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Phosphorylation of the N-Formyl Peptide Receptor Is Required for Receptor Internalization but Not Chemotaxis

1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês

10.1074/jbc.272.47.29426

1083-351X

Matthew Hsu, Stephanie C. Chiang, Richard D. Ye, Eric R. Prossnitz,

Blood Coagulation and Thrombosis Mechanisms

The human N-formyl peptide receptor (FPR) is a member of the family of leukocyte, G protein-coupled, chemoattractant receptors. To determine the role(s) of receptor phosphorylation in FPR processing and formylmethionylleucylphenylalanine (fMLF)-mediated chemotaxis, we utilized U937 cells expressing the recombinant wild type receptor and a mutant form of the FPR. This mutant, which lacks all of the serine and threonine residues in the C terminus of the receptor, ΔST, has recently been shown to produce a receptor capable of fMLF binding and G protein activation but was demonstrated not to undergo fMLF-dependent phosphorylation or desensitization of the calcium mobilization response upon repeated exposure to agonist (Prossnitz, E. R. (1997) J. Biol. Chem. 272, 15213–15219). In this report, we examined the role of receptor phosphorylation in FPR internalization and leukocyte chemotaxis. Whereas the wild type receptor was rapidly internalized upon stimulation, the phosphorylation-deficient mutant was not, remaining entirely on the cell surface. In addition, contrary to the hypothesis that receptor processing and recycling are required for chemotaxis, we found no defect in the ability of the mutant FPR to migrate up a concentration gradient of fMLF. These results indicate that phosphorylation of the FPR is a necessary step in receptor internalization but that receptor phosphorylation, desensitization, and internalization are not required for chemotaxis. The human N-formyl peptide receptor (FPR) is a member of the family of leukocyte, G protein-coupled, chemoattractant receptors. To determine the role(s) of receptor phosphorylation in FPR processing and formylmethionylleucylphenylalanine (fMLF)-mediated chemotaxis, we utilized U937 cells expressing the recombinant wild type receptor and a mutant form of the FPR. This mutant, which lacks all of the serine and threonine residues in the C terminus of the receptor, ΔST, has recently been shown to produce a receptor capable of fMLF binding and G protein activation but was demonstrated not to undergo fMLF-dependent phosphorylation or desensitization of the calcium mobilization response upon repeated exposure to agonist (Prossnitz, E. R. (1997) J. Biol. Chem. 272, 15213–15219). In this report, we examined the role of receptor phosphorylation in FPR internalization and leukocyte chemotaxis. Whereas the wild type receptor was rapidly internalized upon stimulation, the phosphorylation-deficient mutant was not, remaining entirely on the cell surface. In addition, contrary to the hypothesis that receptor processing and recycling are required for chemotaxis, we found no defect in the ability of the mutant FPR to migrate up a concentration gradient of fMLF. These results indicate that phosphorylation of the FPR is a necessary step in receptor internalization but that receptor phosphorylation, desensitization, and internalization are not required for chemotaxis. Neutrophils normally exist in a resting state as they circulate though the body. However, upon interaction with small molecules known as chemoattractants, they rapidly respond with endothelial adhesion followed by emigration from the vasculature and chemotaxis to the site of inflammation (1Zigmond S.H. Curr. Opin. Cell Biol. 1989; 1: 80-86Crossref PubMed Scopus (28) Google Scholar). Chemoattractants activate neutrophils through binding to heptahelical receptors located on the cell surface (2Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1130) Google Scholar, 3Ye R.D. Boulay F. Adv. Pharmacol. 1997; 39: 221-289Crossref PubMed Scopus (105) Google Scholar). These receptors activate heterotrimeric GTP-binding proteins (G proteins) 1The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; FPR, N-formyl peptide receptor; Nle, norleucine; fMLF,N-formyl-methionyl-leucyl-phenylalanine; HBSS, Hanks' buffered saline solution. that initiate numerous elaborate signal transduction cascades, culminating in neutrophil migration and activation. Once at the site of inflammation, neutrophils respond with phagocytosis, superoxide generation, and the release of degradative enzymes (4Snyderman R. Uhing R.J. Gallin J.I. Goldstein I.M. