Partial Phosphorylation of the N-Formyl Peptide Receptor Inhibits G Protein Association Independent of Arrestin Binding
2001; Elsevier BV; Volume: 276; Issue: 52 Linguagem: Inglês
10.1074/jbc.m106414200
ISSN1083-351X
AutoresTeresa Bennett, Terry D. Foutz, Vsevolod V. Gurevich, Larry A. Sklar, Eric R. Prossnitz,
Tópico(s)S100 Proteins and Annexins
ResumoIt is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin. It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin. G protein-coupled receptor fluorescein 5-isothiocyanate formyl-Met-Leu-Phe-Lys N-formyl peptide receptor guanosine 5′-3-O-(thio)triphosphate wild type 1,4-piperazinediethanesulfonic acid G protein-coupled receptors (GPCR)1 comprise the largest class of membrane-expressed receptors. They transduce signals from the extracellular environment via interactions with a wide variety of agonists, including single amino acids, small peptides, hormones, lipids, odorants, and photons of light. The life cycle of the class I, rhodopsin-like GPCR has been well described (1Koenig J.A. Edwardson J.M. Trends Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 2Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 3Zhang J. Ferguson S.S. Barak L.S. Aber M.J. Giros B. Lefkowitz R.J. Caron M.G. Receptors and Channels. 1997; 5: 193-199PubMed Google Scholar). Following synthesis and export to the cell surface, interaction with an agonist induces a conformational change in the receptor resulting in activation of a G protein. Activated receptors are then targeted by a member of the GPCR kinase family (2Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). Once phosphorylated, receptors undergo desensitization and internalization, followed either by recycling of the dephosphorylated receptors to the cell surface or degradation. Phosphorylation of the receptor is believed not to be sufficient to fully desensitize the receptor; it does, however, increase the affinity of the receptor for a family of molecules called arrestins (4Gurevich 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 (340) Google Scholar, 5Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina J. Faure J.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar). The coupling of an arrestin molecule with a phosphorylated receptor sterically inhibits further interaction with G proteins, resulting in termination of G protein activation. For many GPCRs, arrestins also act as accessory molecules for endocytosis, targeting receptors to clathrin-coated pits (6Goodman Jr., O.B. 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 (1179) Google Scholar). Other receptors appear to internalize through alternative routes that are independent of both arrestin and clathrin (7Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (49) Google Scholar, 8Roseberry A.G. Hosey M.M. J. Cell Sci. 2001; 114: 739-746PubMed Google Scholar). A more widespread role for arrestins as scaffolds and adapter proteins has been recently identified, linking them with activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase, in addition to regulating receptor trafficking (9Miller W.E. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (281) Google Scholar). The N-formyl peptide receptor (FPR) is a member of the chemokine/chemoattractant subfamily of GPCR. The FPR is expressed predominantly on granulocytes, specifically recognizes bacterially generated N-formyl peptides, and interacts with a pertussis toxin-sensitive G protein (10Prossnitz E.R. Ye R.D. Pharmacol. Ther. 1997; 74: 73-102Crossref PubMed Scopus (229) Google Scholar). Agonist binding to the FPR activates the granulocyte, initiating several second messenger systems that result in the activation of integrins, cell adhesion to the endothelium, diapedesis and chemotaxis, and ultimately superoxide production and release of granule contents at the site of inflammation (10Prossnitz E.R. Ye R.D. Pharmacol. Ther. 1997; 74: 73-102Crossref PubMed Scopus (229) Google Scholar). These functions constitute the first line of host defense in response to a bacterial challenge. Inappropriate granulocyte activation has also been identified as the major cause of tissue damage in several inflammatory and autoimmune diseases (11Davies E.V. Hallett M.B. Int. J. Mol. Med. 1998; 1: 485-490PubMed Google Scholar, 12Jordan J.E. Zhao Z.Q. Vinten-Johansen J. Cardiovasc. Res. 1999; 43: 860-878Crossref PubMed Scopus (559) Google Scholar, 13Ricevuti G. Ann. N. Y. Acad. Sci. 1997; 832: 426-448Crossref PubMed Scopus (88) Google Scholar). Precise control of receptor inactivation and processing is necessary to limit the response and enable the cell to function effectively. FPR phosphorylation has been demonstrated to be necessary for both receptor desensitization and internalization (14Maestes 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 phosphorylation targets appear to be two clusters of four serine and threonine residues, each preceded by one