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

Abstract

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as cffcctor enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1 -10 mM) was found to induce high-affinity binding of the chcinotactic tripeptidc, N-formyl-methionylleucylphenylalanine (met-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of CTP and its analog, guanosine-S'-O-(3-thiotriphosphate), to rcduce Met-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-aflinity GTPase by Met-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive G,-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of G,-protein-coupled P-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity-binding of isoproterenol to p-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibired by the GTP analog, guanosine-S'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi-and G,-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selectivc action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.

Signal amplification in HL‐60 granulocytes

“…We have recently reported that Mg2 + markedly enhanced the number of high-affinity Met-Leu-Phe-binding sites in HL-60 membranes [27]. This effect was also elicited with other divalent cations such as Ca2+, Ba2' or Sr2+, and was even seen in response to polycations like the aminoglycoside antibiotic neomycin [41]. We now demonstrate that Mg2+ is both absolutely and specifically required to observe chemotactic agonist-stimulated, high-affinity [35S]GTP[S] binding to HL-60 membranes.…”

Neomycin is a potent secretagogue of mast cells that directly activates a GTP-binding protein involved in exocytosis.

“…Our results further demonstrate that caution should be taken when using neomycin as a selective inhibitor of phosphoinositide hydrolysis (Siess and Lapetina, 1986;Nakashima et al, 1987;Polascik et al, 1987;Hermann et al, 1989). When added to intact cells, neomycin earl clearly interact with a membrane component that eventually activates phosphoinositide breakdown and histamine secretion.…”

“…For example, depending on its concentration neomycin wasshown to stimulate secretion, aggregation responses, and arachidonic acid release from semi-permeabilized platelets (Nakashima et al, 1987;Polascik et al, 1987). Neomycin was also shown to modify the activity of human platelet membrane GTPase (Hermann and Jakobs, 1988) and to induce high-affinity agonist binding of G protein-coupled receptors (Hermann et al, 1989). Therefore, to further investigate our hypothesis, in this study we have examined whether neomycin may activate secretion from mast cells in a mechanism that is independent of phosphoinositide breakdown.…”

Interaction between Phosphoinositide-Phospholipase C and Phosphatidylcholine- Phospholipase D in Plasma Membranes of Retinal Capillary Pericytes

“…Under such a condition, neomycin, a PI-PLC inhibitor, significantly inhibited PC-PLD activity. Although neomy cin, an aminoglycoside antibiotic, is generally known for inhibiting PI-PLC by binding to inositol phospholipids [22,23], neomycin modifies the activity of platelet membrane GTPase [24] and induces high-affinity agonist binding of G protein-coupled receptors [25]. It also has been reported that neomycin inhib its GTPyS-induced histamine secretion and phosphatidic acid formation in permeabilized mast cells [26].…”