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• W2011495264 abstract "In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G protein-coupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, ΔST-FPR, which lacks all phosphorylation sites in the carboxyl-terminal domain. We found that activation of the signaling-competent ΔST-FPR resulted in rapid receptor clustering on the plasma membrane independent of Gi protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization. In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G protein-coupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, ΔST-FPR, which lacks all phosphorylation sites in the carboxyl-terminal domain. We found that activation of the signaling-competent ΔST-FPR resulted in rapid receptor clustering on the plasma membrane independent of Gi protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization. A central question in understanding the process of cellular activation is defining the events that localize and restrict signaling activity at the membrane. This is of particular importance in chemotactic cells where cell polarization dictates the direction of movement. Activation of cell surface receptors initiates cellular signaling pathways that ultimately control cell polarity and migration. Polarization correlates with the redistribution of distinct lipid raft domains and their associated signaling molecules to the leading and trailing edges of the cell (1Gomez-Mouton C. Lacalle R.A. Mira E. Jimenez-Baranda S. Barber D.F. Carrera A.C. Martinez A.C. Manes S. J. Cell Biol. 2004; 164: 759-768Crossref PubMed Scopus (191) Google Scholar). However, at what point and even whether chemotactic receptors translocate into raft domains remain controversial (1Gomez-Mouton C. Lacalle R.A. Mira E. Jimenez-Baranda S. Barber D.F. Carrera A.C. Martinez A.C. Manes S. J. Cell Biol. 2004; 164: 759-768Crossref PubMed Scopus (191) Google Scholar, 2Servant G. Weiner O.D. Neptune E.R. Sedat J.W. Bourne H.R. Mol. Biol. Cell. 1999; 10: 1163-1178Crossref PubMed Scopus (194) Google Scholar). Chemoattractant G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; FPR, N-formyl peptide receptor; β2AR, β2 adrenergic receptor, GFP, green fluorescent protein; MEF, mouse embryonic fibroblast; fMLF, N-formyl-methionyl-leucyl-phenylalanine; FITC, fluorescein isothio-cyanate; t-Boc, t-butoxycarbonyl-phenylalanyl-leucyl-phenylalanyl-leucyl-phenylalanine; MβCD, methyl-β-cyclodextrin; mRFP1, mono-meric red fluorescent protein 1; GM1, Galβ1-3GalNAcβ1-4Gal(3-2αNeuAc)β1-4Glcβ1-1Cer. are seven transmembrane receptors that activate numerous cellular functions in part through the stimulation of heterotrimeric GTP-binding (G) proteins. Agonist binding to the GPCR triggers a conformational change within the receptor that facilitates G protein binding and activation. This binding leads to exchange of GDP for GTP on the Gα subunit of the G protein followed by the dissociation of the α subunit from the dimeric β/γ subunit. These G protein subunits then regulate the activity of multiple cellular effectors such as adenylyl cyclase, phosphatidylinositol 3-kinase, ion channels, and phospholipase C (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar). Within seconds to minutes of agonist activation, the receptor becomes phosphorylated by GPCR kinases; the phosphorylated receptor in turn promotes the binding of arrestins. While four isoforms of arrestin have been identified, only two isoforms, arrestin-2 (β-arrestin) and arrestin-3 (β-arrestin-2), are ubiquitously expressed in mammalian cells (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar). Binding of arrestins to phosphorylated receptors can inhibit further association of G proteins and thereby inactivate classical receptor-mediated signaling, a process termed desensitization (4Key T.A. Bennett T.A. Foutz T.D. 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). In addition, arrestins can function as adapters to mediate receptor internalization via clathrin-coated pits (5Goodman Jr., O.B. 