or two acidic residues, in the C-terminal tail domain. The site containing Ser328, Thr329, Thr331, and Ser332 is referred to as the A site, and residues Thr334, Thr336, Ser338, and Thr339 comprise the B site (see Fig. 1). Several FPR phosphorylation-deficient mutants have been previously generated and characterized with regards to Ca2+ mobilization, receptor phosphorylation, desensitization, and internalization (14Maestes 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). This work has demonstrated that although receptor desensitization and internalization are both dependent on phosphorylation, desensitization is not necessary for internalization, indicating that the two mechanisms are independent processes. Protein phosphorylation plays a key role in intracellular signaling, altering the charge and/or conformation of substrate proteins. Increasing the average levels of phosphorylation of the visual GPCR rhodopsin results in an increase in affinity for arrestin and also appears to diminish the affinity of the receptor for G protein (15Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (577) Google Scholar,16Gibson S.K. Parkes J.H. Liebman P.A. Biochemistry. 2000; 39: 5738-5749Crossref PubMed Scopus (68) Google Scholar). However, these results, generated by varying the stimulation time of the receptor, are difficult to interpret because of the heterogeneity of the sites phosphorylated. Numerous other GPCRs have been shown to colocalize with arrestin only in the activated state, leading to the presumption that arrestin binding is necessary for desensitization (2Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 17Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar). In fact, it has become largely accepted that phosphorylation of a GPCR alone is insufficient to block G protein binding. However, evidence in support of this premise is lacking because the effect of phosphorylation on G protein binding to nonvisual GPCRs has not been investigated thoroughly. To explore the effect of FPR phosphorylation on signaling and processing assemblies, we have examined the wild type receptor as well as several phosphorylation-deficient mutants, using both cellular and noncellular approaches. We have examined the ability of these mutants, isolated from both stimulated and nonstimulated cells (i.e.phosphorylated and unphosphorylated states of the FPR), to interact with G protein and arrestin. In vitro spectrofluorometric analysis of solubilized mutant FPR proteins demonstrated that unphosphorylated receptors interacted with G proteins to the same extent as the wild type receptor. Using fluorescence microscopy, we have previously demonstrated that the WT FPR colocalizes with arrestin in response to treatment with agonist (18Bennett 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). Subsequently, utilizing a spectrofluorometric method similar to that used for determining G protein coupling, we have shown that the solubilized phosphorylated FPR specifically binds arrestin-2 and arrestin-3. This study is the first to simultaneously examine the binding of G proteins and arrestins to nonvisual GPCRs with intermediate levels of phosphorylation. Contrary to accepted dogma, our results demonstrate a novel finding suggesting that partial GPCR phosphorylation alone can be sufficient to block G protein binding, thereby desensitizing cellular responses prior to arrestin binding. The generation of FPR mutants and of U937 cells transfected with the FPR have been previously described (19Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. J. 1993; 294: 581-587Crossref PubMed Scopus (42) Google Scholar). Plasticware was obtained from VWR Scientific Company. Chemicals and reagents were obtained from Sigma except where otherwise noted. The cells were grown in tissue culture-treated flasks (Corning) in RPMI 1640 (Hyclone) containing 10% fetal bovine serum (Hyclone), 2 mm l-glutamine, 10 mm HEPES, with 10 units/ml penicillin and 10 μg/ml streptomycin. The cultures were grown at 37 °C with 5% CO2 and passaged from subconfluent cultures every 3–4 days by reseeding at 2 × 105 cells/ml. To expand cells for membrane preparations, 1-liter baffled spinner flasks (Pyrex) were seeded, equilibrated to 5% CO2, then sealed, and incubated at 37 °C. The cells were harvested when densities reached 1 × 109cells/liter. Functional bovine brain G protein was purchased from Calbiochem. Arrestin-2 was expressed in Escherichia coli (strain BL21) and purified by sequential heparin-Sepharose and Q-Sepharose chromatography essentially as described (20Gurevich V.V. Benovic J.L. Methods Enzymol. 