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J. Biol. Chem., Vol. 276, Issue 52, 49204-49212, December 28, 2001

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Regulation of Formyl Peptide Receptor Agonist Affinity by Reconstitution with Arrestins and Heterotrimeric G Proteins^*

T. Alexander Key^§, Teresa A. Bennett^§, Terry D. Foutz^§, Vsevolod V. Gurevich^¶, Larry A. Sklar^§, and Eric R. Prossnitz^**

From the  Department of Cell Biology and Physiology, ^§ Cancer Research and Treatment Center, and Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, the ^¶ Ralph and Muriel Roberts Laboratory for Vision Science, Sun Health Research Institute, Sun City, Arizona 85351, and the  National Flow Cytometry Resource, Los Alamos National Labs, Los Alamos, New Mexico 87545

Received for publication, October 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although heptahelical chemoattractant and chemokine receptors are known to play a significant role in the host immune response and the pathophysiology of disease, the molecular mechanisms and transient macroassemblies underlying their activation and regulation remain largely uncharacterized. We report herein real time analyses of molecular assemblies involving the formyl peptide receptor (FPR), a well described member of the chemoattractant subfamily of G protein-coupled receptors (GPCRs), with both arrestins and heterotrimeric G proteins. In our system, the ability to define and discriminate distinct, in vitro receptor complexes relies on quantitative differences in the dissociation rate of a fluorescent agonist as well as the guanosine 5'-3-O-(thio)triphosphate (GTPS) sensitivity of the complex, as recently described for FPR-G protein interactions. In the current study, we demonstrate a concentration- and time-dependent reconstitution of liganded, phosphorylated FPR with exogenous arrestin-2 and -3 to form a high agonist affinity, nucleotide-insensitive complex with EC₅₀ values of 0.5 and 0.9 µM, respectively. In contrast, neither arrestin-2 nor arrestin-3 altered the ligand dissociation kinetics of activated, nonphosphorylated FPR. Moreover, we demonstrated that the addition of G proteins was unable to alter the ligand dissociation kinetics or induce a GTPS-sensitive state of the phosphorylated FPR. The properties of the phosphorylated FPR were entirely reversible upon treatment of the receptor preparation with phosphatase. These results represent to our knowledge the first report of the reconstitution of a detergent-solubilized, phosphorylated GPCR with arrestins and, furthermore, the first demonstration that phosphorylation of a nonvisual GPCR is capable of efficiently blocking G protein binding in the absence of arrestin. The significance of these results with respect to receptor desensitization and internalization are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The family of chemoattractant/chemokine receptors is of great interest for its role in host immune responses and the pathophysiology of disease. Conclusive links have been drawn between chemoattractants and such diverse processes as inflammation, leukocyte migration, angiogenesis, and tumor metastasis (1). However, despite the ever-expanding characterization of function for chemokine receptors, much remains unknown regarding their activation and regulation, in particular, the precise molecular mechanisms and transient macroassemblies underlying their biochemical behavior. Regulation of chemoattractant/chemokine receptors most likely proceeds in a manner similar to that of the more characterized, classical heptahelical or G protein-coupled receptors (GPCRs),¹ such as rhodopsin and the ₂-adrenergic receptor (2-4). However, comparative efforts to elucidate regulatory processes are as a matter of course limited, because there is such great mechanistic diversity within the family of GPCRs as a whole and in particular across subfamilies.

General themes regarding the biology of GPCRs are well described in the literature (5-7). In response to the binding of their cognate, stimulatory ligand(s), GPCRs undergo a conformational change, activate G protein, and initiate a variety of diverse signaling events. Activated receptors are rapidly phosphorylated by a member of the family of G protein-coupled receptor kinases, triggering desensitization and eventual internalization. It is known that, for certain GPCRs, arrestins mediate these latter processes (8). Studies suggest that the family of arrestin proteins accomplish desensitization primarily through preferential binding to the phosphorylated form of the receptor, thus sterically occluding further G protein binding (9, 10). It has also been shown that arrestins have direct interactions with proteins involved in endocytotic sequestration, including the proteins clathrin and AP-2 (11-13). However, precise analyses of both the sequence and components of transient GPCR complexes have remained for the most part unexplored in the case of chemokine/chemoattractant receptors.

The traditional ternary complex model frames GPCR complexes and states in terms of molecular interactions between ligand, receptor, and G protein (14, 15). Liganded or activated receptor promotes the assembly of a transient receptor complex (LRG) with high agonist affinity, followed by the catalytic release of GDP and ultimate G protein activation through GTP binding. Although the properties of these protein complexes have been well characterized for several heptahelical chemokine receptors, there have been few direct studies of alternative, ternary complexes, i.e. of G protein-uncoupled, putative ligand-receptor-arrestin complexes. A limited number of inquiries into the formation of ligand-receptor-arrestin complexes for classical GPCRs have been conducted (8, 16, 17). Prior studies with the visual receptor rhodopsin have implied a direct connection between the phosphorylation and activation states of the receptor and its interactive ability with arrestin (18, 19). It was suggested that disruption of the polar core of arrestin by the negatively charged phosphates on the receptor is primarily responsible for its activation (20, 21). In contrast, the affinity of G protein for rhodopsin appears to diminish on a linear basis with increasing levels of receptor phosphorylation (22). Notwithstanding our knowledge of some of the factors influencing the formation of certain ligand-receptor-arrestin complexes, the properties of such assemblies in terms of chemoattractant/chemokine receptors are poorly understood.