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 (189) Google Scholar) and to recruit effectors such as Src and mitogen-activated protein kinases, resulting in G protein-independent signaling (6Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar, 7Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar). Understanding the functional consequences of ligand-induced GPCR activation, regulation, and trafficking is complicated by the fact that multiple processes occur simultaneously in the cell following agonist stimulation. These include G protein coupling, cellular activation, receptor phosphorylation, binding of arrestins and associated proteins, receptor clustering, internalization, intracellular trafficking, and recycling of receptors to the cell surface. In particular, understanding the role of receptor clustering, phosphorylation, and arrestin binding in receptor dynamics is a distinct challenge. Activation of many receptors, including GPCRs, in the plasma membrane is thought to be regulated by the trafficking of the receptors into or out of discrete detergent-insoluble, glycolipid-rich microdomains termed rafts (8Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 9Igarashi J. Michel T. J. Biol. Chem. 2000; 275: 32363-32370Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 10Lasley R.D. Narayan P. Uittenbogaard A. Smart E.J. J. Biol. Chem. 2000; 275: 4417-4421Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 11Pike L.J. J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 12Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). These rafts are enriched with cholesterol and sphingomyelin, often contain the protein caveolin, and contribute structural dimension to the membrane. Since the plasma membrane is a two-dimensional, fluid phase structure, the membrane domains where receptor clusters form are thought to control membrane organization during receptor internalization (11Pike L.J. J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). Given the heterogeneity among rafts (11Pike L.J. J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar), it is unclear, however, whether receptor clustering may occur solely to initiate internalization of activated receptors or whether it serves a distinct function in signaling by conformationally activated states of the receptor. Recently the roles of arrestins in GPCR function have been more definitively examined through the use of mouse embryonic fibroblasts (MEFs) that are deficient in either one or both arrestins. Signaling and trafficking of numerous GPCRs, including the β2 adrenergic receptor, angiotensin 1A receptor, protease-activated receptor, and N-formyl peptide receptor (FPR) (13Paing M.M. Stutts A.B. Kohout T.A. Lefkowitz R.J. Trejo J. J. Biol. Chem. 2002; 277: 1292-1300Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14Vines C.M. Revankar C.M. Maestas D.C. LaRusch L.L. Cimino D.F. Kohout T.A. Lefkowitz R.J. Prossnitz E.R. J. Biol. Chem. 2003; 278: 41581-41584Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar), have been investigated in these arrestin-deficient cells. Whereas the β2 adrenergic receptor and angiotensin 1A receptor display a dependence on arrestin for receptor internalization, the protease-activated receptor and FPR do not. However, in wild type cells following ligand activation, the FPR colocalizes with arrestins predominantly on intracellular endosomes. Yet until recently a function for arrestin in the trafficking of the FPR has remained elusive (16Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry (Mosc.). 2001; 40: 3467-3475Crossref Scopus (48) Google Scholar). Recent studies by our laboratory have, however, revealed a role for arrestin in the intracellular trafficking and recycling of the FPR (14Vines C.M. Revankar C.M. Maestas D.C. LaRusch L.L. Cimino D.F. Kohout T.A. Lefkowitz R.J. Prossnitz E.R. J. Biol. Chem. 2003; 278: 41581-41584Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In these studies, MEFs derived from knock-out mice that lacked both arrestin-2 and arrestin-3 were found to internalize the FPR but subsequently mistraffic the FPR, leading to a loss of receptor recycling (14Vines C.M. Revankar C.M. Maestas D.C. LaRusch L.L. Cimino D.F. Kohout T.A. Lefkowitz R.J. Prossnitz E.R. J. Biol. Chem. 2003; 278: 41581-41584Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Furthermore a mutant preactivated arrestin, arrestin 3A, which binds to GPCRs in the absence of receptor phosphorylation, was found to inhibit FPR recycling, confirming a role for arrestins in the intracellular trafficking and recycling of GPCRs. 