©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of Low Affinity N-formyl Peptide Receptors to G Protein
CHARACTERIZATION OF A NOVEL INACTIVE RECEPTOR INTERMEDIATE (*)

Eric R. Prossnitz (1)(§), Ronda E. Schreiber (1) (2), Gary M. Bokoch (1) (2), Richard D. Ye (1)

From the (1) Departments of Immunology and (2) Cell Biology, the Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

G protein-coupled seven-transmembrane-containing receptors, such as the N-formyl peptide receptor (FPR) of neutrophils, likely undergo a conformational change upon binding of ligand, which enables the receptor to transmit a signal to G proteins. We have examined the functional significance of numerous conserved charged amino acid residues proposed to be located within or near the transmembrane domains. Whereas the wild type FPR exhibits a Kfor an agonist of 1-3 nM, which is reduced to 40 nM in the presence of guanosine 5`-3- O-(thio)triphosphate (GTPS), substitution of either Asp or Arg resulted in mutant receptors that bound ligand with only low affinity ( K= 30-50 nM) independent of GTPS. In contrast, substitution of Arg, predicted to be located at a similar depth within the membrane as Asp, had no effect on ligand binding. Replacement of residues Arg-Glu-Arg resulted in an FPR with intermediate ligand binding characteristics. Functional analysis of the mutant receptors revealed that substitution of either Asp or Arg resulted in a mutant receptor that was unable to mediate calcium mobilization, whereas replacement of residues Arg-Glu-Arg yielded a receptor with an EC of 50 nM, compared with 0.5 nM for the wild type FPR. In order to determine the point of the defect in signal transduction, we performed reconstitution of the solubilized receptors with purified G proteins. The wild type FPR displayed a Kfor G protein of 0.6 µM compared with the Arg/Glu/Arg mutant with a Kof approximately 30 µM. A significant physical interaction between the Asp or Arg mutants and G protein was not observed. The implications of these findings for signal transduction mechanisms are discussed.


INTRODUCTION

The activation of cellular processes through the binding of agonists to cell surface membrane receptors is of central importance to numerous aspects of biology. The recent explosion in the cloning of cell surface receptors has demonstrated that a large number of these signal-transducing receptors are coupled to GTP-binding regulatory proteins (G proteins)() (for a review, see Refs. 1 and 2). The class of G protein-coupled receptors is typified by its unique membrane topology of seven-transmembrane-spanning domains. Under physiological conditions, transmembrane signaling is initiated by the binding of ligand to the extracellular surface of the receptor. This is proposed to induce a conformational change in the receptor resulting in the activation of a G protein and the subsequent activation of downstream effectors. Despite recent advances in the study of G protein-coupled receptors, little is known regarding the structural and functional differences between the various states of the receptor, such as the liganded, activated receptor and the unliganded, inactive receptor.

The ternary complex model (TCM) is the most accepted model for describing the activation of G protein-coupled receptors (3) . It describes the active form of the receptor as a ternary complex among ligand, receptor, and G protein formed as a result of the sequential association of ligand and G protein, in either order, with receptor. The more favorable sequence is traditionally thought to involve ligand binding to receptor followed by G protein binding. This simple form of the TCM has been found to be inadequate for the evaluation of recently described experiments (4, 5) . Characterization of constitutively active ( i.e. ligand-independent) mutants of - and -adrenergic receptors has led to the conclusion that the unliganded form of receptors exists in an equilibrium between active and inactive states and that the binding of ligand serves to shift the equilibrium from the inactive to the active state. Only this active state is capable of initiating signal transduction. The TCM of signal transduction has also been investigated by studies of conformational changes in the N-formyl peptide receptor (FPR) through rapid kinetic spectrofluorometric methods using fluorescent chemotactic peptides as ligands (6, 7) . This technique takes advantage of the large difference (approximately 2 orders of magnitude) in dissociation rates between the high and low affinity states of the FPR. Analysis of such kinetic data has shown that the assembly of the ternary complex is rapid, occurring with a half-time of less than 1 s, suggesting that a portion of the receptor population may be precoupled to G protein.

