Evidence for nucleoside diphosphokinase-dependent channeling of guanosine 5'-(gamma-thio)triphosphate to guanine nucleotide-binding proteins.
Agonist binding to guanine nucleotide-binding protein (G protein)-coupled receptors in membranes of myeloid differentiated human leukemia (HL-60) cells is inhibited by guanine nucleotides, most potently by the GTP analog guanosine 5'-(gamma-thio)triphosphate (GTP gamma S). In order to study whether GTP gamma S formed locally from adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and GDP by nucleoside diphosphokinase has any advantage over exogenously added GTP gamma S in binding to and activating G proteins, regulation of complement component 5a (C5a) binding to its receptors, as well as formation of GTP gamma S, was studied in membranes of HL-60 cells. GTP gamma S added to HL-60 membranes potently inhibited binding of 125I-C5a (IC50 about 3 nM), an effect not influenced by addition of either GDP or ATP gamma S. When HL-60 membranes were incubated with the combination of ATP gamma S and GDP, a marked potentiation (up to 300-fold) of the inhibition caused by either GDP or ATP gamma S alone was observed. By measuring nucleoside diphosphokinase-catalyzed formation of GTP gamma S and inhibition of 125I-C5a binding in the presence of GDP and ATP gamma S under identical assay conditions, it was found that formed GTP gamma S inhibited binding of 125I-C5a with an IC50 value of about 0.3 nM, thus being about 10-fold more potent than exogenously added GTP gamma S. These data suggest that the GTP gamma S-forming nucleoside diphosphokinase is closely associated with the C5a receptor-G protein complex and channels the formed GTP gamma S into the G protein.
Wieland
T
T
Institut für Pharmakologie, Universität GH Essen, FRG.
Jakobs
K H
KH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D002478:Cells, Cultured; D015936:Complement C5a / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*
C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015936:Complement C5a; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D019204:GTP-Binding Proteins