The formyl peptide receptor (FPR) is a well described member of the chemoattractant subclass of GPCRs and is expressed predominantly in leukocytes (23). Coupling to a pertussis toxin-sensitive G protein, the FPR is known to modulate several important cell functions, including superoxide formation, degranulation, and chemotaxis via interactions with its ligand, the formyl peptide (24, 25). We have previously described characteristics of G protein-based ternary complex formation in both cell-based and cell-free assays with the FPR using flow cytometry and spectrofluorimetry (26-30). Moreover, we have recently shown in vivo that the FPR colocalizes with arrestin-2 and -3 after agonist stimulation in transfected U937 cells, suggesting an interaction between these proteins (31). Curiously, however, we have found FPR internalization to proceed through an arrestin-independent mechanism (32, 33), similar to results found with the m2 muscarinic receptor (34). In the current study using a solubilized receptor system, we sought to investigate the formation of receptor complexes and the corresponding ligand binding properties of both phosphorylated (R^p) and native FPR in association with G proteins and arrestins. We describe the assembly and real time disassembly of soluble receptor complexes, including the formation of a high ligand affinity, nucleotide-insensitive complex of phosphorylated FPR with arrestins. Our findings represent to our knowledge the first qualitative and quantitative characterization of a solubilized, desensitized receptor complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Chemicals and reagents were obtained from Sigma except where noted. Bovine brain G protein subunit mixtures were purchased from Calbiochem. Arrestin-2 and -3 were expressed in Escherichia coli (strain BL21) and purified by sequential heparin-Sepharose and Q-Sepharose chromatography as previously reported (35). Plasticware was obtained from VWR Scientific Company. Alkaline phosphatase was from Calbiochem. N-Formyl-Met-Leu-Phe-Lys-fluorescein 5-isothiocyanate (fMLFK-FITC) was obtained from Peninsula Laboratories. N-Formyl-Met-Leu-Phe-Phe (fMLFF) was from Commonwealth Biotechnologies.

Cell Culture-- U937 cells were grown in tissue culture-treated flasks (Corning) with RPMI 1640 (Hyclone) containing 10% fetal bovine serum (Hyclone), 2 mM L-glutamine, 10 mM HEPES, 10 units/ml penicillin, and 10 µg/ml streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. U937 cells were stably transfected with the wild type FPR as previously described (36). The cells were passaged from near confluent cultures every 3-4 days by reseeding at 2 × 10⁵ cells/ml, expanded for membrane preparations to sealed, 5% CO₂-equilibrated, 1-liter, baffled spinner flasks (Pyrex), and incubated at 37 °C.

Cell Stimulation and Membrane Preparation-- Spinner flasks containing near confluent FPR-expressing U937 cells were stimulated for 8 min at 37 °C with 10 µM fMLF. As previously reported, stimulation at this ligand concentration for this duration of time results in ~90% maximal receptor phosphorylation (32). Following stimulation, flasks were immediately placed on ice, and an equal volume of ice-cold PBS was added. The preparation was otherwise identical to that for unstimulated cells.

For membrane preparation, the cells were harvested by centrifugation at 200 × g for 5 min and resuspended in cavitation buffer (10 mM HEPES, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl₂, 600 µg/ml ATP, pH 7.3) at a density of 10⁷ cells/ml at 4 °C. The cell suspension was placed in a nitrogen bomb and pressurized to 500 p.s.i. for 15-20 min at 4 °C. Following cavitation, nuclei and cytoplasmic material were separated by centrifugation twice at 1000 × g for 5 min at 4 °C. The membrane fraction was pelleted by centrifugation at 109,000 × g for 30 min and resuspended in HEPES sucrose buffer (200 mM sucrose, 25 mM HEPES, pH 7.0). All membrane preparations received protease inhibitor mixture set I (Calbiochem) and phosphatase inhibitor mixture (Calbiochem) prior to flash freezing. The aliquots were stored until use at 80 °C.

Detergent Solubilization-- The membranes were thawed and diluted to 1-2 × 10⁷ membrane cell equivalents/ml in an extracellular binding buffer (BB: 30 mM HEPES, 100 mM KCl, 20 mM NaCl, 1 mM EGTA, 0.1% (w/v) bovine serum albumin, 0.5 mM MgCl₂). The membranes were isolated by centrifugation at 110,000 × g for 45 min in a Beckman Avanti centrifuge, and the supernatant was discarded. The membrane pellet was resuspended in 200 µl of BB containing protease inhibitor mixture set I, phosphatase inhibitor mixture, and 1% n-dodecyl -D-maltoside (Calbiochem). The suspensions were then gently mixed on a nutator (Clay Adams) for 90 min at 4 °C. The insoluble fraction was removed by centrifugation at 70,000 × g for 5 min in a Beckman Airfuge, and the supernatant containing the solubilized FPR was placed on ice for immediate experimentation. Our prior work indicates that the preceding extraction results in a monodisperse receptor preparation consisting of ~150 nM FPR (26).