The FPR of neutrophils is representative of the class of G protein-coupled receptors. Neutrophils respond to a large number of structurally diverse ligands with functions such as chemotaxis, superoxide production, and degranulation. Many of these chemotactic receptors have recently been cloned, including the receptors for N-formyl peptides (8) , complement component C(9) , platelet-activating factor (10) , and interleukin-8 (11, 12) . These receptors are all members of the class of G protein-coupled receptors, appearing to couple through the pertussis toxin-sensitive G subtype of G protein. Recent work has suggested that the FPR couples to G proteins via its second intracellular loop and carboxyl-terminal domain but that the third intracellular loop is of only minor significance (13, 14) . The carboxyl-terminal domain has also been shown to be a substrate for G protein-coupled receptor kinase 2, suggesting this kinase is involved in phosphorylation and desensitization of the FPR (15) . In this study, we describe the characterization of FPR mutants in the second transmembrane domain, the second intracellular loop, and the carboxyl-terminal domain that are unable to bind ligand with high affinity and are also incapable of signal transduction, suggesting a defect in receptor-G protein interactions. Since it is known that the high affinity, liganded state of the wild type FPR forms a stable physical complex with G protein (16) , we used these mutants to test whether the low affinity state of the FPR is similarly capable of interacting with G protein. The results will provide important mechanistic information regarding the dynamics of G protein-coupled receptors.


EXPERIMENTAL PROCEDURES

Materials

The cDNA that encodes the FPR was obtained from a human HL-60 granulocyte library as described previously (17) . Restriction enzymes, T4 DNA ligase, lipofectamine, G418, and trypsin-free dissociation buffer were from Life Technologies, Inc. Oligonucleotides were obtained from Operon. Mutagenesis was carried out using the Amersham Corp. oligonucleotide-directed mutagenesis system. Sequencing was carried out using Sequenase version 2.0 (U. S. Biochemical Corp.). Bacterial cells were grown in Circlegrow medium (BIO 101). fML[H]P (specific activity, 56 Ci/mmol) was obtained from DuPont NEN, and unlabeled fMLP was purchased from Sigma. Carrier-free sodium [I]iodide was obtained from Amersham Corp. N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and indo-1/AM were obtained from Molecular Probes. Mouse L cell fibroblasts (L2071) were obtained from ATCC. Dulbecco's modified Eagle's medium was from Whittaker Bioproducts; fetal bovine serum was from HyClone. Enzymobeads were from Bio-Rad, and sulfosuccinimidyl-2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionate was from Pierce.

Construction and Expression of Site-directed Mutants

The FPR gene was subcloned into the EcoRI site in the polylinker of M13 mp18 (13, 17) . Single-stranded M13 DNA in conjunction with mutant oligonucleotides (containing the altered nucleotides necessary to generate the desired mutations) was used to introduce mutations into the FPR gene. Plaques were amplified, and the mutations were confirmed by dideoxy sequencing. For expression, the mutated FPR genes were subcloned into the EcoRI site of the vector pSFFV.neo, which contains the selectable marker aminoglycoside transferase. Mutations were reconfirmed by dideoxy sequencing prior to transfection. Mouse L cell fibroblasts were transfected as follows. Approximately 10 cells were plated out in a 25-cm flask 20 h prior to transfection with 10 µg of linearized vectors by lipofectamine. Transfectants were selected by their resistance to G418 sulfate at an active drug concentration of 0.35 mg/ml. For each mutant, approximately 20-50 individual colonies from transfected cells were pooled for analysis.

Flow Cytometry

Mouse L cell fibroblasts were detached from the culture flask with protease-free dissociation buffer, harvested by centrifugation, washed once with phosphate-buffered saline, and resuspended to 10 cells/ml in phosphate-buffered saline. Binding was carried out in 0.5 ml with N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein at 20 nM. Following incubation for at least 15 min on ice, the cells were analyzed on a FACScan flow cytometer (Becton Dickinson) for fluorescent intensity. Debris and dead cells were excluded with a gate on forward and side scatter. Nonspecific binding was determined in the presence of 1 µM N-formyl-Met-Leu-Phe.

Membrane Preparation

Cells were detached from culture flasks with protease-free dissociation buffer, collected at 500 g, and resuspended at 10 cells/ml in 10 mM PIPES (pH 7.3), containing KCl (100 mM), NaCl (3 mM), MgCl (3.5 mM), ATP (0.6 mg/ml), and the protease inhibitors chymostatin, phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate (Sigma). The cells were disrupted by N cavitation (500 p.s.i., 15 min, 4 °C), and the cell nuclei and unbroken cells were removed by centrifugation at 1000 g for 5 min. The membranes were collected by sedimentation at 130,000 g for 45 min, resuspended at a protein concentration of 5 mg/ml in 25 mM HEPES (pH 7.0), 200 mM sucrose, and stored at -80 °C until use.

