RESUMO
Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.
Assuntos
Heme/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores Virais/metabolismo , Animais , Transporte Biológico/fisiologia , Encefalopatias/metabolismo , Encefalopatias/patologia , Gatos , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Heme/toxicidade , Humanos , Proteínas de Membrana Transportadoras/genética , Oócitos/citologia , Oócitos/fisiologia , Gravidez , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Virais/genética , Síndrome , Distribuição Tecidual , Xenopus laevisRESUMO
Golgi beta1,6N-acetylglucosaminyltransferase V (Mgat5) produces beta1,6GlcNAc-branched complex N-glycans on cell surface glycoproteins that bind to galectins and promote surface residency of glycoproteins, including cytokine receptors. Carcinoma cells from polyomavirus middle T (PyMT) transgenic mice on a Mgat5-/- background have reduced surface levels of epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) receptors and are less sensitive to acute stimulation by cytokines in vitro compared with PyMT Mgat5+/+ tumor cells but are nonetheless tumorigenic when injected into mice. Here, we report that PyMT Mgat5-/- cells are reduced in size, checkpoint impaired, and following serum withdrawal, fail to down-regulate glucose transport, protein synthesis, reactive oxygen species (ROS), and activation of Akt and extracellular signal-regulated kinase. To further characterize Mgat5+/+ and Mgat5-/- tumor cells, a screen of pharmacologically active compounds was done. Mgat5-/- tumor cells were comparatively hypersensitive to the ROS inducer 2,3-dimethoxy-1,4-naphthoquinone, hyposensitive to tyrosine kinase inhibitors, to Golgi disruption by brefeldin A, and to mitotic arrest by colcemid, hydroxyurea, and camptothecin. Finally, regulation of ROS, glucose uptake, and sensitivities to EGF and TGF-beta were rescued by Mgat5 expression or by hexosamine supplementation to complex N-glycan biosynthesis in Mgat5-/- cells. Our results suggest that complex N-glycans sensitize tumor cells to growth factors, and Mgat5 is required to balance responsiveness to growth and arrest cues downstream of metabolic flux.