Depletion of Endogenous G Proteins and Arrestins-- Approximately 200 µl of solubilized receptor was incubated with 10 µl of BB and either 10 µl of anti-G_i-1,2,3 antibody (Calbiochem) or 10 µl of a polyclonal mixture of anti-arrestin-2 and -3 (generously provided by Dr. Jeffrey Benovic, Thomas Jefferson University) or 10 µl of each for 45 min on ice with gentle agitation. To remove antibody-substrate conjugates, excess protein A-Sepharose (Calbiochem) was added to the sample and allowed to incubate for 30 min. The samples were then centrifuged at 14,000 × g for 30 s, and the supernatant was removed. Excess protein A was again added, and the samples were respun to ensure complete removal of antibodies. Whole and cleared lysates were run on an SDS acrylamide gel, transferred to nitrocellulose, blotted with relevant antibodies, and exposed using a chemiluminescent detection system to estimate the extent of endogenous protein depletion.

Receptor Reconstitution-- Detergent-solubilized FPR (8-12 µl of receptor preparation) was incubated with either bovine brain G_i/G_o heterotrimer mixture, purified arrestin, or buffer for 15 min at 4 °C with gentle agitation. N-Formyl-Met-Leu-Phe-Lys-fluorescein 5-isothiocyanate (10 nM) was added, and the samples were gently mixed at 4 °C for up to 120 min. The samples in some cases were depleted of endogenous proteins, as described above, or received 100 nM GTPS where indicated. Blocked samples received 1 µM fMLFF, a large excess of nonfluorescent formyl peptide, and were mixed for 15 min prior to fluorescent ligand addition. In the case of alkaline phosphatase treatment, ~200 µl of solubilized FPR was incubated with either 5 units alkaline phosphatase (Sigma) or an equal volume of alkaline phosphatase buffer for 60 min at room temperature, followed by 60 min at 4 °C. Treated preparations were then handled in the aforementioned manner for fluorescence setup and analysis.

Spectrofluorimetric Analysis-- Fluorescence associated with fMLFK-FITC was measured by an SLM 8000 spectrofluorimeter (Spectronics) using the photon counting mode in a slow time-based acquisition mode as described previously (30). The sample holder was fitted with a cylindrical cuvette adapter to permit measurements in stirred volumes of 200 µl using small cylindrical cuvettes (Sienco) and 2 × 5-mm stir bars (Bel-Art). Excitation was fixed at 490 nm, and stray light was reduced with a 490-nm, 10-nm band pass filter (Corion). FITC fluorescence emission was monitored using a 520-nm, 10-nm band pass interference filter (Corion) and a 3-70 orange glass, 500-nm long pass filter (Kopp). Additions during kinetic measurements were made with 10-µl glass syringes (Hamilton) through a microinjection port above the sample holder.

Following protein reconstitution and ligand incubation at 4 °C, the samples were brought up to a volume of 200 µl with room temperature BB containing 0.1% n-dodecyl -D-maltoside and inhibitors. The diluted samples were then placed into the spectrofluorimeter with gentle stirring. The data were acquired for 120 s with a 0.5-s integration time. For the first 20 s, equilibrium fluorescence levels were obtained. At 20 s, 60 nM anti-fluorescein antibody, prepared as previously described (29), was added to the sample. The antibody rapidly binds free fMLFK-FITC with high affinity and results in complete quenching of unbound ligand. At 70 s, 100 µM GTPS (Sigma) was added to assess coupling between receptors and G proteins based on characteristic ligand dissociation rates. All experiments were performed using a detergent concentration slightly above the critical micelle concentration, typically 0.2% throughout (30).

