5T4 Regulates CXCR4 Chemotaxis expression and the location of CXCR4, assessed in these cells. Cell surface expression of CXCR4 is only seen with constructs containing 5T4 TM (viii, xvi, xxiv); the extracellular and cytoplasmic domains of 5T4 are not required. (B), Consistent with this CXCL12 chemotaxis of the retrovirally transduced GFP+ 5T4 MEF with 5T4 extracellular domain, (dark grey), 5T4 extracellular domain CD44 transmembrane and cytosolic domains, (grid) and mock infected, (white) showing no affect whereas full length 5T4, (light grey), 5T4 extracellular and transmembrane domains, (spots) and CD44 extracellular domain 5T4 transmembrane and cytosolic domains, (stripes) show comparable levels to wild-type (black columns). doi:10.1371/journal.pone.0009982.g006 of antibodies to sequences in both the proximal and distal domains of the 5T4 molecules. It is also conceivable that the 5T4-CXCR4 interaction may also influence receptor fate by modulating internalization events controlled by b-arrestin binding to the activated receptor or of targeting for degradation by preventing ubiquination and trafficking through early endosomes to the lysosome or by providing opportunity for recycling to the cell surface through the 5T4 PDZ motif which is a mechanism used by some other G protein binding receptors [27]. Indeed, 5T4 molecules also influence aspects of cytoskeletal organization Figure 7. Effects of cytoskeleton, microtubule and Golgi disruption on the co-localisation pattern of 5T4 and CXCR4. Primary murine embryonic fibroblasts were assessed for their pattern of 5T4 and CXCR4 expression by immunofluorescence following 24 hours disruption of either the cytoskeleton (cytochalasin D), Golgi (brefeldin A) or microtubules (nocodazole) and 1 hour after drug washout. Cell surface expression of 5T4 (green) and CXCR4 (red) with regions of co-localization of the two antigens (seen as yellow) (also shown by co-localization analysis) are depicted. doi:10.1371/journal.pone.0009982.g007 PLoS ONE | www.plosone.org 11 April 2010 | Volume 5 | Issue 4 | e9982