Regulation of Formyl Peptide Receptor Agonist Affinity by Reconstitution with Arrestins and Heterotrimeric G Proteins
2001; Elsevier BV; Volume: 276; Issue: 52 Linguagem: Inglês
10.1074/jbc.m109475200
ISSN1083-351X
AutoresT. Alexander Key, Teresa Bennett, Terry D. Foutz, Vsevolod V. Gurevich, Larry A. Sklar, Eric R. Prossnitz,
Tópico(s)Neuropeptides and Animal Physiology
ResumoAlthough 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 (GTPγS) 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 GTPγS-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. 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 (GTPγS) 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 GTPγS-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. G protein-coupled receptor formyl peptide receptor phosphorylated receptor N-formyl-Met-Leu-Phe-Lys fluorescein 5-isothiocyanate binding buffer N-formyl-Met-Leu-Phe-Phe guanosine 5′-3-O-(thio)triphosphate ligand-receptor-G protein complex 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 (1Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2083) Google Scholar). 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 β2-adrenergic receptor (2Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 3Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 4Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le T.I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4971) Google Scholar). 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 (5Ferguson S.S. Caron M.G. Semin. Cell Dev. Biol. 1998; 9: 119-127Crossref PubMed Scopus (160) Google Scholar, 6Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 7Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar). 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 (8Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). 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 (9Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar,10Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). It has also been shown that arrestins have direct interactions with proteins involved in endocytotic sequestration, including the proteins clathrin and AP-2 (11Ferguson S.S. Downey W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (836) Google Scholar, 12Goodman O.B.J. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1140) Google Scholar, 13Goodman O.B.J. Krupnick J.G. Gurevich V.V. Benovic J.L. Keen J.H. J. Biol. Chem. 1997; 272: 15017-15022Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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 (14De L.A. Stadel J.M. Lefkowitz R.J. J. Biol. Chem. 1980; 255: 7108-7117Abstract Full Text PDF PubMed Google Scholar, 15Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1280) Google Scholar). 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 (8Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 16Schleicher A. Kuhn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (164) Google Scholar, 17Gurevich V.V. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. Onorato J.J. J. Biol. Chem. 1997; 272: 28849-28852Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). 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 (18Kuhn H. Hall S.W. Wilden U. FEBS Lett. 1984; 176: 473-478Crossref PubMed Scopus (270) Google Scholar, 19Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (561) Google Scholar). 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 (20Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 21Vishnivetskiy S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In contrast, the affinity of G protein for rhodopsin appears to diminish on a linear basis with increasing levels of receptor phosphorylation (22Gibson S.K. Parkes J.H. Liebman P.A. Biochemistry. 2000; 39: 5738-5749Crossref PubMed Scopus (62) Google Scholar). 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 (23Prossnitz E.R. Ye R.D. Pharmacol. Ther. 1997; 74: 73-102Crossref PubMed Scopus (228) Google Scholar). 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 (24Gierschik P. Sidiropoulos D. Jakobs K.H. J. Biol. Chem. 1989; 264: 21470-21473Abstract Full Text PDF PubMed Google Scholar, 25Murphy P.M. Eide B. Goldsmith P. Brann M. Gierschik P. Spiegel A. Malech H.L. FEBS Lett. 1987; 221: 81-86Crossref PubMed Scopus (53) Google Scholar). 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 (26Bennett T.A. Prossnitz E.R. Key T.A. Gurevich V.V. Neubig R.R. Sklar L.A. J. Biol. Chem. 2001; 276: 22453-22460Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 27Fay S.P. Posner R.G. Swann W.N. Sklar L.A. Biochemistry. 1991; 30: 5066-5075Crossref PubMed Scopus (84) Google Scholar, 28Nolan J.P. Chambers J.D. Sklar L.A. Babcock G. Robinson P. Cytometric Approaches to Cellular Analysis. Wiley-Liss, New York1998: 19-46Google Scholar, 29Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar, 30Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). 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 (31Bennett T.A. Maestas D.C. Prossnitz E.R. J. Biol. Chem. 2000; 275: 24590-24594Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Curiously, however, we have found FPR internalization to proceed through an arrestin-independent mechanism (32Maestes D.C. Potter R.M. Prossnitz E.R. J. Biol. Chem. 1999; 274: 29791-29795Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 33Prossnitz E.R. Gilbert T.L. Chiang S. Campbell J.J. Qin S. Newman W. Sklar L.A. Ye R.D. Biochemistry. 