Dual Mg2+ control of formyl-peptide-receptor--G-protein interaction in HL 60 cells. Evidence that the low-agonist-affinity receptor interacts with and activates the G-protein.

In neutrophils and several other phagocytic cell types, a pertussis- and cholera-toxin-sensitive form of the guanine-nucleotide-binding protein (G-protein) Gp couples receptors for N-formylmethionine-containing chemotactic peptides to stimulation of phospholipase C. Using membranes of myeloid differentiated HL 60 cells, we have examined the role of Mg2+ and guanine nucleotides in regulating (a) the interaction of the formyl-peptide receptor with the chemotactic agonist N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and (b) the receptor-mediated activation of Gp. Mg2+ markedly enhanced the number of receptors with high affinity for the radiolabeled oligopeptide fMet-Leu-[3H]Phe. At the same time, Mg2+ largely increased the potency of guanosine-5'-(3-O-thio)triphosphate, but not of GDP or guanosine-5'-(2-O-thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mg2+ in eliciting these two effects and analysis of the specificities of the relevant divalent cation sites revealed that Mg2+ interacts with at least two independent sites on the receptor-Gp complex. One site is specific for Mg2+ and exhibits affinity in the micromolar range, the other site interacts with millimolar concentrations of several divalent cations in a non-selective fashion. It is suggested that the former site is located on Gp and that interaction of Mg2+ with this site is necessary for the receptor-mediated G-protein activation, whereas interaction of divalent cations with the latter site is necessary for high affinity agonist binding. The regulation of the formyl-peptide receptor binding properties by guanine nucleotides is independent of Gp activation, since inhibition of peptide binding is achieved by addition of both guanine nucleoside diphosphates and triphosphates and is readily seen both in the presence and in the absence of Mg2+. The latter finding, together with the observation that, at micromolar concentrations of Mg2+, high-affinity GTPase activity is stimulated by fMet-Leu-Phe primarily via low affinity receptors, suggests that, contrary to widely held opinions, (a) divalent cations are not required for a functional receptor--G-protein interaction and (b) high-affinity agonist binding is not a prerequisite for the receptor-mediated activation of the G-protein.