title: G-proteins are not directly involved in the CD3-antigen-mediated production of inositol phosphates in HPB-ALL T-leukaemia cells expressing phospholipase C isoforms gamma 1 and beta 3.
abstract: The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.
authors.affiliation: Department of Immunology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
authors.forename: M
authors.initials: M
authors.lastname: Biffen
authors.forename: M
authors.initials: M
authors.lastname: Shiroo
authors.forename: D R
authors.initials: DR
authors.lastname: Alexander
mesh_terms: D017252:CD3 Complex / Q000502:physiology*; D002463:Cell Membrane Permeability; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D015459:Leukemia-Lymphoma, Adult T-Cell; D016853:Muromonab-CD3 / Q000494:pharmacology; D010740:Phospholipases / Q000302:isolation & purification / Q000378:metabolism; D013601:T-Lymphocytes; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
chemical_list: D017252:CD3 Complex; D007295:Inositol Phosphates; D007527:Isoenzymes; D016853:Muromonab-CD3; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014638:Vanadates; C023427:guanosine 5'-O-(2-thiodiphosphate); D015544:Inositol 1,4,5-Trisphosphate; D010740:Phospholipases; D019204:GTP-Binding Proteins