Photoaffinity Labeling of the FPR

Membranes were photoaffinity-labeled with 20 nM N-formyl-Met-Leu-Phe- N-(2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionyl)-Lys (fMLPK-[I]SASD) as described previously (18, 19) . Briefly, N-formyl-Met-Leu-Phe-Lys, dissolved in dry dimethylformamide, was added to equimolar amounts of sulfosuccinimidyl-2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionate and triethylamine and incubated overnight in the dark at room temperature. fMLPK-SASD was radiolabeled using 5 mCi of NaI and the Enzymobead reagent as described by the manufacturer. The mixture was then chromatographed on a Bio-Gel P-2 (extra fine) equilibrated in 20 mM NaOH to obtain the fMLPK-[I]SASD, which eluted in the void volume. FPR-expressing fibroblasts and their respective membrane preparations were covalently labeled as follows. Cells (0.5 10) were harvested and resuspended in 200 µl of HEPES-buffered saline. Membranes (50 µg) were diluted to 200 µl of HEPES-buffered saline. fMLPK-[I]SASD (5-10 µl of the eluted sample, representing a final concentration of 10-30 nM) was added in the absence or presence of 1 µM unlabeled fMLP to assess nonspecific labeling. Following a 10-min incubation on ice, the radiolabel was covalently incorporated by exposure on ice to UV light in a Rayonet ultraviolet light reactor irradiating at 370 nm for 5-10 min. Labeled samples were centrifuged and washed two times with HEPES-buffered saline prior to further analysis.

Quantitative Radioligand Binding

Ligand binding assays were performed on membrane preparations in a final volume of 0.2 ml. Membranes (30 µg of protein) were suspended in the binding buffer (pH 7.4) consisting of 140 mM NaCl, 1.0 mM KHPO, 5 mM NaHPO, 1.5 mM CaCl, 0.3 mM MgSO, 1 mM MgCl, and 0.2% bovine serum albumin. Binding was started by the addition of various amounts of [H]fMLP. Equilibrium binding was carried out at 23 °C for 45 min and terminated by rapid filtration through Whatman GF/C filters followed by three washes with 0.75 ml of ice-cold binding buffer. Specific binding was calculated as total binding minus nonspecific binding, which was determined in the presence of 50 µM unlabeled fMLP. Each determination was done in duplicate. The amount of bound ligand was estimated by scintillation counting, and the binding data were analyzed by fitting to a double rectangular hyperbola with the nonlinear curve fitting program, SigmaPlot (Jandel Scientific). Measurement of [Ca]-Cells were harvested in dissociation buffer, washed once with phosphate-buffered saline, and resuspended at 5 10 cells/ml in RPMI 1640 medium containing 10% fetal bovine serum. The cells were incubated with 5 µM indo-1/AM for 25 min at 37 °C, washed once with medium, and resuspended to a concentration of approximately 10 cells/ml. The elevation of intracellular Ca by various amounts of fMLP was monitored by continuous fluorescent measurement using an SLM 8000 photon-counting spectrofluorometer (SLM-Aminco) detecting at 400 and 490 nm, respectively, as described (17) . The concentration of intracellular Ca was calculated as described (20) .

Purification of G Proteins

G (containing subtypes G and G) was purified from bovine brain essentially as described (21) . After the heptylamine-Sepharose purification step, resolution of G from G was achieved by purification on a 20-ml DEAE-Sephacel column, which was equilibrated with TENL (25 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.6% Lubrol) and eluted with a 200-ml linear gradient of 0-250 mM NaCl in TENL. G-enriched fractions were pooled and judged by silver stain following SDS-polyacrylamide gel electrophoresis to contain approximately 80% G and 20% G.

FPR/G Protein Reconstitution and Analysis

Reconstitution was performed as described previously (22) . Briefly, membranes, photoaffinity-labeled with fMLPK-[I]SASD, were extracted with 1% octylglucoside in 10 mM HEPES, pH 7.4, 100 mM KCl, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml chymostatin for 1 h on ice. Extracts were incubated with or without G for 2 h at 4 °C and then applied to 700 µl of 5-20% linear sucrose gradients prepared in the extraction buffer. Gradients were centrifuged at 45,000 rpm in an SW 50.1 rotor (Beckman) for 13.3 h and fractionated into 14 50-µl fractions. Fractions were subjected to SDS-polyacrylamide gel electrophoresis, followed by autoradiography to confirm the specific labeling and distribution of the receptor. Gradients containing 20 µg of bovine serum albumin (4.4 S) and rabbit immunoglobulin (7.7 S) were centrifuged in parallel with FPR-containing gradients. Based on the sedimentation of these standard proteins, the migration of 4 S and 7 S proteins was calculated to peak in fractions 6 and 10, respectively.


