To identify novel regulatory components involved in the recycling of the insulin-responsive glucose transporter GLUT4, we have used the yeast two-hybrid system to isolate GLUT4-binding proteins from a rat adipose cell cDNA library. We found a 49-kDa protein (p49/STRAP) that specifically interacts with an acidic amino acid motif (Q7IGSEDG) in the N-terminus of GLUT4. Confocal immunoflourescence microscopy of primary rat adipose cells shows co-localization of myc-p49 with GLUT4 and also with the ER-resident protein calnexin. Insulin stimulation had no effect on GLUT4-binding and subcellular distribution of p49 in adipose cells. However, overexpression of the GLUT4-binding domain of p49 in adipose cells reduces protein synthesis and cell-surface expression of GLUT4, but not of GLUT8. Moreover, cell-surface expression of a p49-binding-deficient GLUT4 mutant (ED/QN) is also reduced. Kinetic analysis of HA-epitope-tagged GLUT4 protein synthesis indicates a possible role of p49 in biosynthesis and/or processing of GLUT4 in adipose cells.? ? GLUT1 was recently described as a receptor for the retrovirus HTLV. The entry of retroviruses into target cells involves the interaction of two envelope (Env) glycoproteins, a surface glycoprotein (SU) and a transmembrane glycoprotein (TM), with at least one specific host cell receptor. Binding of HTLV SU can be detected on all vertebrate cell types examined to date. However virus binding and virus infection are two different steps and host cell-surface proteins which are involved in HTLV SU binding may be different than cell-surface proteins which are involved in the infection process. In this study, we examined the role of GLUT1 in the cell-cell transmission of HTLV-1 virions. To determine the importance of GLUT1 in virus infection, we first established a system which would allow us to overexpress GLUT1 in target cells, and monitor the relative amount of GLUT1 expressed on the cell surface. We transfected COS cells with the parental vector pCIS, or with GLUT1 or a mutant form of GLUT6 (GLUT6(LL/AA)), tagged with hemaglutinin (HA), to determine cell-surface expression by quantitative Western blot analysis, coupled to bis-glucose photolabeling and FACS. We showed that GLUT1 cell-surface expression is almost undetectable in untransfected compared with GLUT1 over-expressing COS cells. Similarly, the cell-surface expression of mutant GLUT6 (LL/AA) is high in transfected COS cells. To study the role of GLUT1 in HTLV-1 infection, we developed a co-culture assay, using live HTLV-1 producing cells, to monitor de novo infection of GLUT1 over-expressing COS cells. We showed that the percentage of infected COS cells is drastically increased on HA-GLUT1 transfected cells (46.4%) in comparison to that on HA-GLUT6 (LL/AA) transfected cells (2.0%). Additionally, tax mRNA expression and HTLV-1 proviral load are increased in COS cells transfected with GLUT1. We show that GLUT1 has an important role in HTLV infection. We propose that GLUT1 mediates infection of the virus binding by mediating vesicular uptake through its subcellular trafficking properties. ? ? Finally, studies using adherent cell lines have shown that glucose transporter-1 (GLUT1) can function as a receptor for human T-cell leukemia virus type 1 (HTLV). In primary CD4(+) T cells, heparin sulfate proteoglycans (HSPGs) are required for efficient entry of HTLV-1. Here, the roles of HSPGs and GLUT1 in HTLV-1 and HTLV-2 Env-mediated binding and entry into primary T cells were studied. Examination of the cell surface of activated primary T cells revealed that CD4(+) T cells, the primary target of HTLV-1, expressed significantly higher levels of HSPGs than CD8(+) T cells. Conversely, CD8(+) T cells, the primary target of HTLV-2, expressed GLUT1 at dramatically higher levels than CD4(+) T cells. Under these conditions, the HTLV-2 surface glycoprotein (SU) binding and viral entry were markedly higher on CD8(+) T cells while HTLV-1 SU binding and viral entry were higher on CD4(+) T cells. Binding studies with HTLV-1/HTLV-2 SU recombinants showed that preferential binding to CD4(+) T cells expressing high levels of HSPGs mapped to the C-terminal portion of SU. Transfection studies revealed that overexpression of GLUT1 in CD4(+) T cells increased HTLV-2 entry, while expression of HSPGs on CD8(+) T cells increased entry of HTLV-1. These studies demonstrate that HTLV-1 and HTLV-2 differ in their T-cell entry requirements and suggest that the differences in the in vitro cellular tropism for transformation and in vivo pathobiology of these viruses reflect different interactions between their Env proteins and molecules on CD4(+) and CD8(+) T cells involved in entry.
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