2T. A. Key and E. Prossnitz, submitted for publication. These results lead to the possibility that FPR clustering, a requisite event in receptor internalization, occurs in the absence of arrestin binding and therefore possibly prior to receptor phosphorylation. In the present study, we investigated whether the stimulation of the FPR would promote redistribution of the receptor in the absence of receptor phosphorylation and arrestin binding. To determine whether receptor clustering represents a unique intermediate involved in cellular activation distinct from internalization, we used a non-phosphorylatable mutant FPR (17Prossnitz E.R. J. Biol. Chem. 1997; 272: 15213-15219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). We observed that the FPR was rapidly clustered on the plasma membrane following ligand binding independently of receptor phosphorylation. This clustering required receptor activation as an FPR mutant that binds ligand but is incapable of transitioning to an activated state failed to cluster. Conversely G protein activation was not required to induce clustering since pertussis toxin failed to prevent FPR clustering. Furthermore we demonstrated that receptor clustering and signaling were cholesterol-dependent processes independent of the phosphorylation state of the receptor. In addition, cholesterol depletion inhibited signaling, clustering, and internalization of the wild type FPR. Finally we showed that the FPR colocalized with GM1 glycolipid-rich raft domains following receptor activation. As many signal transduction components, including G proteins and downstream effectors, are known to reside in rafts (11Pike L.J. J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar), we conclude that the FPR in its activated state translocates to signaling domains to effect cell activation prior to receptor phosphorylation and internalization. Reagents—N-Formyl-methionyl-leucyl-phenylalanine (fMLF), hexapeptide (N-formyl-norleucyl-leucyl-phenylalaninyl-norleucinyl-tyrosinyl-lysine, 6-pep), methyl-β-cyclodextrin and water-soluble cholesterol (catalog number C4951) were purchased from Sigma. ALEXA 546-6-pep (18Vines C.M. Xue M. Maestas D.C. Cimino D.F. Prossnitz E.R. J. Immunol. 2002; 169: 6760-6766Crossref PubMed Scopus (16) Google Scholar) and ALEXA 488-GM1 (19Spiegel S. Skutelsky E. Bayer E.A. Wilchek M. Biochim. Biophys. Acta. 1982; 687: 27-34Crossref PubMed Scopus (23) Google Scholar) were synthesized as described previously. 6-pep-FITC was from Molecular Probes. The FPR antagonist t-butoxycarbonyl-phenylalanyl-leucyl-phenylalanyl-leucyl-phenylalanine (t-Boc) was obtained from Bachem Biosciences. Anti-flotillin-1 antibodies (catalog number SC-16642, C-20) were purchased from Santa Cruz Biotechnology. RPMI 1640 medium, fetal bovine serum, tissue culture reagents, and LipofectAMINE 2000 were purchased from Invitrogen. Cell Lines and cDNA—The cDNA encoding the FPR was obtained from a human HL-60 granulocyte library (20Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1991; 179: 471-476Crossref PubMed Scopus (40) Google Scholar). The ΔST-FPR mutant in which all 11 of the serine and threonine residues have been replaced (S319A, T325G, S328A, T329A, T331A, S332G, T334G, T336G, S338G, T339A, and S342G) (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 (60) Google Scholar) and the D71A mutant (22Prossnitz E.R. Schreiber R.E. Bokoch G.M. Ye R.D. J. Biol. Chem. 1995; 270: 10686-10694Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) have been described previously. The D71A/ΔST-FPR chimeric mutant was generated by digesting both the ΔST and D71A FPR plasmids with BclI, ligating the appropriate fragments, and sequencing the resulting construct to ensure the presence of both mutations. Arresin-2 and -3-green fluorescent protein (GFP) were kindly provided by Dr. Jeffrey Benovic (Thomas Jefferson University). Arrestin-3-mRFP1 was generated by substitution of the GFP cDNA in arrestin-3-GFP with a PCR-amplified cDNA of mRFP1 generously provided by Dr. Roger Tsien (23Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (2006) Google Scholar). Plasmid DNA was stably transfected into U937 cells using Effectene (Qiagen). Briefly cells were plated