RESULTS

G protein-coupled receptors may represent the largest class of signal-transducing molecules, and as such they bind a vast array of ligands and couple to many distinct intracellular effectors via a large collection of G proteins. Although all members of this class contain the canonical seven-transmembrane domain motif, they possess considerable sequence diversity to accommodate the multitude of functions they perform. Despite this diversity, G protein-coupled receptors contain certain amino acid residues that are highly conserved throughout the entire class. Perhaps the most distinct of these is the Asp-Arg-Tyr triplet at the boundary between transmembrane domain 3 (TM3) and the second intracellular loop. Another almost invariant residue, an Asp, is found in the middle of TM2. Highly conserved among the muscarinic, adrenergic, dopaminergic, serotonin, and a number of other G protein-coupled receptors is an Asp in the middle of TM3. In some instances, this and the other conserved Asp residues are replaced by the similarly charged acidic amino acid, Glu. We have also noted a weak homology at the boundary between the TM7 and the carboxyl terminus. This consensus consists of a Phe preceded and/or followed by one or more basic amino acids. Examples of this include FR ( e.g. opsins), FRR ( e.g. -adrenergic), FKK ( e.g. m4 muscarinic), FRK (D2 dopamine), KKFRKH ( e.g. platelet-activating factor), and KFRH ( e.g. interleukin-8). As a model G protein-coupled receptor to study the role of these conserved residues, we have utilized the FPR, which possesses all of these consensus residues. Instead of a Tyr at the third position of the Asp-Arg-Tyr triplet, the FPR contains a Cys, found in a small number of G protein-coupled receptors. The consensus at the TM7/cytoplasm boundary of the FPR consists of the sequence FRER, which is consistent with the pattern of sequences outlined above, although the presence of an acidic residue near the Phe residue is unusual. For the purposes of this study, we altered the conserved amino acids described above as illustrated in Fig. 1. Arg was also mutated because it represents a charged residue with a predicted position in the fourth transmembrane domain at a similar depth to Asp.


Figure 1: Schematic representation of the structure of the FPR and the locations of site-directed mutants. Opencircles represent individual amino acids of the FPR; solidcircles, with the indicated amino acid changes, show the residues that were mutated. Potential N-linked glycosylation sites are also indicated ( CHO). RER/GAG, R309G/E310A/R311G.



Following the generation of the desired mutations by site-directed mutagenesis, the mutant recombinant FPR constructs in the expression vector pSFFV.neo were stably transfected into mouse L cell fibroblasts. We have previously shown that these cells are capable of expressing functional FPR upon transfection with the wild type gene. After selection with G418, the stable transfectants were analyzed for surface expression of the FPR. This was accomplished in two ways: 1) by flow cytometry using a fluorescein-conjugated hexapeptide ligand and 2) by covalent photoaffinity labeling using an iodinated photoactivatable tetrapeptide ligand followed by SDS-polyacrylamide gel electrophoresis. The results are shown in Fig. 2 . Whereas mock-transfected cells show no specific binding of either of the two ligands ( panelA), wild type FPR-transfected cells show binding of both the fluoresceinated ligand, as indicated by the increase in cell-associated fluorescence ( panelB), and the iodinated ligand, as evidenced by the presence of a band of molecular mass 55,000-70,000 kDa upon SDS-polyacrylamide gel electrophoresis ( panelB, inset). In both cases, the binding of the ligand was demonstrated to be specific by the addition of excess fMLP, which competed for the binding of the labeled ligand. The addition of excess unlabeled fMLP to the mock-transfected cells had no effect on the nonspecific background binding of either of the labeled ligands. Analysis of the mutant forms of the FPR revealed that mutants D71A, R123G, R163F, and R309G/E310A/R311G were capable of binding formyl peptide ligands to a similar extent as the wild type receptor. However, the mutants D106A and D122A displayed no specific binding of either ligand, suggesting that either these two mutants were not expressed at the cell surface or that they were expressed at the cell surface but were incapable of binding the ligand.


Figure 2: Cell surface expression of the wild type and mutant forms of the FPR. Expression was evaluated for cells transfected with vector only, the wild type FPR, and each mutant FPR by flow cytometry, plotting cell number versus fluorescent intensity ( panelsA-H). Binding was determined with N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein at 20 nM. The inset within each panel shows the profile of membrane proteins photoaffinity-labeled with approximately 20 nM fMLPK-[I]SASD analyzed on a 12% SDS-polyacrylamide gel. The major labeled protein, representing the FPR, has a molecular mass of 55,000-70,000 kDa. In each assay, binding was determined in the absence (-) and presence (+) of 1 µM N-formyl-Met-Leu-Phe. Data are representative of two to four experiments. RER/GAG, R309G/E310A/R311G.



