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

Neomycin, an inositol‐phospholipid‐binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector 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 chemotactic tripeptide, N ‐formyl‐methionylleucylphenylalanine (fMet‐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 GTP and its analog, guanosine‐5′‐ O ‐(3‐thiotriphosphate), to reduce fMet‐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‐affinity GTPase by fMet‐Leu‐Phe. In close agreement to these findings on the pertussis‐toxin‐sensitive G i ‐protein‐coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high‐affinity agonist binding of G s ‐protein‐coupled β‐adrenoceptors. Similar to formyl peptide receptor binding, high‐affinity binding of isoproterenol to β‐adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine‐5′‐ 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 G i ‐ and G s ‐protein‐coupled receptors, but not in inducing subsequent G‐protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor‐G‐protein interaction.