In Vivo Phosphorylation and Immunoprecipitation-- To assess the phosphorylation status of the receptor, U937 cells transfected with C-terminal His₆-tagged FPR were used. The cells were grown to a density of ~1.25 × 10⁶ cells/ml and washed three times in 10 mM HEPES and 150 mM NaCl (pH 7.4) to remove traces of phosphate, as previously described (32). The cells were resuspended in phosphate-free RPMI 1640 containing 10 mM HEPES (pH 7.4) to a density of 10⁷ cells/ml, and 10 mCi of carrier-free, acid-free [³²P]orthophosphate was added. The cells were loaded for 3 h at 37 °C with 5% CO₂. After loading, the cells were washed two times and resuspended to a density of 10⁸ cells/ml with phosphate-free RPMI. Stimulated cells received 10⁶ M fMLFF at 37 °C for 10 min. The samples were pelleted and subsequently solubilized in ice-cold BB containing 1% DOM and protease inhibitors for a period of 1 h with mild agitation. The insoluble fraction was pelleted via centrifugation, and the supernatant was collected. Solubilized membranes received either 5 units of alkaline phosphatase/10⁶ cells or an equivalent volume of phosphatase buffer and incubated at room temperature for 60 min. 1× RIPA buffer was added, and the entire volume was transferred to 10 mg of protein A precoated with 5 µg of chicken anti-C-terminal FPR (and goat anti-chicken antibodies) or protein G beads precoated with mouse anti-His antibodies and incubated overnight on ice. The beads were washed extensively and resuspended in 2× Laemmli sample buffer, followed by electrophoresis on a 4-20% SDS-polyacrylamide gel (32). The gels were dried, and determinations of relative ³²P content were performed on duplicate samples with a Molecular Dynamics PhosphorImager.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Spectrofluorimetric Assay-- The detergent-based solubilization of GPCRs from membranes, employment of FITC-conjugated ligands, and reconstitution with purified proteins of interest for studying molecular complexes have recently been characterized (26, 30). The system fundamentally hinges on three facts. First of all, ligand dissociation rates vary in accordance with the components comprising the receptor complex. It has been shown for a number of GPCRs, including the FPR, that an FPR-G protein complex has a significantly higher affinity for ligand (i.e. slower ligand dissociation rate) than receptor alone (27, 37). Recent data on the ₂-adrenergic and the m2 muscarinic receptor also suggest that an R^p-arrestin complex has a higher affinity for ligand than R^p alone (17). However, at this point, a direct study of FPR interactions with arrestins in terms of agonist affinity has not been conducted.

Second, the addition of a guanine nucleotide, such as the nonhydrolyzable analogue of GTP, GTPS, facilitates the rapid dissociation of G protein from receptor. This dissociation, in turn, typically leads to a decrease in affinity of ligand for receptor, because FPR-G protein complexes give way to isolated FPR. The subsequent changes in ligand kinetics are directly measurable in our system as a conversion from a high ligand affinity FPR-G protein complex to a low ligand affinity FPR species. The few characterized high ligand affinity ligand-R^p-arrestin complexes, in contrast, are unaffected by the presence of guanine nucleotide analogues and thus may be distinguished from high ligand affinity LRG complexes by their insensitivity to GTPS (17). Hence, discrimination of LRG and ligand-R^p-arrestin high affinity complexes should be possible on this basis.

The third unique aspect of our system involves the ability of the anti-fluorescein antibody to quench only unbound ligand. The ligand utilized is of such size and composition that, when bound to the FPR, the attached fluorescein is sterically unavailable for interacting with anti-fluorescein antibodies (29). It is only upon dissociation from the receptor that ligand can be bound and quenched by antibody. Because of the high concentration of antibody used, the latter process is quite rapid, occurring on the order of less than a second. In that regard, following addition of the antibody, the remaining fluorescence is solely a function of ligand bound to the FPR and therefore provides a direct measure over time of the fluorescent ligand dissociation rate.

Reconstitution with Endogenous Proteins-- We initially sought to examine agonist affinity differences between nonphosphorylated and phosphorylated receptor preparations as obtained from unstimulated and fMLF-stimulated cells, respectively. As shown in Fig. 1A, incubation of fluorescent ligand with the solubilized FPR prepared from unstimulated cells leads to the formation of slowly dissociating, nucleotide-sensitive complexes with nearly maximal effects seen at ~90 min (data not shown for later time points). We have previously reported reconstitution of the native FPR with endogenous G proteins, with similar properties, over this time course (26). However, in the case of the FPR obtained from fMLF-stimulated cells (i.e. phosphorylated FPR), there is also evidence for time-dependent formation of a slowly dissociating complex (Fig. 1B). This high ligand affinity complex is in contrast predominately GTPS-insensitive, unlike the well characterized LRG complex. Thus, although there is evidence of time-dependent complex formation in the case of R^p, its precise makeup is not clear. Given the GTPS insensitivity of the complex, it is likely that the assembly does not involve accessible GTP-binding proteins.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Time-dependent effects on unphosphorylated (A and C) or phosphorylated (B and D) FPR agonist affinity. Samples of detergent-solubilized FPR were incubated with 10 nM FITC-fMLFK, a fluorescent agonist, and mixed at 4 °C for 90 (), 30 (), or 0 () min in the absence (A and B) or presence (C and D) of 100 nM GTPS. Blocked samples (solid line) received 10 µM of an unlabeled agonist, fMLFF, 10 min prior to fluorescent agonist addition. The samples were diluted in buffer to 200 µl and transferred to glass cuvettes immediately prior to analysis in the SLM 8000 spectrofluorimeter. At 20 s, 60 nM anti-fluorescein antibody, which quenches the fluorescence of unbound ligand, was added to the sample through a microinjection port. At 70 s, 100 µM GTPS, which disrupts coupling of receptor and G proteins, was added. The data are plotted as peptide intensity versus time. The normalized means are representative of at least three independent experiments conducted in duplicate.

