title: Receptor-stimulated guanine-nucleotide-triphosphate binding to guanine-nucleotide-binding regulatory proteins. Nucleotide exchange and beta-subunit-mediated phosphotransfer reactions.
abstract: In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins, binding of the GTP analogues, guanosine 5'-[gamma-thio]triphosphate, GTP[S], and guanosine 5'-[beta, gamma-imino]triphosphate, p[NH]ppG, and the regulation of binding by the formyl-peptide-receptor agonist, fMet-Leu-Phe, were studied in membranes of differentiated HL-60 cells. For fMet-Leu-Phe-stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of fMet-Leu-Phe-stimulated binding of GTP[S] and p[NH]ppG were similar for up to approximately 30 s. Thereafter, receptor-stimulated binding of p[NH]ppG rapidly reached equilibrium, whereas the binding of GTP[S] proceeded further. At equipotent concentrations of p[NH]ppG and GTP[S], maximal fMet-Leu-Phe-stimulated binding of GTP[S] was approximately twofold higher than that of p[NH]ppG. Finally, for half-maximal receptor-stimulated binding of GTP[S], approximately fivefold higher concentrations of both Mg2+ and GDP were required than for p[NH]ppG binding. With p[NH]ppG as the competing nucleotide, the extent of receptor-stimulated binding of GTP[S] as well as its Mg2+ requirement and time course were similar to the receptor-stimulated p[NH]ppG binding observed in the presence of GDP. However, with GTP[S] as the competing nucleotide, fMet-Leu-Phe reduced the binding of p[NH]ppG, a reaction further enhanced when GDP was additionally present. Under similar conditions as used in the binding studies, GTP[S] thiophosphorylated a 35-kDa protein, which is most likely a guanine-nucleotide-binding regulatory protein beta subunit [Wieland, T., Nürnberg, B., Ulibarri, I., Kaldenberg-Stasch, S., Schultz, G. & Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111-18118]. The thiophosphorylation state of this protein was regulated by guanine nucleotides, Mg2+ and, most importantly, by activated formyl-peptide receptors. The data thus provide evidence for an essential difference between GTP[S] and p[NH]ppG binding to guanine-nucleotide-binding regulatory proteins and suggest that, in addition to the nucleotide-exchange reaction, a (thio)phosphate-group-transfer process via guanine-nucleotide-binding regulatory protein beta subunits is involved in the receptor-stimulated binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins.
authors.affiliation: Institut für Pharmakologie, Universität GH Essen, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Kaldenberg-Stasch
authors.forename: M
authors.initials: M
authors.lastname: Baden
authors.forename: B
authors.initials: B
authors.lastname: Fesseler
authors.forename: K H
authors.initials: KH
authors.lastname: Jakobs
authors.forename: T
authors.initials: T
authors.lastname: Wieland
mesh_terms: D001667:Binding, Competitive; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology; D018000:Receptors, Peptide / Q000502:physiology
chemical_list: D010710:Phosphates; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D008274:Magnesium