The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction.
In this study, we have used photoaffinity labeling by [32P]azido-GTP as well as [32P]ADP-ribosylation by pertussis toxin (PT) and cholera toxin (CT) to identify GTP-binding proteins associated with mouse T-lymphoma plasma membranes. Our results indicate that GP85 (CD44) can be photoaffinity labeled by [32P] azido-GTP and [32P]ADP-ribosylated by both PT and CT. Using purified GP85 (CD44) obtained by Triton X-100 extraction, wheat germ agglutinin-Sepharose, and anti-GP85 (CD44) antibody affinity chromatographies, we have further characterized GP85 (CD44) as a GTP-binding protein. GP85 (CD44) is found to bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in a time- and dose-dependent manner with a dissociation constant of 0.83 nM. Importantly, GP85 (CD44) appears to display a GTPase activity which hydrolyzes [gamma-32P]GTP at a rate of 0.011 mol of Pi released/mol of GP85 (CD44)/min. This GTPase activity can be readily inhibited by PT- or CT-mediated ribosylation of GP85 (CD44). Most interestingly, GTP binding significantly enhances the interaction of purified GP85 (CD44) with ankyrin, whereas ADP-ribosylation of GP85 (CD44) by PT or CT inhibits the GTP-induced increase in ankyrin binding to GP85 (CD44). In addition to GP85 (CD44) being the first reported transmembrane GTP-binding protein, these results suggest that GTP plays an important role in promoting the interaction between GP85 (CD44) and its underlying membrane cytoskeleton through ankyrin.
Lokeshwar
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Department of Cell Biology and Anatomy, School of Medicine, University of Miami, Florida 33101.
Bourguignon
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LY
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D017487:Ankyrins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D017487:Ankyrins; D046911:Macromolecular Substances; D016201:Receptors, Lymphocyte Homing; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins