title: Contribution of nucleoside diphosphokinase to guanine nucleotide regulation of agonist binding to formyl peptide receptors.
abstract: High-affinity agonist binding to formyl peptide receptors in membranes of myeloid differentiated human leukemia (HL 60) cells is known to be regulated by guanine nucleotides, most potently by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]). Here we analyzed whether nucleoside diphosphokinase present in these membranes and capable of forming GTP[S] from GDP and adenosine-5'-O-(3-thiotriphosphate) (ATP[S]) can contribute to nucleotide regulation of agonist receptor binding. Using GDP and ATP[S] at concentrations causing by themselves only small reductions in receptor binding of the labelled formyl peptide, N-formyl-methionyl-leucyl-phenylalanine ([3H]FMLP), a marked potentiation (up to 30-fold) was observed when both nucleotides were combined. Under conditions in which the combination of GDP and ATP[S] induced 70-90% of maximal inhibition of [3H]FMLP binding, a total concentration of about 7 nM GTP[S] formed was measured. The synergistic effect of GDP and ATP[S] on [3H]FMLP binding was not seen in the presence of UDP (1 mM), which blocked formation of GTP[S] from GDP and ATP[S]. Furthermore, no potentiation was observed when instead of GDP and ATP[S], guanosine-5'-O-(2-thiodiphosphate) and adenylyl-5'-imidodiphosphate, respectively, were used. Finally, regulation of [3H]FMLP binding by ATP[S] plus GDP (or GTP) was a time-dependent process, reaching maximal inhibition after 20-30 min of incubation at 25 degrees C. The data indicate that nucleoside diphosphokinase present in membranes of HL 60 cells can transfer the thiophosphate group of ATP[S] to GDP leading to formation of GTP[S] and that the GTP[S] thus formed efficiently binds to G proteins interacting with formyl peptide receptors and thereby regulates their agonist binding affinity.
authors.affiliation: Pharmakologisches Institut der Universität Heidelberg, F.R.G.
authors.forename: T
authors.initials: T
authors.lastname: Wieland
authors.forename: J
authors.initials: J
authors.lastname: Bremerich
authors.forename: P
authors.initials: P
authors.lastname: Gierschik
authors.forename: K H
authors.initials: KH
authors.lastname: Jakobs
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism* / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007951:Leukemia, Myeloid; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014530:Uridine Diphosphate / Q000378:metabolism / Q000494:pharmacology
chemical_list: D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D006153:Guanosine Diphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014530:Uridine Diphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D019204:GTP-Binding Proteins