We further sought to characterize the time-dependent ligand affinity differences by incubating detergent-solubilized receptor with fluorescent ligand in the absence and presence of 100 nM GTPS. In prior studies, it has been observed that 100 nM nucleotide completely disrupts coupling between receptor and G protein (38). As Fig. 1C demonstrates, incubation of the extract from unstimulated cells with GTPS prior to spectrofluorometric analysis alters the observed dissociation kinetics of ligand; the time-dependent formation of high affinity, nucleotide-sensitive receptors is completely prevented. However, when GTPS is preincubated with the phosphorylated receptor extract, its presence does not inhibit the formation of high ligand affinity, nucleotide-insensitive complexes. It should also be noted that in the absence of GTPS preincubation (Fig. 1B), a small fraction of the high ligand affinity complex of the phosphorylated FPR is nucleotide-sensitive, indicating the presence of a minor amount of G protein complex under these conditions. In contrast, when the phosphorylated FPR sample is preincubated with GTPS, there is no indication of any fraction of the high ligand affinity complex being attributable to G proteins. Thus, these results further support the notion that high ligand affinity complex formation of phosphorylated receptors with endogenous proteins does not fundamentally involve G proteins.

Clearance of Endogenous G Proteins and Arrestins-- Given these findings, we investigated the effects of depletion of both endogenous G_i proteins and arrestins on the ligand dissociation kinetics for both the nonphosphorylated and phosphorylated receptor preparations. FPR extracts were incubated with excess polyclonal antibodies for ~45 min at 4 °C. Antibody conjugates were then removed by two sequential protein A-Sepharose incubations to ensure complete antibody removal. We have previously demonstrated that immunodepletion of G protein prevents the formation of a high ligand affinity complex with the FPR (26). As Fig. 2A demonstrates, depletion of G_i proteins from the nonphosphorylated receptor preparation completely inhibits the formation of the high affinity, nucleotide-sensitive LRG complex. In comparison, G protein depletion from the phosphorylated receptor preparation has little effect on time-dependent agonist affinity changes (Fig. 2B). Thus, G_i depletion has effects on ligand affinity similar to those of nucleotide pretreatment (cf. Figs. 2A and 1C). Arrestin-depletion, on the other hand, has little or no effect on the ligand dissociation kinetics of the nonphosphorylated FPR complex (Fig. 2C), but does completely prevent the formation of a high ligand affinity complex formed by the phosphorylated FPR (Fig. 2D). Furthermore, it appears that arrestin-depletion mildly enhances the nucleotide sensitivity of phosphorylated FPR complexes (cf. Fig. 2, B and D), perhaps signifying a low level of LRG formation in the absence of arrestin. Lastly, the simultaneous depletion of both G_i proteins and arrestins prevents both characteristic LRG and high affinity, nucleotide-insensitive complex formation of the nonphosphorylated and phosphorylated FPR preparations, respectively (Fig. 2, E and F). Therefore, complete removal of both endogenous proteins appears to be sufficient to prevent all time-dependent ligand affinity changes regardless of the phosphorylation state of the receptor. Although these results suggest that high affinity complexes involving the nonphosphorylated and phosphorylated forms of the FPR are at least a function of endogenous G proteins and arrestins, respectively, they do not reveal the exact makeup or mechanisms underlying the assembly of these complexes.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effects of endogenous protein depletion on time-dependent agonist affinity changes. Solubilized preparations of both unphosphorylated (A, C, and E) and phosphorylated (B, D, and F) FPR were incubated with either anti-Gi antibodies (A and B), anti-arrestin-2/3 antibodies (C and D), or both (E and F) and cleared using protein A-Sepharose beads. Western blot analyses of both G protein (G) and arrestin (H) content were performed against purified proteins to confirm depletion. The cleared fractions were incubated with fluorescent agonist and mixed at 4 °C for 90 (), 60 (), or 30 () min prior to SLM query. Injections of anti-fluorescein antibody and GTPS were made at 20 and 70 s, respectively.

Exogenous G Protein Reconstitution-- We have previously reported the ability of G protein-depleted, nonphosphorylated FPR to reconstitute in a detergent-solubilized state with both a purified mixture of G_i/o proteins and individual G_i subunits, with EC₅₀ values in the range of ~1-2 µM (26). In the current study, we evaluated the ability of G protein- and arrestin-depleted, phosphorylated receptor to form a ternary ligand-R^p-G protein complex. The hallmarks of the FPR-G protein complex are slower fluorescent ligand dissociation, with rapid conversion to a swiftly dissociating species upon addition of GTPS. In Fig. 3, it is evident that there are significant differences between the properties of nonphosphorylated FPR (Fig. 3A) and phosphorylated FPR (Fig. 3B) reconstituted with exogenous G protein. In the case of the phosphorylated receptor, G protein addition, even at three times the EC₅₀ concentration for the nonphosphorylated FPR, results in neither high ligand affinity complexes nor nucleotide-sensitive ligand dissociation, suggesting the absence of complex formation.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Reconstitution with heterotrimeric G proteins. Solubilized, unphosphorylated (A) and phosphorylated receptor (B) preparations depleted of both G proteins and arrestins were incubated with either 3 µM of a bovine brain mixture of heterotrimeric G proteins (), buffer alone (), or 10 µM unlabeled ligand (solid line) for 15 min prior to fluorescent ligand addition. The fluorescent samples were then mixed for 90 min at 4 °C and set up for SLM analysis.