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 2nd Ed. Raven Press, New York1992: 421-439Google Scholar). One of the most thoroughly studied chemoattractant receptors is the N-formyl peptide receptor (FPR), which recognizes short N-formylated oligopeptides of bacterial or mitochondrial origin (5Boulay F. Tardif M. Brouchon L. Vignais P. Biochem. Biophys. Res. Commun. 1990; 168: 1103-1109Crossref PubMed Scopus (202) Google Scholar, 6Prossnitz E.R. Ye R.D. Pharmacol. Ther. 1997; 74: 73-102Crossref PubMed Scopus (229) Google Scholar, 7Schiffmann E. Showell H.V. Corcoran B.A. Ward P.A. Smith E. Becker E.L. J. Immunol. 1975; 114: 1831-1837PubMed Google Scholar). Leukocyte chemotaxis has been shown to be dependent on the binding of chemoattractants to their respective receptors (8Zigmond S.H. Am. J. Respir. Cell Mol. Biol. 1989; 1: 451-453Crossref PubMed Scopus (24) Google Scholar, 9Kew R.R. Peng T. DiMartino S.J. Madhavan D. Weinman S.J. Cheng D. Prossnitz E.R. J. Leukocyte Biol. 1997; 61: 329-337Crossref PubMed Scopus (61) Google Scholar). Following binding of the ligand and cellular activation, the receptors undergo desensitization and internalization (10van Epps D.E. Simpson S. Bender J.G. Chenoweth D.E. J. Immunol. 1990; 144: 1062-1068PubMed Google Scholar, 11Wilde M.W. Carlson K.E. Manning D.R. Zigmond S.H. J. Biol. Chem. 1989; 264: 190-196Abstract Full Text PDF PubMed Google Scholar). Once internalized, ligand dissociates from the receptor and is degraded, whereupon the receptor is recycled to the cell surface for additional rounds of activation (10van Epps D.E. Simpson S. Bender J.G. Chenoweth D.E. J. Immunol. 1990; 144: 1062-1068PubMed Google Scholar). Receptor recycling has been suggested to be essential for sustained cellular responses, such as cell chemotaxis (12Perez H.D. Elfman F. Marder S. Lobo E. Ives H.E. J. Clin. Invest. 1989; 83: 1963-1970Crossref PubMed Scopus (31) Google Scholar, 13Perez H.D. Ong R. Khanna K. Banda D. Goldstein I.M. J. Immunol. 1982; 129: 2718-2724PubMed Google Scholar). Inhibition of receptor recycling through exposure to wheat germ agglutinin or by neuraminidase treatment was found to block chemotaxis without affecting receptor-mediated superoxide generation or degranulation. Receptor endocytosis was also demonstrated to proceed normally with the internalized receptor accumulating within the cell and not being re-expressed to the cell surface (12Perez H.D. Elfman F. Marder S. Lobo E. Ives H.E. J. Clin. Invest. 1989; 83: 1963-1970Crossref PubMed Scopus (31) Google Scholar, 13Perez H.D. Ong R. Khanna K. Banda D. Goldstein I.M. J. Immunol. 1982; 129: 2718-2724PubMed Google Scholar). A role for neutral endopeptidase has also been implicated through the use of the inhibitor phosphoramidon (14Painter R.G. Dukes R. Sullivan J. Carter R. Erdos E.G. Johnson A.R. J. Biol. Chem. 1988; 263: 9456-9461Abstract Full Text PDF PubMed Google Scholar). Treatment with this inhibitor blocked degradation of the internalized ligand as well as re-expression of the internalized receptor, suggesting that dissociation of the ligand from the receptor and its subsequent hydrolysis are essential for receptor recycling (15Painter R.G. Aiken M.L. J. Leukocyte Biol. 1995; 58: 468-476Crossref PubMed Scopus (5) Google Scholar). In fact, degradation of internalized ligand was shown to occur at a rate proportional to receptor re-expression, suggesting that the former process may be rate-limiting. Inhibition of receptor recycling through this method specifically blocked chemotaxis but not other neutrophil responses, providing further support for the conclusion that receptor recycling is required for chemotaxis (15Painter R.G. Aiken M.L. J. Leukocyte Biol. 1995; 58: 468-476Crossref PubMed Scopus (5) Google Scholar). We have recently shown that phosphorylation of the FPR is an essential step in the functional desensitization of the receptor. In this report, we investigated the role of receptor phosphorylation in the internalization process as a means to examine the role of receptor processing in chemotaxis. Our results demonstrate that although receptor phosphorylation is absolutely essential to receptor desensitization and internalization, neither phosphorylation nor receptor internalization is required for cell chemotaxis. The cDNA encoding the FPR was obtained from a human HL-60 granulocyte library as described previously (16Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1991; 179: 471-476Crossref PubMed Scopus (40) Google Scholar).N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and indo-1AM were obtained from Molecular Probes. fMLF was purchased from Sigma. Carrier-free, acid-free [32P]orthophosphate was from Amersham Corp. Protein A-Sepharose CL-4B beads were obtained from Pharmacia