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J. Biol. Chem., Vol. 276, Issue 45, 42043-42049, November 9, 2001

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Defective G_i Protein Coupling in Two Formyl Peptide Receptor Mutants Associated with Localized Juvenile Periodontitis^*

Roland Seifert and Katharina Wenzel-Seifert

From the Department of Pharmacology and Toxicology, University of Kansas, Lawrence, Kansas 66045-2505

Received for publication, July 16, 2001, and in revised form, August 30, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The formyl peptide receptor (FPR) is a prototypical chemoattractant receptor expressed in neutrophils. It is well known that the FPR couples to G_i proteins to activate phospholipase C, chemotaxis, and cytotoxic cell functions, but the in vivo role of the FPR in man has remained elusive. Recently, F110S and C126W mutations of the FPR have been associated with localized juvenile periodontitis. We studied FPR-F110S and FPR-C126W in comparison with wild-type FPR (FPR-WT) by coexpressing epitope-tagged versions of these receptors with the G protein G_i212 in Sf9 insect cells. FPRs were efficiently expressed in Sf9 membranes as assessed by immunoblotting using the ₂-adrenoreceptor as a standard. FPR-C126W differed from FPR-WT and FPR-F110S in migration on SDS-polyacrylamide gels and tunicamycin-sensitive glycosylation. FPR-WT efficiently reconstituted high-affinity agonist binding and agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPS) binding to G_i212. In contrast, FPR-F110S only weakly reconstituted agonist-stimulated GTPS binding, and FPR-C126W was completely inefficient. Collectively, our data show almost complete and complete loss of G_i protein coupling in FPR-F110S and FPR-C126W, respectively. The severe functional defects in FPR-F110S and FPR-C126W contrast with the discrete clinical symptoms associated with these mutations, indicating that loss of FPR function in host defense is, for the most part, readily compensated.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Neutrophils play an important role in host defense against bacterial infections and in the pathogenesis of various inflammatory diseases (1, 2). Neutrophils express GPCRs¹ for the chemoattractants fMLP, complement C5a, interleukin-8, leukotriene B₄, and platelet-activating factor (3-6). Chemoattractant receptors couple to G_i proteins to activate phospholipase C2 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 ~11-15 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 epitope-tagged FPR-F110S and FPR-C126W and analyzed coupling of these FPR mutants and FPR-WT to the G protein G_i212 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.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence of FPR-WT and localization of the F110S and C126W mutations. Shown is the two-dimensional structure of FPR-WT (isoform 26) (27). Amino acids are given in one-letter code. The FPR N terminus (top) faces the extracellular space; the FPR C terminus (bottom) faces the cytosol. The transmembrane domains are included in the boxed area. Extracellular consensus sites for N-glycosylation are shown (Y). The positions of the F110S and C126W mutations are indicated (). There is a disulfide bridge between the first and second extracellular loops. Note that the consensus sites for N-glycosylation are not altered in FPR-F110S and FPR-C126W.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- 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 ₁₂ subunits was a kind gift of Dr. P. Gierschik (Department of Pharmacology and Toxicology, University of Ulm, Ulm, Germany). 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. The anti-G_i2 Ig was from Calbiochem. [³⁵S]GTPS (1100 Ci/mmol), [³H]dihydroalprenolol (85-90 Ci/mmol), and [³H]fMLP (56 Ci/mmol) were from PerkinElmer Life Sciences. Unlabeled GTPS and GDP were from Roche Molecular Biochemicals. All restriction enzymes and T4 DNA ligase were from New England Biolabs Inc. (Beverly, MA). Cloned Pfu DNA polymerase was from Stratagene (La Jolla, CA).