Exogenous Arrestin Reconstitution-- We have previously demonstrated that reconstitution of the nonphosphorylated FPR with wild type arrestin-3 has no effect on the ligand dissociation rate or sensitivity to guanine nucleotide of the nonphosphorylated FPR in solution (26). However, reconstitution with a truncated, phosphorylation-independent mutant of arrestin-3 (1), known to bind receptors in a phosphorylation-independent but activation-dependent manner (17), inhibited FPR-G protein formation, suggesting complexing of the arrestin mutant with the nonphosphorylated FPR.

In the current investigation, we sought to determine whether arrestin-depleted R^p could reconstitute with purified, wild type arrestins. As shown in Fig. 4A, the addition of arrestin-3 to the nonphosphorylated FPR preparation has no effect on ligand dissociation kinetics. However, at the same concentration, arrestin-3 promotes a significant change in the ligand dissociation kinetics of the phosphorylated FPR, yielding a slowly dissociating and nucleotide-insensitive receptor complex (Fig. 4B). Moreover, the changes in agonist dissociation characteristics were concentration-dependent (Fig. 5, A and B). Nonlinear regression analyses of the binding curves with arrestin-2/3-depleted receptors revealed an EC₅₀ of 0.9 ± 0.2 µM arrestin-3. Thus, desensitized FPR assemblies involving arrestin-3 appear to impart high agonist affinity to the receptor. Moreover, dissociation of these ligand-R^p-arrestin complexes is insensitive to nonhydrolyzable nucleotide analogues, demonstrating a lack of involvement of G proteins in such complexes.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Reconstitution with arrestins-2 and -3. Solubilized, nonphosphorylated (A and C) and phosphorylated receptor preparations (B and D) were depleted of arrestins and incubated with either 17 µM of arrestin-3 (A and B) or arrestin-2 (C and D) (), buffer alone (), or 10 µM unlabeled ligand (solid line) for 15 min prior to fluorescent ligand addition. Fluorescent samples were then mixed for 90 min at 4 °C and set up for fluorescence analysis. Injections of anti-fluorescein antibody and GTPS were made at 20 and 70 s, respectively.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent reconstitution of phosphorylated receptor with arrestins. Phosphorylated receptor preparations depleted of both arrestins and G proteins were incubated with varying concentrations of arrestin-3 (A) or arrestin-2 (C) prior to fluorescent ligand addition and fluorescence analysis. The data were replotted to express the fraction of high agonist affinity complex formation. The EC50 values of 0.9 ± 0.1 µM (B) and 0.5 ± 0.1 µM (D) were resolved for arrestin-3 and -2, respectively.

We also endeavored to determine whether both FPR and R^p could interact with wild type arrestin-2 by examining kinetic effects resulting from such reconstitutions. In the case of nonphosphorylated FPR (Fig. 4C), no effects on ligand dissociation kinetics were observed upon the addition of arrestin-2. In the case of depleted, phosphorylated receptor (Fig. 4D), as with arrestin-3, the ligand-receptor complex became both slowly dissociating and nucleotide-insensitive in response to the presence of arrestin-2. In addition, agonist affinity changes were both concentration- (Fig. 5, C and D) and time-dependent (data not shown). Nonlinear regression analyses of the binding curves with arrestin-2/3-depleted FPR yielded an EC₅₀ of 0.6 ± 0.2 µM and a _1/2 of 8.9 min (data not shown) for arrestin-2-FPR complex formation.

Quantitative experiments were also repeated with nondepleted receptor preparations. In general, the data were qualitatively similar, although estimates for EC₅₀ values were somewhat increased because of the presence of endogenous arrestins. This suggests that in vitro concentrations of both arrestins and G proteins are not altogether insignificant. However, they were not substantial enough to obscure time-dependent differences upon the addition of exogenous arrestin proteins.

Conversion of the Phosphorylated FPR to Its Nonphosphorylated Form-- We examined the ability of alkaline phosphatase to convert the agonist dissociation kinetics and associated protein coupling characteristics of the activated, phosphorylated receptor to that of the unstimulated, nonphosphorylated receptor in the absence and presence of both arrestin-2 and G proteins. Solubilized membranes were incubated with alkaline phosphatase prior to ligand addition and reconstitution. As evidenced in Fig. 6, the phosphatase-pretreated, stimulated receptor behaves in a similar fashion to untreated, nonphosphorylated receptor, insofar as incubation with exogenous arrestin-2 does not alter the ligand dissociation kinetics of the receptor preparation but the addition of exogenous G protein now results in the formation of the high affinity, nucleotide-sensitive complex characteristic of LRG. Interestingly, pretreatment of the receptor isolated from unstimulated cells with phosphatase results in enhanced formation of LRG (data not shown), as reflected in an increased fraction of high affinity receptors. This suggests that in the unstimulated cell a small percentage of FPR may exist in a phosphorylated state.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Effects of alkaline phosphatase treatment. Solubilized, phosphorylated FPR was incubated with alkaline phosphatase for 90 min at 4 °C. Phosphatase-treated samples received either 3 µM G proteins (), 17 µM arrestin-2 (), 10 µM fMLFF (solid line), or buffer (). For illustrative purposes, the untreated samples were run in parallel with either G proteins () or arrestin-2 (). As shown in C, the phosphorylation status of the receptor was demonstrated under experimental conditions via 32P incorporation, subsequent immunoprecipitation using receptor-specific antibodies, and PhosphorImager analysis. The corresponding inset displays the respective bands of immunoprecipitated material on an SDS-polyacrylamide gel.