overnight, transfected with 1 μg of plasmid DNA, and then selected for 14–21 days in G418. Surviving cells were pooled, and the expression levels were analyzed by flow cytometric analysis of 10 nm 6-pep-FITC binding. U937 cell lines were maintained at 37 °C in 5% CO2 in RPMI supplemented with 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 mm HEPES (pH 7.4), 10% heat-inactivated fetal bovine serum. Mouse embryonic fibroblasts derived from arrestin-2-/-/arrestin-3-/-mice (MEF arr2-/-/arr3-/-) (15Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar) were a generous gift from Dr. R. Lefkowitz (Howard Hughes Medical Institute and Departments of Medicine and Biochemistry, Duke University Medical Center). MEF arr2-/-/arr3-/-cells were maintained at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 mm HEPES (pH 7.4), 10% fetal bovine serum. Receptor Internalization—To examine the internalization of receptor in response to ligand stimulation, U937 cells stably expressing a wild type or mutant FPR were resuspended in serum-free RPMI and allowed to incubate at 37 °C for 10 min prior to stimulation with ligand. Cells were then stimulated with 1 μm fMLF for the indicated time periods. Plunging the cells in ice cold RPMI and incubation on ice for at least 15 min arrested the internalization. Cells were washed three times in cold Hanks' buffered saline solution to remove uninternalized ligand. Receptors remaining on the cell surface were labeled in the presence of 10 nm 6-pep-FITC and analyzed for fluorescence intensity on a FACScan flow cytometer (BD Biosciences). Only viable cells were included in the assay as determined by a gate on forward and side scatter. Nonspecific binding was determined in the presence of 1 μm fMLF. Receptor internalization was expressed relative to the total number of receptors on untreated cells. Colocalization Studies—U937 cells were transiently transfected by electroporation as follows. U937 cells stably expressing the wild type or a mutant of the FPR were pelleted and resuspended in serum-free RPMI at a concentration of 2 × 107 cells/ml. Cells (8 × 106) were transferred to an electroporation cuvette (0.4-mm gap) and pulsed (200 V/2000 microfarads for an exponential decay (t ½) of ∼50 ms) with 25 μg of DNA. Cells were allowed to recover for 10 min before being transferred to 10 ml of complete RPMI. Following electroporation cells were incubated for 12-16 h at 37 °C in 5% CO2. To examine the response of FPR-expressing U937 cells to stimulation with formyl peptides, cells were stimulated with ALEXA 546-6-pep for the indicated time points and plunged immediately into 1 volume of ice-cold 2% paraformaldehyde for 15 min. Cells were subsequently pelleted and resuspended in 2% paraformaldehyde for an additional 15 min on ice. For immunofluorescence staining following fixation, the cells were stained with anti-flotillin antibodies and rinsed three times in 1× phosphate-buffered saline. The cells were then labeled with a secondary FITC-conjugated anti-rabbit antibody. Cells were pelleted and resuspended in Vectashield, transferred to a glass slide, and examined by confocal microscopy. For MEF cell lines, cells were plated on glass coverslips overnight in complete medium at 37 °C in 5% CO2 and then transiently transfected (LipofectAMINE 2000) with pEGFP (Clontech) fused to the carboxyl terminus of wild type FPR, the ΔST mutant of FPR, the D71A mutant of FPR, or the β2 adrenergic receptor. Plated cells were stimulated with ALEXA 546-6-pep for the indicated time periods, and coverslips were flooded with 1 volume of ice-cold 2% paraformaldehyde. Following incubation of at least 15 min on ice, fresh 2% paraformaldehyde was added for an additional 15 min on ice. Cells were mounted in Vectashield, transferred to a glass slide, and examined by confocal microscopy. Confocal images were acquired at room temperature using a Zeiss LSM510 system equipped with argon and HeNe lasers for excitation at 488 and 543 nm. Samples were viewed with the 40 × 1.3 oil immersion objective lens. Calcium Mobilization—To assess functional signaling, calcium mobilization in response to ligand stimulation was measured as described previously (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 (60) Google Scholar). Briefly cells