Construction of FLAG Epitope- and Hexahistidine-tagged FPR-F110S and FPR-C126W-- A DNA sequence encoding the cleavable signal peptide from influenza hemagglutinin followed by the FLAG epitope, which can be recognized by the M1 antibody, was placed 5' of the start codon of the cDNAs of FPR mutants. We also added a hexahistidine 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. To create FPR-F110S, in PCR-1A, the N-terminal portion of the DNA sequence encoding FPR-WT was amplified using a sense primer encoding the last 18 base pairs of the FLAG epitope and an antisense primer introducing the F110S mutation (accompanied by the creation of a new BspEI site) with pGEM-3Z-SF-FPR-WT26 as template. In PCR-1B, the C-terminal portion of the DNA sequence encoding FPR-WT was amplified using a sense primer introducing the F110S mutation and an antisense primer encoding the two C-terminal amino acids of FPR-WT, a hexahistidine tag, the stop codon, and an extra XbaI site with pGEM-3Z-SF-FPR-WT26 as template. In PCR-1C, the products of PCR-1A and PCR-1B were annealed in the region coding for the newly introduced F110S mutation and were amplified with the sense primer of PCR-1A and the antisense primer of PCR-1B. The resulting fragment was digested with AvaI and BseRI and cloned into pGEM-3Z-SF-FPR-WT26 digested with AvaI and BseRI. The DNA encoding FPR-F110S was cloned into the baculovirus expression vector pVL1392 using the SacI site at the 5'-end of the signal FLAG region of the receptor and the XbaI site at the 3'-end of the receptor. To create FPR-C126W, in PCR-2A, the N-terminal portion of the DNA sequence encoding FPR-WT was amplified using a sense primer encoding the last 18 base pairs of the FLAG epitope and an antisense primer introducing the C126W mutation (accompanied by the creation of a new BsgI site) with pGEM-3Z-SF-FPR-WT26 as template. In PCR-2B, the C-terminal portion of the DNA sequence encoding FPR-WT was amplified using a sense primer introducing the C126W mutation and an antisense primer encoding the two C-terminal amino acids of FPR-WT, a hexahistidine tag, the stop codon, and an extra XbaI site with pGEM-3Z-SF-FPR-WT26 as template. In PCR-2C, the products of PCR-2A and PCR-2B were annealed in the region coding for the newly introduced C126W mutation and were amplified with the sense primer of PCR-2A and the antisense primer of PCR-2B. The resulting fragment was digested with AvaI and BseRI and cloned into pGEM-3Z-SF-FPR-WT26 digested with AvaI and BseRI. The DNA encoding FPR-C126W was cloned into the baculovirus expression vector pVL1392 using the SacI site at the 5'-end of the signal FLAG region of the receptor and the XbaI site at the 3'-end of the receptor. PCR-generated DNA sequences were confirmed by restriction enzyme analysis and enzymatic sequencing.

Generation of Recombinant Baculoviruses, Cell Culture, and Membrane Preparation-- Sf9 cells were cultured in 250-ml disposable Erlenmeyer flasks at 28 °C under rotation at 125 rpm in SF 900 II medium (Life Technologies, Inc.) supplemented with 5% (v/v) fetal calf serum (BioWhittaker, Inc., Walkersville, MD) and 0.1 mg/ml gentamycin. Cells were maintained at a density of 1.0-6.0 × 10⁶ cells/ml. Recombinant baculoviruses were generated in Sf9 cells using the BaculoGold transfection kit (Pharmingen, San Diego, CA) according to the manufacturer's instructions. For membrane preparation, cells were sedimented by centrifugation and suspended in fresh medium at 3.0 × 10⁶ cells/ml. Cells were infected with 1:100 dilutions of high-titer baculovirus stocks encoding GPCRs, G_i2, and ₁₂ complex. At this time, we added tunicamycin (10 µg/ml) to some cultures to inhibit N-glycosylation of GPCRs (23). Cells were cultured for 48 h before membrane preparation. Sf9 membranes were prepared as described (24) using 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml benzamidine, and 10 µg/ml leupeptin as protease inhibitors. Membranes were suspended in binding buffer (12.5 mM MgCl₂, 1 mM EDTA, and 75 mM Tris-HCl, pH 7.4) and stored at 80 °C until use.