Biotech Inc. Chemotaxis chambers (48-well) were from Neuroprobe with cellulose nitrate filters from Toyo. RPMI was from Whittaker Bioproducts; fetal bovine serum was from HyClone. The FPR cDNA was subcloned and mutagenized as described (16Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1991; 179: 471-476Crossref PubMed Scopus (40) Google Scholar, 17Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. J. 1993; 294: 581-587Crossref PubMed Scopus (42) Google Scholar). U937 cells were grown in RPMI 1640 supplemented with 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 mm HEPES (pH 7.4), and 10% heat-inactivated fetal bovine serum. For transfection, approximately 4 × 106 cells were harvested and resuspended in 400 μl of RPMI 1640 containing 10 mm glucose and 0.1 mm dithiothreitol (18Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. J. Immunol. 1993; 151: 5704-5715PubMed Google Scholar). Linearized DNA (10 μg in a volume of 10 μl) was added to the cells and preincubated for 5 min at room temperature. The cells were then subjected to a 240-V pulse from a 960-microfarad capacitor (resulting in a pulse time constant of approximately 30 ms) and immediately returned to 5–10 ml of culture medium. The following day, G418 was added to a final active concentration of 1 mg/ml. As the selection proceeded, the cells were centrifuged and resuspended in fresh medium (containing G418) at 4–6-day intervals. Cells were cultured at 37 °C in a humidified atmosphere of 6% CO2 and 94% air. Phosphorylation of the FPR was determined as described (19Prossnitz E.R. J. Biol. Chem. 1997; 272: 15213-15219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, FPR-transfected U937 cells were harvested and washed extensively to remove traces of phosphate. Cells were resuspended in phosphate-free RPMI 1640 containing 1 mCi of carrier-free, acid-free [32P]orthophosphate (10 mCi/ml). Cells were loaded for 3 h at 37 °C and subsequently stimulated with fMLF for 10 min at 37 °C. Cells were lysed by the addition of 0.33 volume of 4 × radioimmune precipitation buffer (40 mm Tris-HCl, pH 7.5, 600 mm NaCl, 4 mm EDTA, 0.4% SDS, 2% deoxycholate, 4% Triton X-100, 4 mm p-nitrophenyl phosphate, 40 mmsodium phosphate, 40 mm NaF, 20 μg/ml soybean trypsin inhibitor, 20 μg/ml leupeptin, 2 mm phenylmethylsulfonyl fluoride, 400 ng/ml aprotinin, and 200 μg/ml pepstatin A). Following lysis, extraction, and removal of insoluble debris, the supernatant was added to 10 mg of Protein A-Sepharose, which had been precoated with 15 μl of a rabbit antiserum directed against the C-terminal 12 amino acids of the FPR, and incubated for 1 h while rotating at 4 °C. The beads were then washed as follows: once with 1 ml of 50 mm Tris-HCl, 500 mm NaCl, 1% Triton X-100, 0.2% SDS, pH 8.0; once with 1 ml of 50 mm Tris-HCl, 500 mm NaCl, 1% Triton X-100, 0.1% SDS, pH 8.0; once with 1 ml of 50 mm Tris-HCl, 500 mm NaCl, pH 8.0; and finally with phosphate-buffered saline. Laemmli sample buffer was added, and the samples were heated at 37 °C for 10 min, followed by electrophoresis on a 12.5% SDS-polyacrylamide gel. Gels were dried, and 32P content was determined with a Molecular Dynamics PhosphorImager. To assess desensitization, the ability of the cells to respond to ligand with a calcium mobilization response was monitored. For calcium determinations, cells were harvested by centrifugation, washed once with phosphate-buffered saline, and resuspended at 5 × 106 cells/ml in Hanks' buffered saline solution (HBSS). The cells were incubated with 5 μm indo-1AM for 25 min at 37 °C, washed once with HBSS, and resuspended to a concentration of approximately 106 cells/ml in HBSS containing 1.5 mm EGTA, pH 8.0. The elevation of intracellular Ca2+ by fMLF was monitored by continuous fluorescence measurement using an SLM 8000 photon-counting spectrofluorometer (SLM-Aminco) detecting at 400 and 490 nm, respectively, as described (16Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1991; 179: 471-476Crossref PubMed Scopus (40) Google Scholar). The concentration of intracellular Ca2+ was calculated as described (20Cobbold P.H. Rink T.J. Biochem. J. 1987; 248: 313-328Crossref PubMed Scopus (425) Google Scholar). For desensitization determinations, cells were first pretreated with 1 μm fMLF, and the response was recorded. The stimulated cells were then removed from the cuvette, washed three times with HBSS at room temperature to remove surface-bound fMLF, and replaced in the cuvette. The response of the washed cells to a second stimulation with 1 μm fMLF was then determined. Receptor internalization was first determined as the loss of FPR from the cell surface