5T4 Regulates CXCR4 Chemotaxis Figure 8. Characterization of m5T4 specific mAbs. (A), Summarizes the IgG subclasses of five m5T4 specific monoclonal antibodies (mAb, made in 5T4KO mice) recognizing distinct epitopes in the proximal and distal 5T4 extracellular LRR containing domains. (B), Shows titration of mAb activity in m5T4 specific ELISA [27]. (C), Shows titration of mAb cell surface labeling of B16m5T4 tumor cells by flow cytometry. (D), Western blot analysis of mAb probing against recombinant m5T4-pIg showing recognition of m5T4 by all mAbs except B1C3. doi:10.1371/journal.pone.0009982.g008 including through the cytoplasmic domain [3,4,5] and these may be an integrated component of chemotactic response/motility. These possibilities require further investigation. We have shown that in differentiating ES cells and MEF, CXCR6 response to CXCL16 is also 5T4 dependent. However, CXCR3 and its response to CXCL10 is not 5T4 dependent and CXCR7 is constitutively expressed at the cell surface in undifferentiated ES cells. Interestingly, the LRR-containing protein LRRC4 has been reported to regulate both the expression and signal conduction of the CXCR4 receptor. Introduction of LRRC4 into glioblastoma cells reduced CXCR4 expression, CXCL12-induced ERK and AKT phosphorylation and matrix metalloproteinase expression [45]. Crucially, the TM regions of 5T4 and LRRC4 are similar but contain significant differences (Figure 12B). Absence of another membrane protein, Robo1, or of its ligand Slit was also shown to up-regulate the CXCL12/ CXCR4 signaling in mammary epithelium [46]. Robo 1 has a distinct TM domain from 5T4. All these observations point to multiple and complex control of CXCL12/CXCR4 signaling which may have developmental stage- or tissue-specific elements. The delineation of embryonic cells with distinct chemotactic responses and where 5T4 expression is one of the controlling factors is consistent with early differentiation of ES cells being representative of events around implantation of the embryo at which point 5T4 expression is first detected [47]. The role of auxiliary proteins in receptor expression and function is not unprecedented. For example certain G protein-coupled receptors associate with receptor activity modifying proteins (RAMPs) which are required for receptor trafficking, ligand binding and receptor specificity [48]. PLoS ONE | www.plosone.org 12 April 2010 | Volume 5 | Issue 4 | e9982