Binding Assays-- [³H]Dihydroalprenolol saturation binding to determine the expression of the ₂-adrenoreceptor (₂AR) was carried out as described (24). For [³H]fMLP saturation binding, Sf9 membranes were thawed, centrifuged at 15,000 × g for 15 min at 4 °C, and suspended in binding buffer. Reaction mixtures (500 µl) contained Sf9 membranes (50-75 µg of protein/tube) in binding buffer supplemented with [³H]fMLP at 0.2-30 nM. Nonspecific binding was determined in the presence of 10 µM unlabeled fMLP. Incubations were conducted for 60 min at 25 °C with shaking at 200 rpm. Bound [³H]fMLP was separated from free [³H]fMLP by filtration through GF/C filters, followed by three washes with 2 ml of binding buffer (4 °C). Filter-bound radioactivity was determined by liquid scintillation counting. For GTPS binding time course studies, Sf9 membranes were suspended in 1500 µl of binding buffer supplemented with 0.05% (w/v) bovine serum albumin, 1 nM [³⁵S]GTPS, 9 nM unlabeled GTPS, and 1 µM GDP in the absence and presence of fMLP (10 µM) or CsH (3 µM). Reactions were conducted at 25 °C with shaking at 200 rpm. Aliquots of 200 µl (containing 15-40 µg of protein) were withdrawn at different time points. Nonspecific [³⁵S]GTPS binding was determined in the presence of 10 µM unlabeled GTPS and was <0.1% of total binding. Bound [³⁵S]GTPS was separated from free [³⁵S]GTPS by filtration through GF/C filters, followed by three washes with 2 ml of binding buffer (4 °C). Filter-bound radioactivity was determined by liquid scintillation counting. For GTPS saturation experiments, reaction mixtures (500 µl) contained Sf9 membranes (15-30 µg of protein/tube), 0.5-2 nM [³⁵S]GTPS plus unlabeled GTPS to achieve final GTPS concentrations of 0.5-20 nM, 1 µM GDP, and 0.05% (w/v) bovine serum albumin in the absence and presence of fMLP (10 µM) or CsH (3 µM). Reactions were conducted for 60 min at 25 °C with shaking at 200 rpm. For studying the effect of NaCl on GTPS binding, reaction mixtures (500 µl) contained Sf9 membranes (15-30 µg of protein/tube), 0.4 nM [³⁵S]GTPS, 1 µM GDP, 0.05% (w/v) bovine serum albumin, and NaCl at various concentrations in the absence and presence of fMLP (10 µM). Reactions were conducted for 60 min at 25 °C with shaking at 200 rpm.

Analysis of FPR and G_i2 Expression in Sf9 Membranes-- Sf9 membranes were analyzed by immunoblotting using the M1 monoclonal antibody (1:1000), which recognizes the N-terminal FLAG epitope of the chemoattractant receptors (20, 24), and anti-G_i2 Ig (1:1000) (21). The acrylamide concentration in gels was 10% (w/v). Proteins were transferred onto Immobilon-P transfer membranes (Millipore Corp., Bedford, MA). Proteins were visualized with peroxidase-coupled sheep anti-mouse IgG (monoclonal antibody M1) and donkey anti-rabbit IgG (anti-G_i2 Ig), respectively, using o-dianisidine and H₂O₂ as substrates.

Miscellaneous-- Protein concentrations were determined using the Bio-Rad DC protein assay kit. Data were analyzed by nonlinear regression using the Prism III program (GraphPAD-Prism, San Diego, CA).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Different Electrophoretic Mobility and Glycosylation of FPR-C126W Compared with FPR-WT and FPR-F110S-- The expression of FLAG epitope-tagged FPR-WT and FPR mutants in Sf9 membranes was examined by immunoblotting with the M1 monoclonal antibody. As a standard, we used FLAG epitope-tagged ₂AR expressed at a level of 3.9 pmol/mg as assessed by antagonist saturation binding. In this way, the expression of FPR-WT and FPR mutants could be determined without relying on the [³H]fMLP binding assay. This was important for two reasons. First, the [³H]fMLP binding assay, as other agonist binding assays (25), largely underestimates the actual GPCR expression level (21). Second, high-affinity [³H]fMLP binding depends on intact FPR/G_i protein coupling (19, 20, 26), but we assumed that G_i protein coupling is defective in FPR-F110S and FPR-C126W.

Fig. 2A compares the electrophoretic mobility of FPR-WT, FPR mutants, and the ₂AR and analyzes the effect of tunicamycin treatment on electrophoretic mobility. Fig. 2B allows for direct comparison of the electrophoretic mobility of fully glycosylated FPR-WT and FPR mutants. The ₂AR migrated as a doublet of 50-52 kDa, representing differently glycosylated forms of the GPCR (24). As reported before (20), FPR-WT migrated as a series of broad bands of 38-45 kDa, reflecting the fact that this GPCR is more extensively glycosylated than the ₂AR. Although the different shapes of the ₂AR and FPR-WT bands render a precise comparison difficult, one can nonetheless estimate that FPR-WT was expressed at a level of ~6-8 pmol/mg.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Analysis of the expression of FPR-WT, FPR-F110S, FPR-C126W, and Gi2 in Sf9 cell membranes by immunoblotting. Sf9 membranes expressing FPR-WT and FPR mutants plus Gi212 or 2AR were prepared. The expression level of 2AR (3.9 pmol/mg) was determined by [3H]dihydroalprenolol saturation binding. For the 2AR, 25, 50, or 100 µg of protein were loaded onto each lane. Otherwise, 100 µg of protein were loaded onto each lane. Membrane proteins were separated by SDS-polyacrylamide electrophoresis and probed with the M1 antibody (anti-FLAG Ig) (A and B) or anti-Gi2 Ig (C) as described under "Experimental Procedures." Numbers on the left indicate molecular masses (in kilodaltons) of marker proteins. Shown are the horseradish peroxidase-reacted Immobilon-P membranes of gels containing 10% (w/v) acrylamide. wt, FPR-WT; F110S, FPR-F110S; C126W, FPR-C126W; c, membranes from control cells (no tunicamycin treatment); t, membranes from cells treated with tunicamycin (10 µg/ml). The immunoblots shown in A and B were performed with different membrane preparations. The immunoblots shown in B and C were performed with the same membrane preparation.

