The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction

Vinata B Lokeshwar, L. Y.W. Bourguignon

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Abstract

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 (GTPγS) 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 [γ-32P]GTP at a rate of 0.011 mol of P(i) 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.

Original languageEnglish (US)
Pages (from-to)22073-22078
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number31
StatePublished - Jan 1 1992
Externally publishedYes

Fingerprint

Ankyrins
Guanine Nucleotides
Guanosine Triphosphate
Lymphoma
Glycoproteins
Carrier Proteins
Cholera Toxin
Pertussis Toxin
GTP-Binding Proteins
Adenosine Diphosphate
GTP Phosphohydrolases
Guanosine 5'-O-(3-Thiotriphosphate)
Affinity chromatography
Wheat Germ Agglutinins
Antibody Affinity
Octoxynol
Cell membranes
Cytoskeleton
Affinity Chromatography
Sepharose

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction",
abstract = "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 (GTPγS) 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 [γ-32P]GTP at a rate of 0.011 mol of P(i) 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.",
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