as follows. FPR-transfected U937 cells were harvested, washed, and resuspended in HBSS. Cells were then stimulated with 1 μm fMLF for 10 min at 37 °C and washed three times with HBSS. Remaining cell surface receptors were determined with 10 nm N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein. Following incubation for at least 15 min on ice, cells were analyzed for fluorescent intensity on a FACScan flow cytometer (Becton Dickinson) with dead cells excluded by a gate on forward and side scatter. Nonspecific binding was determined in the presence of 1 μm N-formyl-Met-Leu-Phe. Receptor internalization was determined relative to cells that had not been treated with fMLF. Internalization was also evaluated as the intracellular accumulation of fML[3H]F as follows. FPR-transfected U937 cells were harvested, washed, and resuspended in HBSS. Cells were incubated with the indicated concentration of fML[3H]F at 37 °C for 60 min. Control cells were incubated on ice (which prevents endocytosis) or with excess unlabeled fMLF to determine nonspecific binding and internalization. After incubation, cell samples were added to 10 volumes of 0.2 m glycine (pH 3.0) containing 0.5m NaCl for 5 min on ice. This incubation removes ligand bound to the cell surface but has no effect on internalized ligand. Cell samples added to 10 volumes of cold Hanks' buffer (as opposed to pH 3.0 glycine) provided total fML[3H]F associated with the cell, both internal and external. Free ligand was separated from cell-associated ligand by rapid filtration through glass fiber filters followed by three washes with cold dilution buffer. Results are expressed as the percentage of saturably bound fML[3H]F that is internalized and normalized to the amount internalized by the wild type FPR. Cell migration was quantitated with a 48-well chemotaxis chamber (Neuroprobe) using 5.0- or 8.0-μm pore size cellulose nitrate filters (9Kew R.R. Peng T. DiMartino S.J. Madhavan D. Weinman S.J. Cheng D. Prossnitz E.R. J. Leukocyte Biol. 1997; 61: 329-337Crossref PubMed Scopus (61) Google Scholar). Cells and fMLF were prepared in HBSS supplemented with 10 mm HEPES and 1% bovine serum albumin. Chemoattractant was placed in the lower chamber and covered with the cellulose nitrate filter. Cells (4 × 106/ml) were placed in the upper chamber and incubated at 37 °C for 2 h. Following the incubation, the filter was fixed with isopropyl alcohol, stained with hematoxylin, and mounted on a microscope slide. The distance migrated by the cells (in μm) was determined by the leading edge technique (21Zigmond S.H. Hirsch J.G. J. Exp. Med. 1973; 137: 387-410Crossref PubMed Scopus (1280) Google Scholar). For each evaluation, five fields per duplicate filter were measured at 400-fold magnification. The data are presented as the distance migrated by the leading front of the cells in a 2-h span. It has been well established that G protein-coupled chemoattractant receptors mediate leukocyte chemotaxis (22Snyderman R. Smith C.D. Verghese M.W. J. Leukocyte Biol. 1986; 40: 785-800Crossref PubMed Scopus (131) Google Scholar). The signal transduction cascade initiated by these receptors has also been shown to be essential since treatment of cells with pertussis toxin, which blocks the interaction of Gi proteins with receptors, completely abolishes leukocyte chemotaxis (23Lad P.M. Olson C.V. Grewal I.S. FEBS Lett. 1986; 200: 91-96Crossref PubMed Scopus (24) Google Scholar, 24Becker E.L. Kermode J.C. Naccache P.H. Yassin R. Marsh M.L. Munoz J.J. Sha'afi R.I. J. Cell Biol. 1985; 100: 1641-1646Crossref PubMed Scopus (81) Google Scholar, 25Spangrude G.J. Sacchi F. Hill H.R. Van Epps D.E. Daynes R.A. J. Immunol. 1985; 135: 4135-4143PubMed Google Scholar). Furthermore, actin polymerization has been demonstrated to be essential, since cytochalasins, which block actin polymerization, also abolish chemotaxis (26Pham Huu T. Biomedicine (Paris). 1979; 30: 121-124PubMed Google Scholar, 27Becker E.L. Davis A.T. Estensen R.D. Quie P.G. J. Immunol. 1972; 108: 396-402PubMed Google Scholar). Much of the signaling between these two events, however, remains unclear. We have sought to determine the role of receptor processing in these events. Previous experiments have suggested that recycling of chemoattractant receptors is essential for chemotaxis to take place. This conclusion was based on a number of experiments, which correlated a block in the re-expression of internalized chemoattractant receptors with a lack of chemotactic ability despite normal ligand binding, cell activation of superoxide generation and degranulation, and normal receptor internalization (15Painter R.G. Aiken M.L. J. Leukocyte Biol. 1995; 58: 468-476Crossref PubMed Scopus (5) Google Scholar, 28Perez H.D. Elfman F. Lobo E. Sklar L. Chenoweth D. Hooper C. J. Immunol. 