Receptor Binding Kinetics and Cellular Responses of Six N-Formyl Peptide Agonists in Human Neutrophils

Abstract

The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.

Human mitochondria-derivedN-formylated peptides are novel agonists equally active on FPR and FPRL1, whileListeria monocytogenes-derived peptides preferentially activate FPR

“…A similar difference between the EC 50 measured by calcium mobilization and superoxide production assays has also been observed in the case of the synthetic peptide WKYMVm [19]. Accordingly, Waller et al [6], studying receptor binding kinetics and cellular responses of several N-formyl peptide agonists in human neutrophils, have shown that the EC 50 for Gprotein activation, in a [ 35 S]GTPcS-binding assay, were two to three orders of magnitude greater than the corresponding EC 50 for actin polymerization and superoxide production assays. It was suggested that the differences in the abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions and that post-receptor events contributed to the amplification of the responses.…”

“…The N-formylated peptide fMLF is the smallest peptide that binds to FPR (K d <40 nM) triggering cellular responses in human neutrophils at low nanomolar concentrations [6]. The N-formyl group was determined as a crucial determinant of ligand binding to FPR [7], although several works have revealed the high potency of non-formylated pentapeptide analogs [8].…”

“…The first ligand shown to bind with high affinity (low nanomolar range) to this receptor was lipoxin A4 (LXA4), an anti-inflammatory lipid mediator [10]. Recently, FPRL1 has emerged as a promiscuous receptor, also activated by the pathogenderived peptide Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) from Helicobacter pylori, by host-derived proteins and peptides such as Ac1-25 from annexin 1, cathelicidin LL37, a fragment of the urokinase receptor, the serum amyloid A, the amyloid b peptide (Ab42), the prion protein fragment PrP(106-126), the neuroprotective peptide humanin, and by different synthetic peptides including the hexapeptide Trp-LysTyr-Met-Val-D-Met-NH 2 (WKYMVm) [11,12]. FPRL2 does not respond to fMLF [13], but instead is activated by some non-formylated chemotactic peptides identified as agonists for FPRL1 [14,15].…”

Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils

“…These are followed by a gradual decrease in free [Ca 2ϩ ] i , as redistribution within the cell occurs, to an eventual level up to 100 nM above that of the naïve PMN, accompanied by a gradual rise in pH i to 0.2 to 0.3 units higher than its pH 0 of 7.05 to 7.08, as the Na ϩ /H ϩ antiport opens (2,26,27). We also confirm here, as has been reported previously (3,6,7,13,15,25,39,(48)(49)(50), that in unprimed naïve PMN in suspension, prepared as described in Materials and Methods, no detectable oxidative products or lytic enzymes are released when the cells are exposed to concentrations less than or equal to 10 Ϫ7 M. Since PMN moving up a chemoattractant gradient must recognize that chemoattractant repeatedly as its concentration rises, we mimicked the effect by injecting a second dose of fMLP (10 Ϫ7 M final concentration) 3, 5, or 10 min after the first exposure to this dose. As there was no difference between the 5-and 10-min intervals (data not shown), we performed the remainder of our studies with a 5-min time lapse between the first and second additions of stimuli.…”

“…Chemoattractants released by an organism or by the opsonins that coat it, such as N-formylmethionyl-leucyl-phenylalanine (fMLP), are recognized by their PMN surface receptors. Naïve PMN have been shown to possess two classes of fMLP receptors (fPR1 and fPR2, of high and low affinity, respectively) (25, 40-43), which, when liganded with up to 100 nM peptide, mediate the activation of signals, including a rapid transient rise in cytoplasmic Ca 2ϩ ([Ca 2ϩ ] i ), due largely to release from intracellular stores, and a simultaneous drop in cytoplasmic pH (pH i ) (14,19,26,27,37); when the fMLP concentrations are above 1 M, the loweraffinity fMLP receptor also initiates some release of bactericidal entities (7,25,42,43,49). The initial rapid cytoplasmic signals initiated by fPR1 have been studied extensively; they are followed within less than 1 min by a redistribution such that a final [Ca 2ϩ ] i up to 100 nM above the resting [Ca 2ϩ ] i ([Ca 2ϩ ] i 0 ) of naïve PMN as well as a pH i 0.2 to 0.3 units higher than its resting pH i (pH 0 ) of 7.05 to 7.07 are attained (2, 15, 37).…”