were harvested by centrifugation, washed once in phosphate-buffered saline, and resuspended at 5 × 106 cells/ml in Hanks' buffered saline solution. The cells were incubated with 5 μm Indo1-AM for 30 min at 37 °C, washed once with Hanks' buffered saline solution, resuspended at 106 cells/ml in Hanks' buffered saline solution, and stored on ice. Cells were allowed to equilibrate at 37 °C for 2 min, stimulated with fMLF, and monitored by continuous fluorescence measurement using a Quantimaster QM 2000-6 spectrofluorometer (Photon Technologies International) detecting at 400 and 490 nm. Cholesterol Depletion/Reconstitution—Cells were incubated in the presence of 10 mm methyl-β-cyclodextrin (MβCD) at 37 °C for 30 min to deplete the cells of cholesterol. To reconstitute cellular cholesterol following cholesterol depletion, cholesterol was restored by incubation with 10 mm water-soluble cholesterol for 30 min at 37 °C. For calcium measurements, cells were loaded with Indo1-AM as described above following treatments with MβCD. Cholesterol-depleted or cholesterol-reconstituted cells were assayed for calcium mobilization as described above. Activation of seven transmembrane receptors is initiated by ligand binding followed by receptor clustering and internalization. As a result of receptor phosphorylation, arrestins bind to receptors and contribute to the termination of the signaling event. It is unclear whether the clustering of GPCRs at sites of internalization is dependent upon arrestin binding or whether receptor clustering takes place prior to the recruitment of arrestins. In the case of the CCR5 receptor (24Fraile-Ramos A. Kohout T.A. Waldhoer M. Marsh M. Traffic. 2003; 4: 243-253Crossref PubMed Scopus (91) Google Scholar) and the well characterized β2 adrenergic receptor arrestin binding to the receptor mediates interactions with internalization machinery (clathrin and the adapter AP-2), suggesting that arrestin is directly involved in the recruitment and clustering of receptors to clathrin-coated pits (25Santini F. Gaidarov I. Keen J.H. J. Cell Biol. 2002; 156: 665-676Crossref PubMed Scopus (93) Google Scholar). Whether clustering of the β2 adrenergic receptor can take place in the absence of arrestins is unknown. However, as the FPR can internalize in the absence of arrestins (14Vines C.M. Revankar C.M. Maestas D.C. LaRusch L.L. Cimino D.F. Kohout T.A. Lefkowitz R.J. Prossnitz E.R. J. Biol. Chem. 2003; 278: 41581-41584Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), it should therefore be capable of clustering in the absence of arrestin binding. To determine whether FPR clustering is a distinct event from receptor internalization and whether the binding of arrestins precedes or follows FPR clustering in cells that express endogenous arrestins, we examined the kinetics of FPR clustering simultaneously with membrane localization of arrestin-2 or arrestin-3 in the U937 promonocytic leukemia cell line. Following incubation with fluorescently labeled ligand at 37 °C, the activated receptors began to form distinct clusters within 20–40 s that persisted as the receptor internalized (Fig. 1, A and B). In contrast, significant colocalization of arrestin did not take place until 3–4 min after receptor stimulation well after the t½ for internalization (∼2 min, Fig. 2). These results suggest that substantial arrestin binding may not occur until more than 50% of the receptor is internalized and that arrestin binding may not be required to promote clustering of the receptor or internalization.Fig. 2Internalization of wild type and mutant forms of the FPR. U937 cells, stably transfected with wild type or mutant FPR (ΔST, D71A, or D71A/ΔST), were incubated in the presence of 1 μm fMLF for the indicated time periods. Following incubation, cells were transferred to ice-cold medium and rinsed three times to remove fMLF remaining on the surface. Receptors remaining on the cell surface were labeled with 6-pep-FITC and quantified by flow cytometry. Data shown are mean ± S.E. of three experiments.View Large Image Figure ViewerDownload (PPT) To determine whether receptor clustering took place in response to receptor phosphorylation, we used a mutant form of the FPR, ΔST-FPR, which lacks all of the serine and threonine residues in the