High Constitutive Activity of the Human Formyl Peptide Receptor

Wenzel-Seifert, Katharina; Hurt, Carl M.; Seifert, Roland

doi:10.1074/jbc.273.37.24181

Cited by 92 publications

(108 citation statements)

References 85 publications

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“…CsH blocks FPR-evoked responses (21,29,30). Accordingly, CsH blocked the chemotactic activity of FMLP on basophils, but had no effect on the response evoked by uPA.…”

Section: Discussionmentioning

confidence: 99%

Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2

Paulis

Montuori

Prevete

et al. 2004

The Journal of Immunology

100

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Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10−12–10−9 M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84–95 (uPAR84–95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84–95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84–95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84–95, which is an endogenous ligand for FPRL2 and FPRL1.

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“…CsH blocks FPR-evoked responses (21,29,30). Accordingly, CsH blocked the chemotactic activity of FMLP on basophils, but had no effect on the response evoked by uPA.…”

Section: Discussionmentioning

confidence: 99%

Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2

Paulis

Montuori

Prevete

et al. 2004

The Journal of Immunology

100

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“…Effects of Na ϩ , Li ϩ , and K ϩ on GTP␥S binding to CHO cell membranes were determined using a buffer containing 75 mM Tris-Cl, pH 7.4, 12.5 mM Mg 2ϩ , 1 mM EDTA, 3 M GDP (Buffer B) to maintain the conditions previously described by Wenzel-Seifert et al (26) and Gierschik et al (27). Incubations were carried out at 30°C for 10 min, and GTP␥S binding never exceeded more than 10% of the total added to assure that binding was in the linear range.…”

Section: Methodsmentioning

confidence: 99%

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

Mills

Miettinen

Cummings

et al. 2000

Journal of Biological Chemistry

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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...

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“…CsH was donated by Novartis (Basel, Switzerland). The generation of the baculovirus encoding FPR-WT (isoform 26) was described previously (20). fMLP, tunicamycin, and the anti-FLAG Ig (monoclonal antibody M1) were from Sigma.…”

Section: Methodsmentioning

confidence: 99%

“…histidine tag to the C terminus to allow future purification of FPR mutants and to provide additional protection against proteolysis (20). FPR mutants were generated by sequential overlap-extension PCRs.…”

Section: Methodsmentioning

confidence: 99%

Defective Gi Protein Coupling in Two Formyl Peptide Receptor Mutants Associated with Localized Juvenile Periodontitis

Seifert

Wenzel-Seifert

2001

Journal of Biological Chemistry

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Neutrophils play an important role in host defense against bacterial infections and in the pathogenesis of various inflammatory diseases (1, 2). Neutrophils express GPCRs 1 for the chemoattractants fMLP, complement C5a, interleukin-8, leukotriene B 4 , and platelet-activating factor (3-6). Chemoattractant receptors couple to G i proteins to activate phospholipase C␤2 and phosphoinositide 3-kinase-␥, with subsequent stimulation of chemotaxis, oxygen radical formation, and granule release (3, 7-9). Disruption of the FPR gene in mice increases susceptibility to infection by Listeria monocytogenes (10). However, although at cellular and molecular levels, the human FPR is one of the most extensively studied GPCRs (3-6, 8, 11), the in vivo role of the FPR in man has remained elusive.LJP is a defined periodontal disease that begins at ϳ11-15 years of age, affects the permanent incisors and/or first molars, and is associated with the presence of Actinobacillus actinomycetemcomitans in subgingival pockets. LJP is not associated with other diseases, and there is evidence that LJP is caused by a genetic defect (12, 13). It has been known for a long time that neutrophils from LJP patients show reduced binding of the agonist fMLP and reduced fMLP-induced chemotaxis (14 -16). Genetic analysis of 30 LJP patients revealed that 29 of those patients possess a F110S and/or C126W mutation in the FPR gene (17). In contrast, none of 31 patients with adult periodontitis and none of 20 control subjects possess a F110S or C126W mutation (17). The F110S mutation resides in the third transmembrane domain, whereas the C126W mutation resides in the second intracellular loop (Fig. 1). The second intracellular loop of the FPR is important for G i protein coupling (18), and a C126S mutation uncouples the FPR from G i proteins (19). Based on all these findings, we developed the hypothesis that the underlying cause of LJP is defective G i protein coupling of the FPR. To test this hypothesis, we engineered FLAG epitopetagged FPR-F110S and FPR-C126W and analyzed coupling of these FPR mutants and FPR-WT to the G protein G␣ i2 ␤ 1 ␥ 2 using Sf9 insect cells as the expression system. In previous studies, we already showed that Sf9 insect cells are a very sensitive system for studying G i protein coupling of chemoattractant receptors (20 -22). Here we report that FPR-F110S and FPR-C126W show an almost complete defect and a complete defect in G i protein coupling, respectively. The discrete clinical symptoms associated with the defective FPR indicate that the loss of FPR function in host defense is, for the most part, readily compensated. EXPERIMENTAL PROCEDURESMaterials-The baculovirus encoding G␣ i2 was kindly provided by Dr. A. G. Gilman (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX). Recombinant baculovirus encoding the unmodified versions of the G protein ␤ 1 ␥ 2 subunits was a kind gift of Dr.

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High Constitutive Activity of the Human Formyl Peptide Receptor

Cited by 92 publications

References 85 publications

Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2

Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2

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

Defective Gi Protein Coupling in Two Formyl Peptide Receptor Mutants Associated with Localized Juvenile Periodontitis

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