To assess the relative phosphorylation status of the FPR in the phosphatase-treated samples, we utilized a C-terminal hexahistidine-tagged form of the FPR stably expressed in U937 cells. In parallel experiments, receptor was solubilized directly from agonist-stimulated or unstimulated whole cells and used either for reconstitution experiments with arrestins or for immunoprecipitation to assess the degree of protein phosphorylation. Detergent extract isolated from agonist-stimulated cells was treated with either phosphatase or buffer as described for membrane-derived receptors. For experiments involving immunoprecipitation of the receptor to assess phosphorylation, the cells were incubated in [³²P]orthophosphate for 3 h prior to stimulation. Our results demonstrated not only that FPR extracted from stimulated whole cells could be reconstituted with arrestin in a phosphatase-sensitive manner (data not shown) but also that phosphatase treatment of the ³²P-labeled extract reduces phosphorylation levels of immunoprecipitated material to those of the unstimulated samples (Fig. 6C).

Competitive Assays-- Because our system permits only inferences of protein-protein interactions from ligand affinity data and is not a direct biochemical measure, we sought to discriminate between a lack of an agonist affinity shift and a lack of binding in the reconstitution assays through the use of competitive assays. We investigated whether preincubation of solubilized receptor with ligand and either exogenous G proteins or arrestins would preclude subsequent agonist affinity shifts associated with reconstitution. In the case of the stimulated receptor, concentrations of G proteins up to 3 µM did not inhibit agonist affinity changes because of incubation with arrestins (data not shown). Similarly, in the case of the nonstimulated FPR, concentrations of arrestins up to 17 µM did not prevent agonist-dependent affinity changes because of G proteins (data not shown). Thus, our data indicate that an absence of the ligand affinity shift in the preceding reconstitution experiments is the result of a lack of protein binding to the receptor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Summary of Solubilized FPR Reconstitution-- In the current study, we evaluated the ability of the FPR to undergo noncellular reconstitution with both G proteins and arrestins by measuring ligand dissociation characteristics of receptor complexes in real time. Our data indicate that detergent extracts from FPR-transfected U937 cell membranes yield functional receptor capable of reconstituting with both G proteins and arrestins. Initial experiments revealed that extracts from unstimulated cells contained sufficient G protein to permit spontaneous although limited recoupling of the FPR with endogenous G proteins, as indicated by alterations in the ligand dissociation kinetics upon the addition of GTPS. Similarly, extracts from fMLF-stimulated cells contained sufficient quantities of arrestins to permit reassociation with the FPR to yield a high ligand affinity complex that was insensitive to GTPS. Both of these interactions could be eliminated by immunodepletion of the extract with the appropriate anti-G_i protein and/or anti-arrestin antibodies, leaving the remaining FPR in a low ligand affinity, uncomplexed state. We also demonstrated that G protein addition was unable to alter the ligand affinity of the phosphorylated FPR as it did for the nonphosphorylated FPR. However, reconstitution of the solubilized receptor with submicromolar concentrations of either arrestin-2 or arrestin-3 was able to convert the phosphorylated FPR, but not the nonphosphorylated FPR, to a high ligand affinity complex. As with G protein interactions, ligand binding was necessary to induce arrestin coupling to the FPR, because preincubation of the protein with receptor prior to ligand addition had no discernible effects on the time course of high agonist affinity complex formation. Both G proteins and the arrestins therefore appear to recognize only the active conformation of the receptor. Lastly, we demonstrated that the properties of the phosphorylated FPR are converted to those of the nonphosphorylated FPR by treatment of the receptor preparation with alkaline phosphatase, suggesting that the phosphorylation status of the FPR was critical in regulating these properties. To our knowledge, these data represent the first reported reconstitution of a detergent-solubilized, phosphorylated GPCR with arrestins.

Regulation of FPR Function by Arrestins-- The role of an FPR-arrestin interaction came into question recently with our observation that partial phosphorylation-deficient mutants of the FPR internalize in the absence of apparent arrestin binding (31), despite the fact that both receptor desensitization and internalization do require receptor phosphorylation (32). Certain FPR mutants lacking a subset of the potential phosphorylation sites were unable to undergo desensitization despite being fully competent for internalization. This result indicated that if arrestins did indeed interact with the FPR, they were not likely involved in both processing events. Further evidence supporting this conclusion has been obtained from the disruption of clathrin-dependent internalization pathways utilized by many GPCRs (33, 39). Numerous agents that inhibit clathrin-dependent internalization were found to have no effect on the internalization of the FPR. Interestingly, evidence in support of an FPR-arrestin interaction came from confocal microscopy studies demonstrating that, following receptor activation, arrestin colocalized with the FPR in punctate structures on the cell surface and within the cytoplasm on endosomes (31). Thus, despite not being required for internalization, arrestins do appear to maintain some interactions with the internalized FPR. In fact, we have observed a correlation between the ability of an FPR phosphorylation-deficient mutant to undergo desensitization and the ability of arrestin to colocalize with the FPR. This result suggests that arrestins, although not required for FPR internalization, may be involved in receptor regulation.

