Characterization of the Binding Site on the Formyl Peptide Receptor Using Three Receptor Mutants and Analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu

Abstract

The formyl peptide receptor (FPR) 1 is a chemoattractant G protein-coupled receptor found on the surface of phagocytes. It is thought to play an important role in allowing phagocytic cells to recognize the presence of bacteria (1), which are a source of formyl peptides (2, 3). In addition, it recognizes and is activated by peptides derived from the GP-41 envelope protein of the human immunodeficiency virus type I (HIV-1) (4, 5). Recent studies with FPR-deficient mice indicate that they exhibit an increased susceptibility to infection with Listeria monocytogenes and that neutrophils from these knockout mice fail to exhibit chemotaxis in response to fMLF (6).The formyl peptide receptor was originally identified based on its ability to bind the formylated peptide, fMLF (1). Six different chemotactic peptides have been isolated from Escherichia coli, including fMLF (3), but the only similarity between them was an NH 2 -terminal formyl methionine, suggesting that this moiety is highly important in binding to FPR. Studies using NH 2 -terminal analogs of MLF had indicated that the formyl group had significant effects on the ligand binding affinity for neutrophil FPR. Free amino, desamino, and acetylated derivatives of MLF were all 3000-fold lower in affinity than fMLF (7). Substitution of the formyl group of fMLF with a tert-butyloxycarbonyl group made the ligand an antagonist of low affinity (8). However, the formyl group may be less essential than originally thought. N-Butyloxycarbonyl MLF exhibits agonist activity (9) with an affinity similar to fMLF, and phenyl and tolyl isourea derivatives of MLF exhibit activity similar to or greater than fMLF (10). On the other hand, most aliphatic isourea derivatives of MLF exhibit low affinity antagonist activity similar to what is observed with tert-butyloxycarbonyl-MLF (10). This difference indicates that FPR exhibits a high degree of specificity for NH 2 -terminal modifications of MLF, and that the specificity for the formyl group is not absolute. In addition, other reports have indicated that non-formylated pentapeptides can activate FPR. Both MNleLFF and MMWLL are effective activators of FPR (11,12), and acetyl-MNleLFF is more potent than fMLF (12), indicating that these pentapeptides exhibit somewhat different NH 2 -terminal specificities than does MLF.We have previously shown that the formyl peptide binding site maps to several membrane-spanning regions (13-14). Ten residues which affect fMLF binding have been mapped to transmembrane domains II-VII (13), including residues Leu-78 (II-17, helix II, residue 17 of 26 transmembrane-spanning residues in the nomenclature used throughout this text), Asp-106 (III-8), Leu-109 (III-11), Thr-157 (IV-18), Arg-201 (V-2), Ile-204 (V-5), , , and Phe-291 (VII-11). In addition, photo-cross-linking data suggest that the leucine side chain of fMLF is probably located close to FPR 93 VRK 95 , which is at the COOH terminus of helix II (14).Human FPR is one of three receptors in the human FPR family, which includes, FPR, the lipoxin A 4 re...

Gastrin-Releasing Peptide/Neuromedin B Receptor Antagonists PD176252, PD168368, and Related Analogs Are Potent Agonists of Human Formyl-Peptide Receptors

“…Thus, because amino acids (in particular Tyr-220) in transmembrane region 5 (TM-5) of the neuromedin receptor (BB 1 ) play a major role in PD168368 binding (Tokita et al, 2001), it can be hypothesized that the binding site for PD168368, PD176252, and analogs may lie in the TM-5 region of FPR2. Indeed, the amino sequence 201 RGIIR 205 is conserved in TM-5 of most species variants for both FPR1 and FPR2 (Alvarez et al, 1996), and site-directed mutagenesis supports the role of residues Arg-201 and Arg-205 in positioning fMLF in the FPR1-binding pocket (Mills et al, 2000). Although bombesin-related receptors and FPRs are phylogenetically quite far from each other in the human rhodopsin receptor family (Gloriam et al, 2009), it seems that conserved residues in these GPCRs results in ligand promiscuity among unrelated receptor targets.…”

Functional Characterization of Three Mouse Formyl Peptide Receptors

“…Notably, some important residues known to be critical for the interaction with formylated peptides at their C-terminus (Mills et al, 2000), such as Arg205, are not present in all receptors. In human FPR3, a histidine takes place at position 205, but whether this substitution is sufficient to alter ligand binding specificity remains unclear because an arginine is found at the same position in all three mFprs tested.…”

Integration of Virtual Screening with High-Throughput Flow Cytometry to Identify Novel Small Molecule Formylpeptide Receptor Antagonists

“…2D). Data from cross-linking and mutagenesis studies suggest that the FPR ligand binding site is located between the transmembrane helices, near the extracellular face of the membrane (Mills et al, 2000). Several weak micromolar binders (phenylbutazone and related pyrazolidinedione drugs) and strong nanomolar binders: cyclosporins and formylpeptide derivatives had been described previously (Dalpiaz et al, 2002;Bae et al, 2003).…”

“…Data from cross-linking and mutagenesis studies suggest that the FPR ligand binding site is located between the transmembrane helices, near the extracellular face of the membrane (Mills et al, 2000). The crystal structure of bovine rhodopsin (Protein Data Bank code 1F88) was used as starting point for binding site modeling.…”