To examine the glycosylation of FPR-WT in more detail, we treated Sf9 cells with an inhibitor of N-glycosylation, tunicamycin (23), during the protein expression period. In membranes from tunicamycin-treated Sf9 cells, FPR-WT migrated as a single 38-39-kDa band. This size corresponds well to the predicted molecular mass of unmodified FPR-WT (27), indicating that FPR-WT is exclusively N-glycosylated. However, the intensity of the FPR-WT band in membranes from tunicamycin-treated Sf9 cells was much lower than the band intensities in control membranes, suggesting that N-glycosylation is important for proper membrane targeting of FPR-WT. The importance of N-glycosylation for membrane targeting has been reported for other GPCRs (28, 29).

In terms of expression level, apparent molecular mass, glycosylation pattern, and tunicamycin sensitivity of glycosylation, FPR-F110S was indistinguishable from FPR-WT. The data also indicate that membrane targeting of FPR-WT and FPR-F110S is similar. Moreover, there was no evidence for structural instability of FPR-F110S since such a property would have resulted in decreased expression levels relative to FPR-WT (30).

The expression level of FPR-C126W seemed to be in a range similar to that of the expression levels of FPR-WT and FPR-F110S, but it was difficult to quantify FPR-C126W expression precisely because the migration of FPR-C126W on SDS-polyacrylamide gels was very different from the migration of FPR-WT and FPR-F110S. Specifically, in addition to the multiple 38-45-kDa bands that were observed for FPR-WT and FPR-F110S, we also observed multiple bands in the 28-32-kDa region and, less prominently, in the 45-100-kDa region. Although the majority of FPR-C126W migrated as 28-32-kDa proteins, i.e. well below the expected molecular mass of non-glycosylated FPR-WT (38-39 kDa), this discrepancy does not automatically imply that FPR-C126W represents an aggregated GPCR. Abnormal migration on SDS-polyacrylamide gels was also observed for wild-type GPCRs (23). The 38-45-kDa bands in Sf9 membranes expressing FPR-C126W were less intense than the corresponding bands in membranes expressing FPR-WT and FPR-F110S. In membranes from tunicamycin-treated Sf9 cells, the size of the 28-32-kDa proteins decreased somewhat; but overall, tunicamycin had little effect on the migration of FPR-C126W. In contrast to the data obtained with FPR-WT and FPR-F110S, tunicamycin did not have an inhibitory effect on the expression of FPR-C126W. Taken together, our data clearly demonstrate that the C126W mutation has a profound impact on electrophoretic mobility and glycosylation of the FPR, whereas the F110S mutation is without effect.

Defective High-affinity [³H]fMLP Binding to FPR-F110S and FPR-C126W-- As already stated above, high-affinity [³H]fMLP binding to FPR depends on the expression of G_i proteins (19, 20, 26). In previous studies on various GPCRs including the FPR, we already showed that G_i2 is expressed at high levels (~200-450 pmol/mg) in Sf9 membranes (21, 22, 31). Using Sf9 membranes expressing G_i2 at ~300 pmol/mg and FPR-WT as a standard, we confirmed that G_i2 was expressed at similar levels in membranes expressing FPR-F110S and FPR-C126W (Fig. 2C). Thus, we can estimate that there is an ~40-50-fold excess of FPR-WT and FPR mutants relative to G_i2 in Sf9 membranes. This ratio should provide excellent conditions for detecting GPCR/G protein coupling in terms of high-affinity agonist binding and GDP/GTP exchange (22).