1986; 136: 1803-1812PubMed Google Scholar). In this study, we have used a unique approach to investigate the role of receptor processing in chemotaxis. We have previously demonstrated that U937 myeloid cells stably transfected with chemoattractant receptors such as the FPR are capable of migrating up a chemotactic gradient (9Kew R.R. Peng T. DiMartino S.J. Madhavan D. Weinman S.J. Cheng D. Prossnitz E.R. J. Leukocyte Biol. 1997; 61: 329-337Crossref PubMed Scopus (61) Google Scholar). This chemotactic response is indistinguishable from the response observed with differentiated U937 cells or neutrophils. To evaluate the role of receptor processing, we compared U937 cells transfected with the wild type FPR to U937 cells transfected with a mutant form of the FPR, ΔST, in which all of the serine and threonine residues in the C terminus have been converted to alanines and glycines. Fig. 1 demonstrates that, whereas the wild type FPR becomes phosphorylated upon stimulation with fMLF, the ΔST mutant does not. That the mutant form of the FPR was capable of responding to fMLF was demonstrated by monitoring the mobilization of intracellular calcium. Upon fMLF stimulation, both the wild type and ΔST mutant underwent similar degrees of calcium mobilization (Fig. 2). This indicated that in the absence of receptor phosphorylation, ligand binding and G protein activation remained intact. We examined desensitization of the fMLF-initiated calcium mobilization response by taking cells that had been exposed to a saturating concentration of fMLF yielding a maximal calcium mobilization response, washing them extensively to remove bound ligand, and determining their responsiveness to a second exposure to fMLF. As expected for the wild type FPR, the amount of calcium mobilization following a second exposure to agonist was minimal (Fig.2). However, exposing fMLF-treated ΔST cells to a second dose of ligand yielded a calcium response of the same magnitude as that observed for the first exposure. These results confirm that receptor phosphorylation is an essential step in the functional desensitization of the receptor-mediated response.Figure 2Desensitization of calcium mobilization of the wild type FPR and ΔST mutant. FPR desensitization was determined as the decrease in fMLF-stimulated elevation of intracellular calcium in response to a second exposure to fMLF. Cells expressing the wild type (WT) and ΔST mutant form of the FPR were loaded with indo-1AM and stimulated (pretreated) with 1 μm fMLF. The cells were then washed extensively to remove bound ligand and re-assessed for calcium mobilization in response to a second stimulation with 1 μm fMLF. Data represent mean ± S.E. of four experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine what mechanism may underlie this phenomenon, we next evaluated the ability of the receptor to undergo ligand-stimulated internalization. Receptor internalization provides possible mechanisms for desensitization by removing occupied receptors from the cell surface to intracellular endosomes. We initially evaluated receptor endocytosis by determining the amount of receptor remaining on the cell surface following a period of exposure to fMLF. Cell surface receptors were quantitated by flow cytometry usingN-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein as a specific probe for cell surface-localized FPR. When U937 cells transfected with the wild type FPR were exposed to fMLF for 10 min at 37 °C and assayed for cell surface receptors, they exhibited a 75% decrease in the number of cell surface receptors compared with cells that had not been exposed to fMLF (Fig.3 A). However, when the same comparison was made with U937 cells expressing the ΔST FPR mutant, which express almost equal numbers of receptors, there was no decrease in the number of cell surface receptors following a pretreatment with fMLF. To confirm that the decrease in cell surface receptors was the result of ligand-mediated receptor internalization, we also measured uptake of tritiated ligand. Increasing concentrations of fML[3H]F were incubated with wild type and mutant cells for 1 h at 37 °C. The cells were then transferred to a low pH buffer, which causes dissociation of ligand bound to the cell surface but has no effect on internalized ligand. Wild type FPR-transfected U937 cells demonstrated significant uptake of fML[3H]F during the course of the assay (Fig. 3 B). On the contrary, cells expressing the ΔST mutant internalized almost no ligand, even