To examine the ligand binding characteristics of the mutants in greater detail, we performed fML[H]P binding assays using membranes prepared from the transfected cells. Analysis of the wild type FPR revealed the expected properties, high affinity binding representing 2.3 pmol/mg protein with a Kof 2.7 nM, which could be converted to low affinity binding by the addition of 2 µM GTPS, yielding a Kof approximately 40 nM with a receptor density of 1.6 pmol/mg of protein (Fig. 3 A, ). The low affinity state of the receptor generated in the presence of GTPS is presumed to be the result of the irreversible activation and dissociation of G proteins from receptors and results in an apparent reduction in the receptor density as determined by filtration binding. Scatchard analysis as well as nonlinear curve fitting of the binding data revealed the presence of a single site in both the presence and absence of GTPS (). Of the mutant receptors observed to bind the fluoresceinated ligand, only the R163F mutant displayed binding affinities characteristic of the wild type receptor, although in the absence of GTPS both the high and low affinity sites were observed (approximately 1 nM and 20 nM, respectively). Binding of fMLP to the wild type FPR of human neutrophils and HL60 cells routinely displays both high and low affinity sites in the absence of GTPS, with the receptor population in the high affinity state being sensitive to GTPS, as observed here with the R163F mutant.


Figure 3: Ligand binding characteristics of the FPR mutants. Specific fML[H]P binding to isolated membranes from cells expressing the wild type ( panelA) and the following mutant forms of the FPR: D71A ( panelB), R123G ( panelC), R163F ( panelD), and R309G/E310A/R311G ( RER/GAG) ( panelE). Binding was performed in the absence () or presence () of 2 µM GTPS. Binding data were fit with a one-site model, except for the R163F mutant in the absence of GTPS, which was better fit with a two-site model. Data are representative of three to five binding assays.



Two of the mutants, D71A and R123G, exhibited fMLP binding with a Kof approximately 30-50 nM. This binding was unaffected by the presence of GTPS, suggesting that even in the absence of GTPS the FPR was unable to exist in the high affinity state. The receptor densities for the D71A and R123G mutants were 0.4 and 0.9 pmol/mg protein, respectively. Although these values represent a small percentage of the number of sites determined for the wild type FPR in the absence of GTPS, they represent up to 60% of the sites determined for the wild type FPR in the presence of GTPS, a more valid comparison since this state of the FPR represents the uncoupled form of the receptor. Analysis of the R309G/E310A/R311G mutant revealed that its binding properties were intermediate between that of the wild type FPR and mutants D71A and R123G. In the absence of GTPS, this mutant predominantly displayed a Kof 40 nM with a receptor density of 1.9 pmol/mg of protein (representing 97-100% of the receptor sites). Scatchard analysis did reveal the presence of a very small population of receptors with high affinity (1.2 ± 0.9 nM) representing only 2-3% of the receptor sites. In the presence of GTPS, no high affinity binding was observed, but the number of low affinity sites was reduced to 1.2 pmol/mg of protein with an unaltered K. This level of reduction in the apparent number of fML[H]P binding sites assayed in the presence of GTPS, is similar to that seen with the wild type FPR.

To examine the functional capabilities of the mutant forms of the FPR, we measured the ability of the transfected cells to mobilize calcium. As we have previously reported, addition of fMLP to L cells transfected with the wild type FPR resulted in a dose-dependent increase in intracellular levels of calcium with an EC of 0.5 nM (Fig. 4). Similar results were obtained with cells expressing the R163F mutant. Cells expressing the R309G/E310A/R311G mutant were also able to initiate calcium mobilization upon the addition of fMLP; however, higher concentrations of ligand were required (EC 50 nM), and the maximal response was less than that observed for the wild type receptor. Mutant D71A did not display any calcium mobilization up to fMLP concentrations of 10 µM; however, at 100 µM fMLP a small but reproducible amount of calcium mobilization was observed. The remaining mutant capable of binding fMLP, R123G, was unable to mobilize calcium even at the highest concentration of fMLP, 100 µM. Higher concentrations of fMLP could not be used due to solubility limits of this hydrophobic molecule. The remaining two mutants created in this study, namely D106A and D122A, were also tested for calcium mobilization. Since our filtration binding assays can determine binding only up to approximately 500 nM, we considered the possibility that either or both of these mutants might not display detectable binding but, if functional, might exhibit signal transduction at the very high fMLP concentration possible in our functional assay. This was, however, not the case since both mutants displayed no calcium mobilization at 100 µM fMLP.