It has previously been observed that following ligand binding and cellular activation the FPR enters an inactive state with high affinity for ligand (37). It was suggested that this state represented a desensitized form of the receptor. More recently, studies of other GPCRs, such as the ₂-adrenergic receptor and muscarinic cholinergic receptor, demonstrated that these receptors exist in ternary, ligand-receptor-arrestin complexes displaying high affinity for ligand (17). Thus, it seemed likely that reconstitution of the FPR with arrestins would result in the high ligand affinity state previously observed in vivo. Our results confirm this prediction. We demonstrated that it is the stimulated and therefore phosphorylated form of the FPR that is capable of interacting with arrestins, resulting in the formation of a high ligand affinity complex. Although the nonphosphorylated FPR and exogenously added G proteins form a complex with similar affinity, this complex, unlike an arrestin one, is sensitive to the addition of GTP analogues, which activate and cause dissociation of G proteins. We were also able to demonstrate the selectivity of these interactions by virtue of the fact that the nonphosphorylated receptor did not interact with arrestins nor phosphorylated receptor with G proteins. Phosphorylation of the FPR greatly lowers its affinity for G protein while increasing its affinity for arrestin.

This latter result, that the phosphorylated FPR does not observably couple to G proteins, differs from findings with the ₂-adrenergic receptor. Reconstitution of the ₂-adrenergic receptor in phospholipid vesicles, followed by in vitro phosphorylation by G protein-coupled receptor kinase 2, resulted in a receptor state that still coupled to G proteins (40). This coupling, however, could be blocked by the addition of arrestins. Our results indicate that in vivo phosphorylation of the FPR is sufficient to prevent G protein coupling. This may be due to inherent differences between the two receptors in the extent and mode of phosphorylation (in vitro versus in vivo), in the G proteins involved (G_s versus G_i), or in the receptor environment (membrane-bound versus solubilized). Although our data indicate that the maximally phosphorylated FPR does not interact efficiently with G proteins, it is possible that partially phosphorylated states of the FPR may interact with both G proteins and arrestins. Under these conditions, the receptor may be only partially desensitized. This is consistent with our observation that less than maximal phosphorylation is sufficient to permit receptor internalization while resulting in diminished desensitization (32). Although a physiological role for high ligand affinity complex formation is unclear at the present moment, we envision that the high affinity complex of ligand-receptor-arrestin may serve to ensure completion of receptor processing events and removal of the ligand from the extracellular milieu.

Receptor Scaffolds-- A growing body of evidence suggests that arrestins bound to phosphorylated receptors act as scaffolds within the confines of the cell (41). Studies have implicated direct interactions of arrestins with such diverse proteins as extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling cascade components, Src, Raf-1, the endocytotic proteins clathrin, and AP-2, in addition to phosphorylated GPCRs (42-44). It has been hypothesized that arrestins function in part by bringing signaling modules under the control of certain GPCRs and thereby provide increased specificity to enzymatic pathways. At this point, the possible scaffolding functions of a desensitized FPR-arrestin complex are unclear. We have demonstrated, however, using a phosphorylation-deficient mutant of the FPR, that arrestin binding is not required for activation of MAPK pathways by the FPR (45). Whether FPR-bound arrestins also function as scaffolds for other signaling molecules remains to be determined.

Molecular Assemblies and Drug Discovery-- The ability to characterize and ultimately discriminate molecular assemblies in solution is of great import in the study of GPCR-mediated signal transduction pathways and the emerging field of GPCR-based drug discovery. The use of noncellular assays permits a more extensive, more specific, and less time-consuming foray into the molecular dynamics of cellular processes. As receptor assemblies increase in number and complexity over time, tools appropriate for their analysis become more necessary. The methodology elucidated in this study should provide a basic format for studies involving these receptor complexes. A fluorescence-based approach utilizing assemblies in solution might lend itself directly to future inquiries of the core components and biology of signal transduction in general and both desensitization and internalization in particular.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AI36357 and AI43932 (to E. R. P.), GM60799 (to L. A. S.), and EY11500 and GM63097 (to V. V. G.) and New Mexico Cancer Research Fund Grant RR01315 (to L. A. S.).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 Cell Biology and Physiology, University of New Mexico, Albuquerque, NM 87131. Tel.: 505-272-5647; Fax: 505-272-1421; E-mail: eprossnitz@salud.unm.edu.

Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M109475200

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; FPR, formyl peptide receptor; R^p, phosphorylated receptor; fMLFK, N-formyl-Met-Leu-Phe-Lys; FITC, fluorescein 5-isothiocyanate; BB, binding buffer; fMLFF, N-formyl-Met-Leu-Phe-Phe; GTPS, guanosine 5'-3-O-(thio)triphosphate; LRG, ligand-receptor-G protein complex.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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