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.