Figure 4: Calcium mobilization by the wild type and mutant forms of the FPR. Cells expressing the wild type () and mutant forms of the FPR (D71A (), D106A (), D122A (), R123G (), R163F () and R309G/E310A/R311G (RER/GAG) ()) were analyzed for fMLP-stimulated elevation of intracellular calcium. The mutants D106A, D122A, and R123G exhibited no measurable calcium mobilization even at 100 µM fMLP. The extent of the increase in intracellular calcium is expressed as a percentage of the maximal response for each cell type at saturating concentrations of the heterologous ligand, ATP. Data are representative of three experiments.



The data presented to this point suggest that the mutants D71A and R123G are functionally uncoupled from G proteins. The nature of this uncoupling cannot be determined from the experiments already described. A number of possible mechanisms exist to explain this phenomenon: 1) G protein can bind to the FPR, but this binding neither results in conversion of the FPR to the high affinity state nor signal transduction; 2) G protein cannot bind to the receptor because the mutants created exist at the interaction site(s) between the FPR and G protein, and 3) the mutant forms of the FPR are trapped in the low affinity state, which is inherently incapable of binding G protein. In order to gain more insight into the mechanism of uncoupling we performed quantitative physical reconstitution between the wild type and mutant forms of the FPR and purified G protein. This assay determines the extent of complex formation between the FPR and G protein by sucrose density sedimentation. Briefly, FPR is photoaffinity-labeled with fMLPK-[I]SASD and extracted from membranes with octylglucoside. The extracted receptor is incubated with varying amounts of purified G protein and layered onto a 5-20% sucrose gradient. Following centrifugation, the gradients are fractionated, and the position of the FPR within the gradient is determined by analyzing the fractions on SDS-polyacrylamide gel electrophoresis. In the absence of G protein, the FPR is found in fractions 5-7, sedimenting as a 4 S species (Fig. 5). With a saturating amount of G protein, the FPR is found in fractions 9-11, sedimenting as a 7 S species. At intermediate amounts of G protein the sedimentation profile appears to represent a combination of the complexed and uncomplexed forms of the FPR. The EC for formation of the 7 S complex was determined to be 0.6 µM. Previous reports of this value have ranged from 0.2-4 µM, the variation possibly being due to the preparation of G protein. That the shift of the FPR from 4 S to 7 S results from a specific binding interaction between the FPR and G protein was confirmed by the addition of GTPS, which activates and dissociates G protein from the receptor, resulting in the conversion of the 7 S species to the 4 S species despite the presence of G protein.


Figure 5: Conversion of the wild type FPR from the 4 S to the 7 S form by reconstitution with G protein. Fractions from gradients containing photoaffinity-labeled wild type FPR reconstituted with increasing amounts of G protein as indicated were solubilized in sample buffer and applied to SDS-12% polyacrylamide gels. GTPS was added to a final concentration of 2 µM. The positions of 4 S and 7 S standards are indicated.



Mutant forms of the FPR capable of binding ligand were similarly analyzed for their ability to complex with G protein (Fig. 6). All of the mutants sedimented as 4 S species in the absence of added G protein. In contrast to the wild type FPR (Fig. 6 A), the mutants D71A (Fig. 6 B) and R123G (Fig. 6 C) failed to form a complex with 4 µM G protein, a quantity sufficient to convert essentially all of the wild type FPR to the 7 S species. At very high concentrations of G protein (30 µM), the mutants underwent only a minimal shift in their sedimentation profiles, similar to the shift observed with the wild type FPR at a concentration of G protein of about 0.1 µM. However, the addition of GTPS to the samples containing 30 µM G protein resulted in no change of the sedimentation profile, suggesting that this receptor species was not a result of an active G protein complex. The mutant R163F, which displayed high affinity ligand binding and almost wild type signal transducing properties, formed a GTPS-sensitive complex with purified G protein with an EC similar to that of the wild type FPR, 1 µM G protein (Fig. 6 D). The R309G/E310A/R311G mutant, like mutants D71A and R123G, also displayed little change in the sedimentation profile at 4 µM G protein; however, at higher G protein concentrations, this mutant displayed a partial shift in its sedimentation profile (Fig. 6 E). The profile at 30 µM G protein appeared to represent an equal distribution between complexed and uncomplexed receptor, similar to that seen with the wild type FPR at G protein concentrations of 0.4-0.8 µM (Fig. 7). That the complex of the R309G/E310A/R311G mutant with 30 µM G protein represented a wild type-like complex was assessed by determining the guanine nucleotide sensitivity of the complex. Addition of GTPS to the sedimentation assay converted the complexed portion of the R309G/E310A/R311G mutant to an uncomplexed 4 S species, suggesting the complexed species is a functional complex.


