Dual Mg²⁺ control of formyl‐peptide‐receptor–G‐protein interaction in HL 60 cells

Abstract

In neutrophils and several other phagocytic cell types, a pertussis-and cholera-toxin-sensitive form of the guanine-nucleotide-binding protein (G-protein) G, 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 formylpeptide receptor with the chemotactic agonist N-formylmethionyl-leucyl-phenylalanine (Met-Leu-Phe) and (b) the receptor-mediated activation of G,. Mg2+ markedly enhanced the number of receptors with high affinity for the radiolabeled oligopeptide fMet-Le~-[~H]Phe. At the same time, Mg" largely increased the potency of guanosine-5'-(3-@thio)triphosphate, but not of GDP or guanosine-5'-(2-O-thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mgz+ 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-G, 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 G, 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 G, 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 Met-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.

Neomycin induces high‐affinity agonist binding of G‐protein‐coupled receptors

“…Furthermore, in contrast to ME*+, neomycin did not provide agonist-stimulated GTPase activity of G-proteins, but even at high concentrations inhibited receptor-stimulated GTP hydrolysis. As reported previously in membranes of differentiated HL 60 cells, for the latter two actions low concentrations of Mg2+ are required which are about two orders of magnitude lower than those required for induction of high-affinity agonist receptor binding [26]. Furthermore, whereas the action of Mg2+ at millimolar concentrations can be mimicked by various other divalent cations, the high-affinity Mg2+ actions, which are probably caused by a direct action on the G-proteins, appear to be much more specific for this divalent cation [26].…”

“…In order to determine whether an intact G-protein is also required for neomycin's action on formyl peptide receptor binding, fMet-Le~-[~H]Phe binding was studied in membranes of control and pertussis-toxin-treated cells. As shown in [26], and that neomycin, although inducing high-affinity fMet-Le~-[~H]Phe binding in a G-protein-dependent manner, does not increase, but even somewhat decreases, the potency of guanine nuclcotides to regulate fMet-Le~-[~H]Phe receptor binding.…”

“…At 10 mM concentration of these antibiotics, the increase in binding was identical to that seen with 10 mM MgC12. We have recently shown that binding of fMet-Le~-[~H]Phe in membrancs of differentiated HL 60 cells is best described by two affinity states with Kd values o f 0.6-1 nM for the high-affinity state and about 30 nM for the low-affinity state and that Mgz+ increases binding of m e tLeu-['H]Phe essentially by enhancing the number of receptors exhibiting high affinity for the labeled agonist [26]. When similar binding saturation experiments were performed in the presence of neomycin (6mM) in comparison to MgC12…”

“…In order to examine the interaction of Mg2+ and neomycin with regard to guanine nucleotide regulation of agonist binding to formyl peptide receptors, Met-Leu-['HIPhe receptor binding was measured in the presence of 0.1 pM GTP[S], a concentration without effect on neomycininduced high-affinity agonist receptor binding but with almost maximal effect in the presence of Mg2+. As illustrated in + required for inducing highaffinity agonist receptor binding [26].…”

Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells

“…1 mM ph,"nylmethylsulfotayl fluoride, and 2/ag/ml soybean trypsin inhibitor) and lysed by nitrogen cavitation as described in [18], except that cells were pressurized with N2 at 4 MPa for 30 mi:, at ~." '7 Removal of unbroken cells and nuclei and preparation of a crude membrane and a cyto~olic fraction from the cavitate was performed as described [18,19]. Whole cell ly'~ates were prepared by scraping the cells into ice-cold extraction buffer (20 mM Tris-HCI, pH 7.5:5 mM EDTA, 10 mM EGTA, 37 mM sodium chelate, 3 mM benzamidine, 43 mM 2-mercaptoethanol, and 100 ,tiM phenylmethylsulfon:tl flapride), incubated for 1 h with gentle vortexing every 10 rain, and then centrifuged at 15,000 x g lbr 20 rain.…”