As reported before (20), in membranes expressing FPR-WT, we readily detected high-affinity [³H]fMLP binding. FPR-WT bound [³H]fMLP with a K_d of 3.2 ± 1.0 nM and a B_max of 0.90 ± 0.10 pmol/mg (Fig. 3A). The K_d for high-affinity [³H]fMLP binding to FPR-WT in Sf9 membranes agrees with the K_d values obtained for the FPR expressed in native systems (32, 33). It should also be noted that nonspecific [³H]fMLP binding in Sf9 membranes expressing FPR-WT was <10% of total [³H]fMLP binding, ensuring high sensitivity for detecting high-affinity agonist binding. However, despite the sensitivity of Sf9 membranes for analyzing high-affinity [³H]fMLP binding (Fig. 3A) and despite the fact that FPR-F110S, FPR-C126W, and G_i2 were efficiently expressed (Fig. 2), we failed to detect high-affinity agonist binding with the two FPR mutants (Fig. 3, B and C).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   [3H]fMLP saturation binding in Sf9 cell membranes expressing FPR-WT, FPR-F110S, or FPR-C126W plus Gi212. Membranes expressing FPRs plus Gi212 were prepared. [3H]fMLP saturation binding experiments in membranes expressing FPR-WT plus Gi212 (A), FPR-F110S plus Gi212 (B), and FPR-C126W plus Gi212 (C) were carried out as described under "Experimental Procedures." Total [3H]fMLP binding is shown (). Nonspecific binding was determined in the presence of 10 µM unlabeled fMLP (). Data shown are the means ± S.D. of three experiments performed in triplicates. Binding data were analyzed by nonlinear regression and were best fitted (F test) to monophasic saturation curves.

Inefficiency of FPR-F110S and FPR-C126W in Stimulating GTPS Binding to G_i Proteins-- The results of the [³H]fMLP binding studies strongly suggest that G_i protein coupling is defective in FPR-F110S and FPR-C126W. However, these studies cannot rule out the possibility that fMLP binds to the FPR mutants with low affinity and that this low-affinity fMLP binding induces a conformational change in GPCRs that enables them to promote GDP/GTP exchange. Indeed, even for FPR-WT, low-affinity fMLP binding to the FPR results in efficient stimulation of GDP/GTP exchange (20, 21). Additionally, FPR-WT is constitutively active, i.e. even agonist-free FPR-WT efficiently stimulates GDP/GTP exchange at G_i proteins. This constitutive activity of FPR-WT is unmasked by strong inhibitory effects of the inverse agonist CsH on basal GDP/GTP exchange (20, 21).

To address the possible activation of FPR mutants by low-affinity fMLP binding and to determine the constitutive activity of FPR-F110S and FPR-C126W, we analyzed GDP/GTPS exchange. In addition to its hydrolysis resistance, GTPS possesses an ~100-fold higher affinity for G_i proteins than does GTP (21). These properties render [³⁵S]GTPS a suitable probe for analyzing guanine nucleotide exchange in a binding assay. We used fMLP at 10 µM, i.e. a concentration that is sufficient to saturate both high- and low-affinity binding to the FPR (20, 21). Over the entire time period of 5-180 min, fMLP stimulated GTPS binding to G_i212 in membranes expressing FPR-WT, whereas CsH reduced GTPS binding (Fig. 4A). The stimulatory effects of fMLP were particularly large at earlier time points of the binding reaction (up to 150% stimulation above basal levels), whereas the inhibitory effects of CsH were particularly large at later time points of the binding reaction. fMLP decreased the t_1/2 of GTPS binding by ~3-fold from 16.1 ± 3.4 to 5.6 ± 0.8 min, whereas CsH increased the t_1/2 to 36.1 ± 7.2 min. In membranes expressing FPR-F110S, fMLP increased the t_1/2 of GTPS binding by ~1.5-fold from 22.9 ± 3.0 to 14.7 ± 2.6 min, with the stimulatory effect of fMLP amounting to ~10-20% (Fig. 4B). CsH was without inhibitory effect on GTPS binding in membranes expressing FPR-F110S, indicating that this FPR mutant, in contrast to FPR-WT, is not constitutively active. Although with FPR-F110S minimal fMLP-stimulated GDP/GTPS exchange at G_i212 could be detected (Fig. 4B), we failed to detect agonist-stimulated GTPS binding with FPR-C126W (Fig. 4C). Moreover, CsH had no inhibitory effect on GTPS binding to G_i212 with FPR-C126W. Taken together, these data indicate that FPR-F110S exhibits an almost complete G_i protein coupling defect and that FPR-C126W exhibits a complete defect.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Time course of GTPS binding in Sf9 cell membranes expressing FPR-WT, FPR-F110S, or FPR-C126W plus Gi212. Membranes expressing FPRs plus Gi212 were prepared. [35S]GTPS binding experiments in membranes expressing FPR-WT plus Gi212 (A), FPR-F110S plus Gi212 (B), and FPR-C126W plus Gi212 (C) were carried out as described under "Experimental Procedures." Membranes were incubated for the periods of time indicated in the presence of solvent (basal; ), 10 µM fMLP (), or 3 µM CsH (). The total GTPS concentration was 10 nM (1 nM [35S]GTPS plus 9 nM unlabeled GTPS). Reaction mixtures also contained 1 µM GDP. Data shown are the means ± S.D. of three experiments performed in triplicates. Binding data were analyzed by nonlinear regression and were best fitted (F test) to monophasic saturation curves.