Figure 6: Physical reconstitution of the mutant forms of the FPR with G protein. Sedimentation profiles were determined for photoaffinity-labeled membranes from cells expressing the wild type FPR ( panelA) and the following mutant forms of the FPR: D71A ( panelB), R123G ( panelC), R163F ( panelD), and R309G/E310A/R311G ( panel E). The G protein concentration used for each reconstitution is indicated.




Figure 7: Quantitative analysis of complexed wild type and mutant R309G/E310A/R311G FPR as a function of G protein concentration. The fraction of complexed () and uncomplexed () receptor was evaluated as a function of G protein concentration for the wild type FPR ( panelA) and the R309G/E310A/R311G mutant ( panelB). Uncomplexed receptor was taken as that receptor in fractions 5, 6, and 7, whereas complexed receptor was assessed in fractions 9, 10, and 11. In the presence of 2 µM GTPS, the fraction of complexed () and uncomplexed () receptor was also determined.




DISCUSSION

In this study, we have utilized site-directed mutants of a well characterized G protein-coupled receptor, the FPR, to investigate mechanisms of G protein coupling. We hypothesized that amino acid residues strictly conserved between virtually all G protein-coupled receptors would be critical to receptor function. Of the six mutants generated, two appeared not to be functionally expressed at the cell surface (D106A and D122A), whereas the remaining four were not only expressed at the cell surface but were capable of binding ligand. Detailed ligand binding analyses revealed that only one of these four mutants (R163F) demonstrated high affinity, GTPS-sensitive fMLP binding as seen with the wild type receptor. The remaining three mutants (D71A, R123G, and R309G/E310A/R311G) did not exhibit high affinity, GTPS-sensitive fMLP binding. Functional analysis showed that of these three mutants, only the R309G/E310A/R311G mutant was capable of signal transduction, although at fMLP concentrations approximately 100-fold higher than that required for the wild type receptor.

These results indicated that the D71A, R123G, and R309G/E310A/R311G mutant FPRs were incapable of a productive interaction with G protein. The data, however, did not address the nature of the defect. To test whether the mutant forms of the FPR were capable of physically interacting with G protein, the formation of a receptor-G protein complex was determined. Formation of a physical complex correlated well with the functional abilities of the mutant receptors. These results suggest that either 1) the mutations occur at the receptor-G protein interface, preventing their interaction, and as a result the receptor exists in a low affinity state or 2) the mutations prevent the receptor from switching to the high affinity conformation and that this prevents the interaction with G protein. The locations of the mutations provide some insight into the interpretation of the results. The R123G and R309G/E310A/R311G mutations exist at the membrane-cytoplasm interface of the receptor. It is therefore possible that either of the two mechanisms is responsible for the uncoupling observed with these mutants. However, the D71A mutation occurs in the middle of the second transmembrane domain, making it unlikely that this site interacts directly with G protein. As a result, the most probable mechanism of uncoupling for this mutant is the stabilization of the low affinity state (or conversely destabilization of the high affinity state) of the FPR.

Our results suggest that the unliganded form of the wild type FPR may be similarly inefficient at binding G protein. We propose that only upon ligand binding, with its subsequent conformational change, can the receptor bind G protein with high affinity. These results require an additional intermediate to be introduced into the ternary complex model (Fig. 8). The receptor (R) exists in an inactive state (R), which has a low affinity for both ligand and G protein. In this model, the binding of ligand follows a two-step process, first forming an inactive complex (LR), which can proceed to the activated ligand-receptor complex (LR). Through the cross-linking of a photoactivatable ligand to the receptor, we are able to generate a stable form of the wild type LR state, or LR state in the case of the mutant receptors. This wild type state of the receptor exhibits a high binding affinity for G protein, which binds to form the active ternary complex (LRG). It is our contention that mutants, such as the D71A FPR, prevent the conversion of the LR to the LR state, preventing association with G protein. Previous mutagenesis studies of other G protein-coupled receptors have characterized nonfunctional receptors capable of ligand binding; however, these studies could not identify the nature of the receptor coupling defect (reviewed in Ref. 23).


Figure 8: Revised model for the interaction of seven-transmembrane receptors with ligand and G protein. In this model, inactive receptor (R) binds ligand (L), which leads to the formation of an inactive intermediate (LR) with its subsequent conversion to an active ligand-receptor complex (LR). This form then interacts with and activates G proteins (G), leading to cellular activation. Also shown is the equilibrium between inactive (R) and spontaneously active receptor (R).



