5T4 Regulates CXCR4 Chemotaxis course shown). (C), Western blot analysis of PAGE separated reduced WT or 5T4KO-ES cells either undifferentiated (U) or differentiating (D) probed with CXCR4 antibody. (D), Murine CXCL12 specific ELISA of conditioned medium from undifferentiated (white columns) and differentiating (black columns) WT and 5T4KO-ES cells. (E), Undifferentiated WT and 5T4KO-ES cells (white columns) exhibit no CXCL12 chemotaxis. Differentiating (black columns) WT, but not 5T4KO-ES cells, acquire significant chemotaxis. (F), CXCL12 chemotaxis in differentiating WT-ES cells (black columns) is blocked by an antibody against CXCL12; undifferentiated ES cells (white columns) show no chemotaxis. (G), Chemotaxis of differentiating WT-ES cells, (black columns) is blocked by a 2hr pre-incubation with 10 mM AMD3100; with no effect on undifferentiated WT-ES cells (white columns). (H), Undifferentiated (white columns) or differentiating (black columns) 5T4KO-ES cells show no change in chemotactic response in the presence of the CD26 inhibitor diprotin A (DPA, 10 mM). doi:10.1371/journal.pone.0009982.g001 at least some 5T4 and CXCR4 molecules co-localize to lipid rafts in differentiating WT but not 5T4KO differentiating ES cells where CXCR4 remains intracellular. However, when differenti- ating 5T4KO-ES cells are infected with RAd-m5T4, CXCR4 can be detected at the cell surface co-localized with 5T4 molecules (Figure 3B). RAd-m5T4 infected undifferentiated WT-ES cells show only limited CXCR4 and 5T4 surface expression in a few outer cells of undifferentiated ES colonies. These are most likely spontaneously differentiating cells suggesting that differentiation is a necessary cofactor for co-localization/expression of CXCR4 and 5T4 at the cell surface. In differentiating 5T4KO-ES cells, CXCR4 accumulated in the Golgi and to a lesser extent in smooth endoplasmic reticulum (Figure 3C). These data are consistent with 5T4 molecules being necessary for the surface expression of the CXCR4 receptor and chemotaxis to CXCL12 in differentiating ES cells. These properties were further explored using MEF derived from WT and 5T4KO mice. CXCR4 cell surface expression, CXCL12 mediated ERK signaling and chemotaxis are 5T4 dependent in mouse embryo fibroblasts A 5T4 dependency for CXCR4-mediated chemotaxis is also apparent in MEF as shown by: (1) a 5T4 gene dose influence on CXCL12 chemotaxis in WT, heterozygote and 5T4KO MEF (Figure 4A); (2) the restoration of the chemotactic response of 5T4KO MEF by RAd-m5T4 (Figure 4B); and (3) the co- localization of some CXCR4 molecules with typical 5T4 cell surface expression in WT MEF while 5T4KO MEF show only intracellular CXCR4 (Figure 4C) that can be rescued at the cell surface by RAd-m5T4 (Figure 4D). Detailed analysis of individual cells in this experiment documented 98% of WT MEF showing all CXCR4 at the cell surface. By contrast, 76% of 5T4KO MEF showed either CXCR4 perinuclear or cytoplasmic labeling and only 2% with any apparent membrane associated labeling; in 22% 5T4KO MEF CXCR4 was not detected. CXCL12 activates the MAPK/ERK signal transduction pathway. We therefore examined the requirement for 5T4 in the activation of this pathway in WT and 5T4KO MEF (Fig. 5). Stimulation of WT MEF with the chemokine CXCL12 induced the phosphorylation of the key intracellular effector ERK1/2. The activation of ERK1/2 by CXCL12 was prevented by using a specific inhibitor of either an upstream kinase MEK (PD98059; Figure 5, Lane M) or of CXCR4 (AMD3100; Figure 5, Lane M) but not by an inhibitor of PI3K (LY294002; Figure 5, Lane P) in a different signaling pathway, demonstrating that signal transduc- tion was dependent upon CXCR4 mediated activation of the MAPK/ERK pathway. In 5T4KO MEF, CXCL12 activation did not lead to ERK1/2 phosphorylation, however this was not due to an overall disruption of the MAPK/ERK pathway as Phorbol 12- Myristate 13-Acetate, (PMA) stimulation led to a MEK but not CXCR4 dependent phosphorylation of ERK1/2 in both geno- types. These data demonstrate that in WT MEF a classical signal transduction pathway evoked by CXCL12 mediated activation of CXCR4 is functional. However, in the absence of 5T4, the chemokine receptor is no longer able to activate this pathway and the phosphorylation status of ERK1/2 is not responsive to CXCL12. The transmembrane domain of 5T4 is necessary for CXCR4 cell surface expression In the embryonic cells investigated it appears that cell surface expression of, and chemotactic response through, CXCR4 can be regulated by 5T4 expression. To examine the role of the extracellular, transmembrane and cytoplasmic domains of 5T4 molecules in CXCR4 surface expression, a series of murine 5T4 gene plasmid constructs were generated and cloned into a retrovirus also encoding eGFP as a reporter gene. 5T4KO MEF were infected with the retroviral constructs and cells were examined for both eGFP expression and CXCR4 localization by immunofluorescence (Figure 6). 5T4KO fibroblasts (controls, Figure 6A, panel i-iv) infected with retroviruses encoding full- length 5T4 (Figure 4a, panels v-viii) showed surface expression of CXCR4. However, the 5T4 extracellular domain was insufficient (Figure 6A, panels ix-xii) and the cytoplasmic domain unnecessary (Figure 6A, panels xiii-xvi) for CXCR4 expression on the cell surface. To test whether the 5T4 transmembrane domain (TM) was necessary and sufficient for cell surface CXCR4 expression, chimeric constructs of mouse 5T4/CD44 molecules with recipro- cally exchanged TM and cytoplasmic domains were engineered. CD44 gene was selected for this experiment since, similarly to 5T4, it is a transmembrane glycoprotein, involved in adhesion and motility. It is constitutively expressed in MEF with no effect on surface CXCR4 expression. Importantly, cells infected with the retrovirus encoding the 5T4 extracellular domain fused to the transmembrane and cytoplasmic region of CD44 exhibited no cell surface expression of CXCR4 (Figure 6A, panels xvii-xx), whereas the reciprocal construct did promote cell surface expression of CXCR4 (Figure 6A, panels xxi-xxiv). Similar results were obtained with transfection of the plasmid constructs, confirming that these results were not an artifact of the viral infections (not shown). More importantly, when the biological function of these cells were assessed there was a clear correlation between the ability of the constructs to promote cell surface expression of CXCR4 and their ability to migrate towards CXCL12 (Figure 6B). Only constructs which contained the TM of 5T4 exhibited both cell surface expression of CXCR4 and CXCL12 mediated chemotaxis. Together these observations suggest that the transmembrane region of the 5T4 glycoprotein is required for the surface expression of CXCR4 in MEF and consequently their ability to respond to CXCL12 chemotactically. Effects of cytoskeleton, microtubule and Golgi disruption on the co-localization pattern of 5T4 and CXCR4 To document the basic components of trafficking, primary WT MEF were treated for 24 hours with cytochalasin D, brefeldin A or nocodazole to disrupt the cytoskeleton, Golgi or microtubules respectively and the pattern of 5T4 and CXCR4 expression was determined before and after washout of the drugs. The PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e9982