carboxyl-terminal domain that are normally phosphorylated in response to receptor activation. This mutant, when activated, fails to bind arrestins or become desensitized as assessed by calcium mobilization (17Prossnitz E.R. J. Biol. Chem. 1997; 272: 15213-15219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Characterization of this receptor in U937 cells revealed that while the FPR has a t½ for internalization of ∼2 min, the ΔST-FPR failed to internalize (Fig. 2) as we have demonstrated previously (17Prossnitz E.R. J. Biol. Chem. 1997; 272: 15213-15219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Similar to the wild type receptor, the ΔST-FPR mutant receptor formed distinct clusters on the membrane following exposure to ligand for ∼2 min (Fig. 3, A and B). In contrast to the wild type FPR, the ΔST-FPR exhibited no colocalization with arrestin even at extended times. The somewhat slower kinetics of ΔST-FPR clustering as compared with wild type FPR clustering could be the result of wild type receptor phosphorylation, which may serve to accelerate its clustering and internalization. These results suggested that even in the absence of a phosphorylated tail and receptor internalization the FPR is capable of forming clusters within the plasma membrane. We next questioned whether G protein activation was required for clustering of the FPR or whether clustering occurred as a function of receptor activation prior to the activation of G proteins. To this end, we pretreated ΔST-FPR cells with pertussis toxin, which leads to ADP-ribosylation of the Gαi subunit of the heterotrimeric G protein, rendering the G protein unable to interact with and be stimulated by ligand-activated GPCRs. Under these conditions, stimulation of the wild type and ΔST-FPR resulted in receptor clustering (Fig. 4, A and B). The effectiveness of the pertussis toxin in completely blocking fMLF-induced calcium mobilization was determined in parallel (data not shown). Therefore, we concluded that G protein activation and therefore cellular signaling were not required for clustering of the FPR. Since G protein activation and the resulting downstream signaling events are not required to induce receptor clustering, we postulated that the FPR in its ligand-bound, conformationally active state was capable of clustering in the membrane. To determine whether conversion of the FPR to an activated state was required to induce clustering, we examined the clustering of the D71A FPR mutant stably expressed in U937 cells. Asp-71 of the FPR is located in the second transmembrane domain and represents a conserved site within many GPCRs believed to be intimately involved in receptor activation (26Prossnitz E.R. Gilbert T.L. Chiang S. Campbell J.J. Qin S. Newman W. Sklar L.A. Ye R.D. Biochemistry (Mosc.). 1999; 38: 2240-2247Crossref Scopus (40) Google Scholar, 27Bennett 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). While the D71A FPR mutant is capable of binding fMLF, it does not bind to or activate G proteins and does not undergo ligand-mediated phosphorylation, arrestin binding, or internalization (22Prossnitz E.R. Schreiber R.E. Bokoch G.M. Ye R.D. J. Biol. Chem. 1995; 270: 10686-10694Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 26Prossnitz E.R. Gilbert T.L. Chiang S. Campbell J.J. Qin S. Newman W. Sklar L.A. Ye R.D. Biochemistry (Mosc.). 1999; 38: 2240-2247Crossref Scopus (40) Google Scholar, 27Bennett 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). It therefore bears all the hallmarks of a receptor trapped in the inactive conformation. Examination of the membrane distribution of the ligand-bound D71A mutant revealed that the receptor failed to form clusters on the membrane, remaining diffuse over the entire cell membrane (Fig. 4C). This suggested that receptor activation was indeed essential for FPR clustering. It should be noted that there existed some minor heterogeneity i" @default.
• W2011495264 created "2016-06-24" @default.
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• W2011495264 date "2004-10-01" @default.
• W2011495264 modified "2023-10-15" @default.
• W2011495264 title "N-Formyl Peptide Receptors Cluster in an Active Raft-associated State Prior to Phosphorylation" @default.
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