at fMLF concentrations 10-fold higher than that required to demonstrate significant uptake with the wild type receptor. These results suggest that receptor phosphorylation is required for internalization as well as desensitization of the FPR and that the two processes may be interdependent. Having defined a form of the FPR that is capable of binding ligand and initiating signal transduction but incapable of being internalized and thus recycled to the cell surface, we were now able to test the hypothesis that chemotaxis requires recycling of the receptor. Chemotaxis was evaluated using a 48-well chemotaxis chamber with the lower chamber containing chemoattractant separated from the upper chamber containing cells by a 120-μm thick convoluted pore cellulose nitrate filter. This method allows the distinction to be made between simple migration though an "open hole" thin polycarbonate filter and true chemotaxis as revealed with this method. Only myeloid cells are capable of migrating through thick convoluted pore filters whereas many cell types including endothelial cells and fibroblasts, for example, can traverse straight open hole pore filters. The ability of the FPR-transfected U937 cells to undergo chemotaxis in response to a gradient is demonstrated in Table I. Only in the presence of a ligand gradient, with the higher concentration of ligand in the lower chamber, was significant migration into the filter observed. The pattern of migration is consistent with the cells responding in a chemotactic manner, as opposed to a chemokinetic manner, where the presence of ligand in the upper chamber (or both chambers) induces increased random movement of the cells and therefore migration into the filter. Chemotaxis of undifferentiated U937 cells is dependent only upon the introduction of a chemoattractant receptor, such as the FPR, with vector-transfected cells showing no response (Fig. 4). When we tested the ΔST FPR mutant, we found that the chemotactic potential of this mutant was identical to that of the wild type receptor, demonstrating that receptor processing and recycling are not required for chemotaxis to take place.Table ICheckerboard analysis of FPR-transfected U937 cell chemotactic activityfMLF in lower chamberfMLF in upper chamber01 nm10 nm100 nm000001 nm12010010 nm46321110100 nm5339220Data are expressed as the distance of migration (in microns) in a 2-h period as described under "Experimental Procedures." Values are means of five measurements on each of duplicate filters and are representative of three experiments. Open table in a new tab Data are expressed as the distance of migration (in microns) in a 2-h period as described under "Experimental Procedures." Values are means of five measurements on each of duplicate filters and are representative of three experiments. The results of this study have demonstrated for the first time that receptor phosphorylation, internalization, desensitization, and recycling are not required for chemotaxis to occur. Our result is contrary to the conclusions based on inhibitors of receptor recycling, which result in inhibition of neutrophil chemotaxis. It is unclear, however, what additional side effects these inhibitors might have upon cell function. Furthermore, it is possible that only in the presence of internalized receptor is receptor re-expression required for chemotaxis to occur. This would indicate that under normal circumstances, replenishing of cell surface receptors is essential to provide the receptors necessary to propagate migration; however, as our results demonstrate, in the absence of receptor depletion through internalization, chemotaxis can be initiated and continue for a prolonged period of time. This demonstrates that signal transduction mediated by a functional chemoattractant receptor in the absence of receptor desensitization can control the spatial and temporal aspects of the signal transduction cascade that are involved in the remodeling of the actin cytoskeleton that propels the cell forward during chemotaxis. Although receptor desensitization and internalization are not required for chemotaxis, these processes are involved in preventing chronic activation of leukocytes at sites of inflammation. To compensate for the possible lack of receptors during the relatively long periods of chemotaxis, it appears that re-expression of internalized receptors has evolved as a mechanism to ensure sufficient cell surface receptors. In conclusion, although receptor recycling occurs during chemotaxis, it is not an essential component of the chemotactic phenomenon. We thank Dr. Richard Kew for expertise in analyzing chemotaxis results.