In an effort to enhance the relative stimulatory effects of fMLP on GTPS binding in membranes expressing FPR-F110S and FPR-C126W, we studied the effect of NaCl on GTPS binding. Na⁺ stabilizes the FPR and other chemoattractant receptors in an inactive state. As a result, Na⁺ reduces basal GTPS binding and substantially enhances the relative stimulatory effect of chemoattractants (20, 22, 34). In membranes expressing FPR-WT, NaCl strongly reduced basal GTPS binding and enhanced the relative stimulatory effect of fMLP from ~1.5-fold in the absence of NaCl up to almost 6-fold with 150 mM NaCl (Fig. 5, A and B). In contrast, even at concentrations as high as 200 mM, NaCl had almost no inhibitory effect on basal GTPS binding and no enhancing effect on GTPS binding in the presence of fMLP in membranes expressing FPR-F110S (Fig. 5, C and D) or FPR-C126W (E and F). These data corroborate the notion that FPR-F110S and FPR-C126W exhibit defective G_i protein coupling and are not constitutively active.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of NaCl on basal and fMLP-stimulated GTPS binding in Sf9 cell membranes expressing FPR-WT, FPR-F110S, or FPR-C126W plus Gi212. Membranes expressing FPRs plus Gi212 were prepared. [35S]GTPS binding experiments in membranes expressing FPR-WT plus Gi212 (A and B), FPR-F110S plus Gi212 (C and D), and FPR-C126W plus Gi212 (E and F) were carried out as described under "Experimental Procedures." Reaction mixtures contained 1 µM GDP, 0.4 nM [35S]GTPS, and solvent (basal; ) or 10 µM fMLP (). In addition, reaction mixtures contained NaCl at the concentrations indicated. Data shown in A, C, and E are the means ± S.D. of three experiments performed in triplicates and represent absolute GTPS binding values. Data were analyzed by nonlinear regression (best fit to a monophasic exponential decay function, F test). To obtain the relative stimulatory effects of fMLP on GTPS binding (; B, D, and F), we divided the GTPS binding values in the presence of fMLP by the GTPS binding values in the presence of solvent for each NaCl concentration.

To allow comparison with previous studies from our laboratory (20-22), we conducted the time course studies of GTPS binding with a high GTPS concentration (10 nM), whereas the experiments investigating the effects of NaCl were conducted with a subsaturating concentration of GTPS (0.4 nM) (see "Experimental Procedures"). When comparing the GTPS binding values in the absence of NaCl in the two types of experiments, we noticed that the maximum values with fMLP in the time course experiments were similar for all three FPRs (Fig. 4); but in the NaCl experiments, the maximum GTPS binding values for FPR-F110S and FPR-C126W were much lower than those for FPR-WT (Fig. 5). Considering that the expression levels of G_i2 were similar for all three FPRs (Fig. 2C), these findings suggested the hypothesis that G_i2 exhibits a higher GTPS affinity in membranes expressing FPR-WT than in membranes expressing FPR-F110S and FPR-C126W.

