was sufficient in the context of CD44 molecules to allow functional surface CXCR4 expression and chemotaxis after introduction to 5T4KO MEF. Although dependent on the TM of the 5T4 molecule, the precise nature and stability of the interaction has not yet been ascertained. There is evidence that CXCR4 molecules form homo- and heterodimers [36] and LRR domains of the 5T4 molecules provide the biochemical basis for formation of multimers [37] so the stoichiometry is complicated to predict. It is possible that lipid rafts and/or additional molecules may also be a component part of a ‘‘functional’’ complex. The lifespan of the primary MEF have limited availability for biochemical studies. Attempts to detect a physical complex of 5T4 and CXCR4 have been successful in PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e9982