2000; 315: 422-437Crossref PubMed Google Scholar). Anti-arrestin-2 and anti-arrestin-3 rabbit polyclonal antisera were generously supplied by Dr. Jeffrey Benovic (Thomas Jefferson University). Texas Red-conjugated goat anti-rabbit antibody was from Vector Laboratories. The cells were harvested, and the preparations to be stimulated were resuspended in phosphate-buffered saline, equilibrated to 37 °C, and then incubated in the presence of 10 μm formyl-Met-Leu-Phe (Sigma) for 8 min. Stimulation at this ligand concentration for this duration results in ∼90% maximal receptor phosphorylation (21Hsu M.H. Chiang S.C. Ye R.D. Prossnitz E.R. J. Biol. Chem. 1997; 272: 29426-29429Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Following stimulation, an equal volume of ice-cold phosphate-buffered saline was added, and tubes were immediately placed on ice. For membrane preparation, all harvested cells were centrifuged at 200 × g for 5 min and resuspended in cavitation buffer (10 nm PIPES, 100 mm KCl, 3 mm NaCl, 3.5 mmMgCl2, 600 μg/ml ATP) at a density of 107cells/ml at 4 °C. The cell suspension was placed in a nitrogen bomb and pressurized to 450 p.s.i. using N2 gas for 20 min at 4 °C. Nuclei and cytoplasmic material were separated by centrifugation at 1000 × g for 5 min at 4 °C. The supernatant containing the membranes was washed twice by centrifugation at 135,000 × g for 30 min at 4 °C, then resuspended in HEPES sucrose buffer (200 mm sucrose, 25 mmHEPES, pH 7), aliquoted, and stored until use at −80 °C. Membranes were thawed and diluted to 1–2 × 108 membrane cell equivalents/ml in binding buffer (30 mm HEPES, 100 mm KCl, 20 mm NaCl, 1 mm EGTA, 0.1% (w/v) bovine serum albumin, 0.5 mm MgCl2). Preparations were maintained at 4 °C throughout the extraction process. The membranes were centrifuged at 135,000 × g for 30 min and resuspended in 150 μl of binding buffer containing protease inhibitor mixture I and 1% n-dodecyl β-d-maltoside (Calbiochem). The preparations were incubated for 60 min at 4 °C with agitation. The insoluble fraction was separated by centrifugation at 70,000 × g for 5 min in a Beckman Airfuge. The supernatant containing the solubilized fraction was removed, and this extract was used for experimentation within 6 h. Solubilized FPR (in 1% n-dodecyl β-d-maltoside prepared as above) was incubated with 1 μm bovine brain G protein or with 6 μmarrestin-2 for 2 h on ice in the presence of 10 nmformyl-Met-Leu-Phe-Lys-FITC (Sigma). Blocked samples were incubated with 1 μm formyl-Met-Leu-Phe-Phe (CBI) for 15 min prior to the addition of fMLFK-FITC. The samples were prepared in small volumes (typically 15 μl) to maximize receptor concentration. The control samples were prepared in the presence of appropriate buffer(s). The G protein buffer contained 50 mm HEPES, 1 mm EDTA, 1 mm dithiothreitol, 0.1% Lubrol (pH 7.6). The arrestin buffer contained 10 mm Tris, 100 mm NaCl, 2 mm EDTA, 2 mm EGTA, 2 mm phenylmethylsulfonyl fluoride, 10 μmleupeptin, 0.7 μg/ml pepstatin A, 10 μm chymostatin (pH 7.5). The reconstitution incubations were carried out in the presence of 0.6–0.7% detergent (final concentration). Fluorescence associated with fMLFK-FITC was measured by a SLM 8000 spectrofluorometer (SLM Instruments, Inc.) using the photon counting mode in acquisition. 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 to the samples during kinetic measurements were performed using 10-μl glass syringes (Hamilton) adding reagents through an injection port on the SLM 8000 above the sample cuvette. Following preparation at 4 °C, the samples were brought up to a volume of 200 μl with binding buffer plus 0.1% n-dodecyl β-d-maltoside (to maintain a detergent concentration slightly above the critical micelle concentration), equilibrated to 22 °C, and placed into the spectrofluorometer with constant stirring. The data were acquired for 100 s in 1-s intervals. Typically, total fluorescence was obtained for the first 10 s, and then an anti-fluorescein antibody was added to the sample. The antibody binds free fMLFK-FITC with high affinity and results in essentially complete quenching of the fluorescence associated with free ligand (22Sklar 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). Thus, the remaining fluorescence represents the receptor-bound ligand. In G protein experiments, GTPγS (100 μm) sensitivity was used to assess the coupling between receptors and G proteins based on characteristic ligand dissociation rates. Membrane preparations were solubilized in detergent as outlined above. The sample was then incubated with 10 μl of anti-Gαi-1,2,3 antibody (Calbiochem) or 10 μl of a polyclonal mixture of anti-arrestin-2 and -3 or both for 45 min on ice with gentle agitation. To remove antibody-substrate conjugates, 100 μg of prehydrated protein A-agarose was added to the sample. After a 30-min incubation, the sample was centrifuged at 14,000 ×g for 30 s, and the supernatant was removed. The protein A-agarose step was then repeated. WT and mutant receptor expressing cells were incubated with 10 nm FMLFK-FITC for 8 min at either 37 or 0 °C. Each of the samples was then fixed with 2% paraformaldehyde for 30 min and permeabilized with 0.02% saponin. Arrestin-2 was detected by incubating cells with