1999; 38: 2240-2247Crossref PubMed Scopus (40) Google Scholar), similar to results found with the m2 muscarinic receptor (34Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). 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 (Rp) 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. 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 inEscherichia coli (strain BL21) and purified by sequential heparin-Sepharose and Q-Sepharose chromatography as previously reported (35Gurevich V.V. Benovic J.L. Methods Enzymol. 2000; 315: 422-437Crossref PubMed Google Scholar). 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. 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 (36Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. J. 1993; 294: 581-587Crossref PubMed Scopus (41) Google Scholar). 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. 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 (32Maestes D.C. Potter R.M. Prossnitz E.R. J. Biol. Chem. 1999; 274: 29791-29795Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). 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 mmNaCl, 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. 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 mmMgCl2). 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 β-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 (26Bennett T.A. Prossnitz E.R. Key T.A. Gurevich V.V. Neubig R.R. Sklar L.A. J. Biol. Chem. 2001; 276: 22453-22460Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Approximately 200 μl of solubilized receptor was incubated with 10 μl of BB and either 10 μl of anti-Gαi-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. Detergent-solubilized FPR (8–12 μl of receptor preparation) was incubated with either bovine brain Gi/Go 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 nmGTPγS where indicated. Blocked samples received 1 μmfMLFF, 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. 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 (30Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). 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 β-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 (29Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar), 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 GTPγS (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 (30Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). 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 (32Maestes D.C. Potter R.M. Prossnitz E.R. J. Biol. Chem. 1999; 274: 29791-29795Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). 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 10−6m 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 (32Maestes D.C. Potter R.M. Prossnitz E.R. J. Biol. Chem. 1999; 274: 29791-29795Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The gels were dried, and determinations of relative32P content were performed on duplicate samples with a Molecular Dynamics PhosphorImager. 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 (26Bennett T.A. Prossnitz E.R. Key T.A. Gurevich V.V. Neubig R.R. Sklar L.A. J. Biol. Chem. 2001; 276: 22453-22460Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 30Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). 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 (27Fay S.P. Posner R.G. Swann W.N. Sklar L.A. Biochemistry. 1991; 30: 5066-5075Crossref PubMed Scopus (84) Google Scholar, 37Sklar L.A. Mueller H. Omann G. Oades Z. J. Biol. Chem. 1989; 264: 8483-8486Abstract Full Text PDF PubMed Google Scholar). Recent data on the β2-adrenergic and the m2 muscarinic receptor also suggest that an Rp-arrestin complex has a higher affinity for ligand than Rp alone (17Gurevich V.V. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. Onorato J.J. J. Biol. Chem. 1997; 272: 28849-28852Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). 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, GTPγS, 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-Rp-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 GTPγS (17Gurevich V.V. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. Onorato J.J. J. Biol. Chem. 1997; 272: 28849-28852Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Hence, discrimination of LRG and ligand-Rp-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 (29Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar). 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. 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 (26Bennett T.A. Prossnitz E.R. Key T.A. Gurevich V.V. Neubig R.R. Sklar L.A. J. Biol. Chem. 2001; 276: 22453-22460Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). 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 GTPγS-insensitive, unlike the well characterized LRG complex. Thus, although there is evidence of time-dependent complex formation in the case of Rp, its precise makeup is not clear. Given the GTPγS insensitivity of the complex, it is likely that the assembly does not involve accessible GTP-binding proteins. 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 nmGTPγS. In prior studies, it has been observed that 100 nmnucleotide completely disrupts coupling between receptor and G protein (38Sklar L.A. Bokoch G.M. Button D. Smolen J.E. J. Biol. Chem. 1987; 262: 135-139Abstract Full Text PDF PubMed Google Scholar). As Fig. 1C demonstrates, incubation of the extract from unstimulated cells with GTPγS 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 GTPγS 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
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