FLVCR, a member of the major facilitator superfamily of transporter proteins, is the cell surface receptor for feline leukemia virus, subgroup C. Retroviral interference with FLVCR display results in a loss of erythroid progenitors (colony-forming units-erythroid, CFU-E) and severe anemia in cats. In this report, we demonstrate that human FLVCR exports cytoplasmic heme and hypothesize that human FLVCR is required on developing erythroid cells to protect them from heme toxicity. Inhibition of FLVCR in K562 cells decreases heme export, impairs their erythroid maturation and leads to apoptosis. FLVCR is upregulated on CFU-E, indicating that heme export is important in primary cells at this stage. Studies of FLVCR expression in cell lines suggest this exporter also impacts heme trafficking in intestine and liver. To our knowledge, this is the first description of a mammalian heme transporter.

MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSHbeads or anti-Myc linked to magnetic beads MK21 mediates several p38␣,␤ MAP kinase-dependent processes (for a review, see Ref. 1), demonstrated most clearly by results from targeted disruption of the MK2 gene in mice (2). MK2 (Ϫ,Ϫ) mice have suppressed stress responses. Cellular studies show deficits in motility, chemotaxis, and cytokine production. Macrophages taken from MK2 (Ϫ,Ϫ) mice exhibit normal TNF mRNA induction in response to endotoxin but do not release TNF protein. Cellular TNF protein was markedly decreased in MK2 (Ϫ,Ϫ) macrophages, suggesting a block in production of TNF from TNF mRNA (2). TNF expression is regulated both via mRNA stability and translation (3, 4) but is not completely understood.The p38␣,␤ MAP kinase pathway regulates stability of mRNAs that contain AU-rich elements in their 3Ј-untranslated regions. Examples include TNF, COX-2, interleukin-6, and interleukin-1␤ (4 -6). Evidence chiefly comes from mRNA stabilization caused by transfection of mutationally activated MEK3/MEK6 or by the addition of agents that activate p38 MAPK and conversely from destabilization caused by the addition of a p38␣,␤ MAP kinase inhibitor. Since p38␣,␤ is required to activate MK2, these experiments do not dissect contributions from MK2. Studies in MK2 (Ϫ,Ϫ) cells suggest that MK2 regulates stability of some cytokine mRNAs (2, 4). Lasa et al. (5) first reported that expression of a mutant of MK2 with constitutive activity stabilized COX-2 mRNA in the presence of SB2035780 and that expression of a kinase-defective MK2 blocked the stabilization induced by activated MEK6, arguing that MK2 is necessary and sufficient to induce stabilization of at least the COX-2 mRNA (5). How MK2 regulates cytokine production post-transcriptionally is unknown. Mahtani et al. (7) reported that tristetraprolin (TTP) is an in vitro substrate for MK2, motivating the detailed studies we describe.TTP (for a review, see Ref. 8) destabilizes class II AU-rich elements and is the prototype for a non-zinc finger class of nucleic acid-binding proteins. Destabilization requires integrity of the TTP tandem Cys 3 His RNA binding domains that coordinate zinc in a disklike structure (9 -11). TTP-null mice exhibit many defects including inflammatory arthritis and systemic lupus erythematosis-like symptoms attributed to increased production of TNF (12).TTP is phosphorylated in cells treated with growth factors or cytokines. Phosphorylation occurs at more than one site evident by the appearance of two distinct slower migrating forms of TTP on gels after stimulation that are reversed by phosphatase treatment (7, 13). TTP under...

A2A adenosine receptors (A2AAR) inhibit inflammation, although the mechanisms through which adenosine exerts its effects remain unclear. Although the transfer of regulatory Th cells blocks colitis induced by pathogenic CD45RBhigh Th cells, we show that CD45RBlow or CD25+ Th cells from A2AAR-deficient mice do not prevent disease. Moreover, CD45RBhigh Th cells from A2AAR-deficient mice were not suppressed by control CD45RBlow Th cells. A2AAR agonists suppressed the production of proinflammatory cytokines by CD45RBhigh and CD45RBlow T cells in association with a loss of mRNA stability. In contrast, anti-inflammatory cytokines, including IL-10 and TGF-β, were minimally affected. Oral administration of the A2AAR agonist ATL313 attenuated disease in mice receiving CD45RBhigh Th cells. These data suggest that A2AAR play a novel role in the control of T cell-mediated colitis by suppressing the expression of proinflammatory cytokines while sparing anti-inflammatory activity mediated by IL-10 and TGF-β.

Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens. For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second Cys 3 His repeat. The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first Cys 3 His sequence. On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal-binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain. This structure is unlike that of any previously described class of metal binding domain.