The CXCR4 chemokine receptor is a G i protein-coupled receptor that triggers multiple intracellular signals in response to stromal cell-derived factor 1 (SDF-1), including calcium mobilization and p44/42 extracellular signal-regulated kinases (ERK1/2). Transduced signals lead to cell chemotaxis and are terminated through receptor internalization depending on phosphorylation of the C terminus part of CXCR4. Receptor endocytosis is also required for some receptors to stimulate ERK1/2 and to migrate through a chemokine gradient. In this study, we explored the role played by the 3 intracellular loops (ICL1-3) and the C terminus domain of CXCR4 in SDF-1-mediated signaling by using human embryonic kidney (HEK)-293 cells stably expressing wild-type or mutated forms of CXCR4. ICL3 of CXCR4 is specifically involved in G i -dependent signals such as calcium mobilization and ERK activation, but does not trigger CXCR4 internalization after SDF-1 binding, indicating that ERK phosphorylation is independent of CXCR4 endocytosis. Surprisingly, ICL2, with or without the aspartic acid, arginine, and tyrosine (DRY) motif, is dispensable for G i signaling. However, ICL2 and ICL3, as well as the C terminus part of CXCR4, are needed to transduce SDF-1-mediated chemotaxis, suggesting that this event involves multiple activation pathways and/or cooperation of several cytoplasmic domains of

The chemokine SDF-1␣ transduces G i -dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a G i -independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1␣ binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr 135 in the DRY motif at the N terminus of ICL2 nor the Tyr 65 and Tyr 76 in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/ STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1␣ binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling.

ZAC is a recently isolated zinc finger protein that induces apoptosis and cell cycle arrest. The corresponding gene is imprinted maternally through an unknown mechanism and maps to 6q24 -q25, within the minimal interval harboring the gene responsible for transient neonatal diabetes mellitus (TNDM) and a tumor suppressor gene involved in breast cancer. Because of its functional properties, imprinting status, and expression pattern in mammary cell lines and tumors, ZAC is the best candidate so far for both disease conditions. In the present work, we delineated ZAC genomic organization and mapped its transcriptional start site. It is noteworthy that the ZAC promoter localized to the CpG island harboring the methylation imprint associated with TNDM and methylation of this promoter silenced its activity. These data indicate that the methylation mark may have a direct effect on the silencing of the ZAC imprinted allele. Our findings further strengthen the hypothesis that ZAC is the gene responsible for TNDM and suggest a novel mechanism for ZAC inactivation in breast tumors.