anti-arrestin antibody followed by a goat anti-rabbit secondary antibody conjugated to Texas Red. After three washes, the cells were resuspended in Vectashield (Vector Laboratories) and placed on a slide. Fluorescence images were acquired on a Zeiss LSM 510 confocal microscope to localize both the FPR (green) and arrestin-2 (red) in FPR transfected U937 cells. Several FPR phosphorylation-deficient mutants have been previously generated, replacing potential phosphorylation sites (serine or threonine residues) with either alanine or glycine residues (14Maestes 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). Four of these mutant receptors were used in the work presented here. Mutant ΔA (S328A,T329A,T331A,S332G) and mutant ΔB (T334G,T336G,S338G,T339A) represent mutations of two clusters of Ser and Thr residues (the A site and the B site, respectively) that each follow acidic residues. Mutants ΔC (S328A,T329A) and ΔD (T331A,S332G) each add back two potential phosphorylation sites altered in mutant ΔA (Fig. 1). Previous characterization demonstrated that these four mutants induced Ca2+ mobilization and internalized to the same extent as the wild type receptor (14Maestes 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). However, they differ in their levels of phosphorylation and their ability to desensitize (TableI and Ref. 14Maestes 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).Table ICharacterization of mutant receptor responses in whole cell assays as compared to the wild type FPRReceptorPhoshorylationCa2+mobiliziationDesensitizationInternalizationWT++++++++++++++++ΔA++++++−++++ΔB+++++++/−++++ΔC+++++++++++++++ΔD+++++++++++++++ Open table in a new tab To further examine the physical interactions of these mutant receptors, we moved beyond cell-based functional assays to a cell-free reconstitution system using solubilized receptor preparations (23Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar, 24Bennett T.A. Key T.A. Gurevich V.V. Neubig R. Prossnitz E.R. Sklar L.A. J. Biol. Chem. 2001; 276: 22453-22460Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These assays incorporate spectrofluorometric analysis that follows ligand dissociation from receptor complexes in real time, allowing us to determine the state of the FPR and determine the affinity of protein-protein interactions. To assess the effect of receptor phosphorylation with respect to G protein coupling, we first characterized the ability of the mutant receptors to bind purified G protein in their unstimulated (putatively unphosphorylated) state. We will refer to the receptor preparations from unstimulated cells as unphosphorylated and those from the stimulated cells as phosphorylated. Two distinct rates of ligand dissociation can be observed dependent on whether a G protein is bound to the receptor (25Fay S.P. Posner R.G. Swann W.N. Sklar L.A. Biochemistry. 1991; 30: 5066-5075Crossref PubMed Scopus (85) Google Scholar, 26Posner R.G. Fay S.P. Domalewski M.D. Sklar L.A. Mol. Pharmacol. 1994; 45: 65-73PubMed Google Scholar, 27Sklar L.A. Mueller H. Omann G. Oades Z. J. Biol. Chem. 1989; 264: 8483-8486Abstract Full Text PDF PubMed Google Scholar). The receptor-G protein complex has a higher affinity for ligand than receptor alone. Additionally, the high ligand affinity state of the receptor (G protein-coupled) is sensitive to guanine nucleotide (GTPγS), which uncouples the G protein from the receptor. Therefore, the addition of GTPγS results in a rapid change in the observed dissociation rate. Solubilized receptor preparations were incubated with 1 μm bovine brain G protein and 10 nmfMLFK-FITC for 90 min. As seen in Fig. 2(a and b), the phosphorylation-deficient mutant receptors interacted with exogenously added G proteins to the same extent as the wild type receptor. The mutant receptors were also able to form the high affinity complex by reconstituting with endogenous G proteins. An example is shown in Fig. 2c with the ΔB receptor. The ratio of receptors in the high ligand affinity state to those in the low ligand affinity state increased with increased G protein concentration. Thus, the high level of receptor-G protein coupling that was observed in Fig. 2 (a and b) was the result of the combination of endogenous G proteins and the exogenously added bovine brain G protein. Removal of the endogenous G proteins by immunodepletion results in all of the receptors displaying the low ligand affinity state characteristic of the uncoupled WT FPR (Fig. 2d). It has been reported that for rhodopsin and the β2adrenergic receptors, phosphorylation decreases the affinity of the receptor for G protein but is not sufficient to fully desensitize the receptor (15Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (577) Google Scholar, 28Benovic J.L. Kuhn H. Weyand I. Codina J. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8879-8882Crossref PubMed Scopus (372) Google Scholar). To examine the effect of phosphorylation on the FPR-G protein interaction, we produced membrane preparations from cells that had been treated with agonist prior to cavitation. As we have previously demonstrated, this agonist stimulation results in phosphorylation of the FPR (21Hsu M.H. Chiang S.C. Ye R.D. Prossnitz E.R. J. Biol. Chem. 1997; 272: 29426-29429Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). These receptor preparations were immunodepleted of endogenous arrestins, because arrestins also bind to phosphorylated receptors and would compete with G protein coupling. The samples were incubated with up to 3 μm bovine brain G protein and 10 nm fMLFK-FITC for 90 min. The phosphorylated WT receptor was unable to couple to G protein (Fig.3a). In contrast, both the ΔA and ΔB receptors displayed the high affinity receptor state indicative of G protein coupling, and the ligand dissociation rate was sensitive to guanine nucleotide (Fig. 3, b andc). The phosphorylated ΔC and ΔD receptors, however, were indistinguishable from wild type. Incubation of ΔC and ΔD receptors with G protein did not alter the ligand dissociation rate compared with samples incubated with a control buffer (Fig. 3,d and e). The effect of the decreased levels of phosphorylation in the mutant receptors altered only one of the cellular processes previously examined: the ability of the ΔA and ΔB receptors to become desensitized (14Maestes 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). Using confocal microscopy imaging, we have demonstrated that arrestin colocalizes with the active WT FPR in vivo (18Bennett 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). Additionally, purified arrestin was able to bind to the solubilized, phosphorylated WT receptor in our fluorometric assays (42Key T.A. Bennett T.A. Gurevich V.V. Sklar L.A. Prossnitz E.R. J. Biol. Chem. 2001; 276: 49204-49212Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We incorporated both of these approaches to further evaluate the role of phosphorylation in receptor desensitization. First, we examined the ability of arrestin to colocalize with the phosphorylation-deficient mutants using confocal fluorescent microscopy techniques. Cells expressing either WT or mutant receptors were stimulated with a fluorescent agonist to identify and track the FPR as it was processed. Arrestin-2 was detected with an anti-arrestin antibody followed by a secondary antibody conjugated to Texas Red. Both the FPR and arrestin-2 initially appear in a diffuse pattern in the control sample, where cells were treated with the fluorescent agonist but kept at 0 °C (Fig.4). The FPR was concentrated mainly at the cell surface, whereas the arrestin was distributed throughout the cytoplasm. The 0 point control for each of the mutant receptors was identical to the wild type FPR (data not shown). Following an 8-min incubation with the fluorescent agonist at 37 °C, the wild type FPR was localized to punctate clusters. Arrestin also translocated to clusters that colocalized with the FPR. All of the phosphorylation-deficient mutant receptors displayed a similar ability to form similar clusters of the FPR after stimulation with agonist. However, the arrestin distribution varied between these cells. In the ΔA and ΔB cells, each lacking four potential phosphorylation sites, the arrestin did not redistribute but remained dispersed throughout the cytoplasm. The ΔB cells displayed a low level of arrestin colocalization with the receptor, but only at a few of the densest receptor clusters could this be observed (data not shown). The ΔC and ΔD expressing cells, with only two potential phosphorylation sites missing, displayed the same FPR-arrestin colocalization as the wild type receptors. To quantitatively assess the ability of the mutant receptors to interact with arrestin, we utilized the in vitroreconstitution assays using solubilized receptor preparations and purified arrestin-2. Arrestin binding to a phosphorylated receptor induces a high ligand affinity state similar to G protein coupling (29Gurevich 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 (155) Google Scholar). The difference between the two high ligand affinity states is discerned by sensitivity to guanine nucleotide. The slow ligand dissociation from the receptor-arrestin complex is not altered by the addition of GTPγS as it is for receptor-G protein complexes. We have previously demonstrated that solubilized WT receptor preparations from cells that were not stimulated with agonist display no ability to bind arrestin (42Key T.A. Bennett T.A. Gurevich V.V. Sklar L.A. Prossnitz E.R. J. Biol. Chem. 2001; 276: 49204-49212Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This also proved true for each of the mutant receptors. Nonstimulated receptors depleted of G protein and then incubated with up to 40 μm arrestin-2 did not display the high ligand affini
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