Mutations of the -adrenergic receptor that result in its ligand-independent ( i.e. constitutive) activation have recently been described (4, 5) . To accommodate these findings, the basic TCM was modified in such a way as to introduce a step representing the spontaneous conversion of R to R, resulting in ligand-independent activation of G proteins (see Fig. 8). The assumption was made that only the activated form of the receptor, R, could activate G protein. In the case of the wild type receptor, a small fraction of receptor would exist in the active state, resulting in basal activity. Although the energy of activation of the receptor should be sufficiently low that the binding of a small ligand can provide sufficient energy to alter the conformation of the receptor into its active state, the barrier to activation should also be sufficiently high so that in the absence of ligand the receptor spends little time in the spontaneously active R state. It was concluded that the effect of the mutations was to mimic the effect of ligand binding and to convert the receptor to an active state. It has since been shown that this constitutively active mutant is also recognized by receptor kinases, further suggesting that the conformation of the mutant receptor is that of the active state (24) . Despite these advances, little is known regarding the nature of receptor activation and the differences between the liganded and unliganded receptor and how these forms of the receptor interact with G protein.

Sklar and co-workers (6, 7) have proposed a model based on kinetic measurements in which the N-formyl peptide receptor population exists in two states: an uncoupled or slowly coupling state and a G protein precoupled or rapidly coupling state representing up to 50% of the total receptor. In these studies, comparable rates for the formation of the LR binary complexes and LRG ternary complexes were observed. Fitting of the experimental data to the TCM was significantly improved by allowing precoupling of the G protein to the receptor. The results however could not distinguish rapid coupling (half-times of less than 1 s) from precoupling. Our data suggest that the inactive receptor (LR and presumably R) has a low affinity for G protein, at least 2 orders of magnitude lower than that of the ligand-activated receptor (LR). This makes the existence of precoupled high affinity receptor questionable and supports the alternative conclusion that two forms of G protein may exist, one rapidly coupling and the other slowly coupling. One possible explanation is that a fraction of G protein is maintained in close proximity to receptors, by the subunits for example, but that only upon ligand binding to the receptor does the subunit of the G protein interact with the receptor to generate the high affinity ligand binding state. Such a mechanism could provide for extremely rapid formation of the ternary complex.

In this study, we have shown that inactive receptor mutants are unable to bind G protein in a manner similar to that of the liganded wild type receptor, suggesting the low affinity inactive state of the wild type receptor has a significantly reduced affinity for G protein. Furthermore, we suggest that the ligand-induced change in the wild type receptor conformation is critical for the receptor-G protein binding interaction to take place. Constitutively inactive receptor mutants should provide a valuable tool for studying the molecular properties of both the inactive and the active receptor state.

  
Table: Ligand binding parameters of wild type and mutant FPRs

Ligand binding assays were performed as described under ``Experimental Procedures'' in either the presence or absence of 2 µM GTPS as indicated. The data were analyzed using a two-site model. Where one of the sites represented less than 1% of the total number of sites (-), it was taken to indicate that only a single site existed. Site 1 is arbitrarily taken as the high affinity site and site 2 as the low affinity site. The Kvalues are given in nM, whereas the B values are given in fmol/mg of protein.



FOOTNOTES

*
This research was supported by National Institutes of Health Grants AI36357 (to E. R. P.), GM46572 and AI33503 (to R. D. Y.), and GM39434 and TRDRP 3RT-0189 (to G. M. B.). We also acknowledge the contribution of the General Clinical Research Center of the Scripps Research Institute (NIH M01RR00833). This is publication 9030-IMM from the Department of Immunology, the Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Immunology, IMM12, the Scripps Research Inst., 10666 N. Torrey Pines Rd., La Jolla CA 92037. Tel.: 619-554-4549; Fax: 619-554-8476.

The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; FPR, N-formyl peptide receptor; fMLP, N-formyl-methionyl-leucyl-phenylalanine; TCM, ternary complex model; PIPES, piperazine- N, N`-bis(2-ethanesulfonic acid); fMLPK-[I]SASD, N-formyl-Met-Leu-Phe- N-(2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionyl)-Lys; TM, transmembrane domain; GTPS, guanosine 5`-3- O-(thio)triphosphate; R, receptor; LR, ligand-receptor; G, G protein.


ACKNOWLEDGEMENTS

We acknowledge Larry Sklar for helpful discussions and Stacey Cavanagh and Daniel Cheng for excellent technical support.


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