To address this hypothesis, we conducted GTPS saturation binding studies. fMLP stimulated GTPS binding to G_i212 in membranes expressing FPR-WT with a K_d of 0.9 ± 0.3 nM (Fig. 6A), whereas the K_d for fMLP-stimulated GTPS binding to G_i212 in membranes expressing FPR-F110S was 3.5 ± 0.5 nM. These data indicate that agonist-occupied FPR-WT stabilizes a conformation in G_i212 that confers an ~4-fold higher GTPS affinity for the G protein than the G_i212 conformation stabilized by fMLP-occupied FPR-F110S. GPCR-specific regulation of the GTPS affinity of G_i2 was reported before for the ₂AR and several wild-type chemoattractant receptors (20-22, 31). Thus, our data indicate that the higher GTPS affinity of G_i212 in membranes expressing FPR-WT relative to membranes expressing FPR-F110S and FPR-C126W accounts for the higher fMLP-stimulated GTPS binding values in the former system when a subsaturating GTPS concentration is used (Fig. 5, A-C).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   GTPS saturation binding studies in Sf9 cell membranes expressing FPR-WT plus Gi212 or FPR-F110S plus Gi212. Membranes expressing FPRs plus Gi212 were prepared. [35S]GTPS binding experiments in membranes expressing FPR-WT plus Gi212 (A) or FPR-F110S plus Gi212 (B) were carried out as described under "Experimental Procedures." Reaction mixtures contained 1 µM GDP, 0.5-2 nM [35S]GTPS plus unlabeled GTPS to achieve final GTPS concentrations of 0.5-20 nM (as indicated), solvent (basal), and 10 µM fMLP or 3 µM CsH. For each GTPS concentration, basal GTPS binding was subtracted from GTPS binding in the presence of fMLP to obtain fMLP-stimulated GTPS binding (). GTPS binding in the presence of CsH was subtracted from basal GTPS binding to obtain CsH-inhibited GTPS binding (). The dashed lines represent extrapolations of basal GTPS binding. Data shown are the means ± S.D. of three experiments performed in triplicates. Binding data were analyzed by nonlinear regression and were best fitted (F test) to monophasic saturation curves.

The GTPS saturation binding studies also allowed us to answer the question of how many G_i proteins a single FPR molecule activates. Recent studies (21, 22) have shown that chemoattractant receptors, in contrast to the previously held view (33, 34), do not activate G_i proteins catalytically, but rather linearly. The B_max for ligand-regulated GTPS binding, i.e. the difference between minimum CsH-inhibited and maximum fMLP-stimulated GTPS binding, with FPR-WT was 6.3 pmol/mg (Fig. 6A). Relating this number to the expression level of FPR-WT (~6-8 pmol/mg) (Fig. 2A), one can estimate that one FPR-WT molecule activates approximately one G_i212 molecule. The B_max for ligand-regulated GTPS binding with FPR-F110S was only 0.7 pmol/mg (Fig. 6B). Given the fact that FPR-WT and FPR-F110S are expressed at about the same level (Fig. 2, A and B), the GTPS saturation binding data show that FPR-F110S couples to G_i proteins with an efficiency that amounts to only ~10% of that of FPR-WT. The lack of inhibitory effect of CsH in the GTPS saturation binding studies with membranes expressing FPR-F110S (Fig. 6B) confirms the conclusion that FPR-F110S is not constitutively active.

Conclusions-- Our data show that the F110S mutation in the third transmembrane domain of the FPR and the C126W mutation in the second intracellular loop of the FPR (Fig. 1) result in an almost complete defect and a complete defect in G_i protein coupling, respectively. The defect in G_i protein coupling of these FPR mutants is associated with a loss of constitutive activity. It is conceivable that the exchange of a hydrophobic phenylalanine against a hydrophilic serine in a transmembrane domain involved in fMLP binding (35) interferes with proper helix formation and thereby reduces agonist affinity of the FPR. Nonetheless, FPR-F110S still binds fMLP with low affinity, inducing a conformational change in the GPCR that allows the FPR mutant to catalyze GDP/GTP exchange (Figs. 4 and 6). However, the fMLP-induced conformational change in FPR-F110S must be different from the fMLP-induced conformational change in FPR-WT because fMLP-occupied FPR-F110S is much less efficient than agonist-occupied FPR-WT in activating G_i proteins in terms of GTPS affinity of G_i2 and the total number of G_i2 molecules activated (Fig. 6).

The fact that the electrophoretic mobility and glycosylation of FPR-C126W are very different from the corresponding parameters of FPR-WT and FPR-F110S (Fig. 2, A and B) points to a fundamental alteration in the overall structure and processing of this FPR mutant. It is possible that FPR-C126W is inserted into membrane compartments not enriched in G_i proteins. Such mistargeting could account for the fact that the G_i protein coupling defect in FPR-C126W was complete (Figs. 2-5). A G_i protein coupling defect in FPR-C126W was not unexpected since the second intracellular loop is involved in G_i protein coupling (18) and since FPR-C126S exhibits a similar functional phenotype as FPR-C126W (Figs. 2-6) (19).

FPR-F110S and FPR-C126W have been identified in a number of LJP patients, but not in patients with adult periodontitis or in control subjects (17). Therefore, it is likely that defective G_i protein coupling of the FPR constitutes the molecular basis for the clinical symptoms in LJP patients with the F110S and C126W mutations. Our present data, together with clinical data (12, 13, 17), suggest that the human FPR plays an essential role in host defense against A. actinomycetemcomitans, a bacterium involved in the pathogenesis of LJP. Possibly, A. actinomycetemcomitans produces chemoattractant receptor antagonists that prevent activation of compensatory GPCRs.

Apparently, an ~90% loss of G_i protein coupling as observed with FPR-F110S is sufficient to result in LJP (17). This is consistent with the fact that even partial inactivation of G_i proteins by pertussis toxin is sufficient to efficiently block most fMLP-induced responses in neutrophils (36, 37). In agreement with these data, there is no evidence that the C126W mutation, leading to a complete loss of G_i protein coupling (Figs. 2-5), results in more severe clinical symptoms than the F110S mutation (17). We did not study the F110S/C126W double mutation; but most likely, FPR-F110S/C126W exhibits a complete G_i protein coupling defect, too. Again, there is no evidence that the double mutation is associated with more severe clinical symptoms than either of the single mutations (17).

Although our present data provide an explanation for LJP in patients with proven F110S and/or C126W mutations of the FPR (17), these mutations cannot be the only cause of LJP. Specifically, in certain LJP patients, high-affinity [³H]fMLP binding is reduced by only 50% (14), whereas with FPR-F110S and FPR-C126W, high-affinity agonist binding is abolished (Fig. 2). In addition, several fMLP-induced responses in neutrophils from LJP patients such as increases in cytosolic calcium concentration and lysosomal enzyme release are robust (15, 16, 38). These fMLP responses are incompatible with an almost complete loss and a complete loss of G_i protein coupling in FPR-F110S and FPR-C126W, respectively. Thus, it is possible that additional, as yet unidentified FPR mutations, resulting in less severe defects in G_i protein coupling than the F110S and C126W mutations, exist in certain LJP patients.

Compared with FPR-WT and FPR-F110S, FPR-C126W showed grossly altered electrophoretic mobility and glycosylation (Fig. 2, A and B). Altered electrophoretic mobility of the FPR from an LJP patient relative to the FPR from a control subject was observed previously (15). Thus, it is tempting to speculate that the LJP patient studied by Perez et al. (15) expressed FPR-C126W. However, the electrophoretic results obtained by Perez et al. and us cannot be directly compared because FPR glycosylation in Sf9 cells and neutrophils is intrinsically different (20, 39).

It is somewhat surprising that severe G_i protein coupling defects of the FPR result only in a very localized infectious disease that is not associated with susceptibility to infectious diseases in general (13). These data indicate that the human organism readily compensates, to a very large extent, for the loss of FPR function. It is possible that the formyl peptide-like receptor that binds fMLP with low affinity (11, 40, 41) takes over FPR functions. In addition, the complement C5a receptor, interleukin-8 receptor, leukotriene B₄ receptor, and platelet-activating factor receptor, all of which are expressed in neutrophils and induce similar cellular responses as the FPR, may contribute to FPR substitution (3-6).

	ACKNOWLEDGEMENT

We acknowledge the help of C. Houston with the immunoblots.

	FOOTNOTES

^* This work was supported by National Institutes of Health Center of Biomedical Research Excellence Award 1-P20-RR15563 and matching support from the State of Kansas and the University of Kansas.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, University of Kansas, 5003 Malott Hall, Lawrence, KS 66045-2505. Tel. and Fax: 785-864-3536; E-mail: kwenzel@falcon.cc.ukans.edu.

Published, JBC Papers in Press, September 14, 2001, DOI 10.1074/jbc.M106621200

	ABBREVIATIONS

The abbreviations used are: GPCRs, G protein-coupled receptors; fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; FPR, formyl peptide receptor; FPR-WT, wild-type formyl peptide receptor; LJP, localized juvenile periodontitis; CsH, cyclosporin H; GTPS, guanosine 5'-O-(3-thiotriphosphate); PCR, polymerase chain reaction; ₂AR, ₂-adrenoreceptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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