Dissociation of Chemotaxis from Agonist-induced Receptor Internalization in a Lymphocyte Cell Line Transfected with CCR2B

To investigate the role of the carboxyl-terminal region (52 amino acids) of the monocyte chemoattractant protein 1 receptor (CCR2B) in chemotaxis, we created a series of mutants and expressed them in a murine pre-B lymphocyte cell line. Truncation of the cytoplasmic carboxyl tail to 20 amino acids had little or no effect on chemotaxis or signal transduction, but further truncation resulted in marked functional defects. Upon incubation with monocyte chemoattractant protein 1, CCR2B underwent rapid and extensive internalization, and this was impaired progressively as the carboxyl tail was truncated from 52 to 8 amino acids. Mutation of all of the serine and threonine residues in the carboxyl tail to alanine also resulted in markedly impaired receptor internalization but did not affect signaling or chemotaxis. We conclude that the membrane-proximal portion of the cytoplasmic carboxyl tail of CCR2B is critically involved in chemotaxis and signal transduction, but neither phosphorylation of carboxyl serines or threonines nor internalization of the receptor is required for robust chemotaxis. To investigate the role of the carboxyl-terminal region (52 amino acids) of the monocyte chemoattractant protein 1 receptor (CCR2B) in chemotaxis, we created a series of mutants and expressed them in a murine pre-B lymphocyte cell line. Truncation of the cytoplasmic carboxyl tail to 20 amino acids had little or no effect on chemotaxis or signal transduction, but further truncation resulted in marked functional defects. Upon incubation with monocyte chemoattractant protein 1, CCR2B underwent rapid and extensive internalization, and this was impaired progressively as the carboxyl tail was truncated from 52 to 8 amino acids. Mutation of all of the serine and threonine residues in the carboxyl tail to alanine also resulted in markedly impaired receptor internalization but did not affect signaling or chemotaxis. We conclude that the membrane-proximal portion of the cytoplasmic carboxyl tail of CCR2B is critically involved in chemotaxis and signal transduction, but neither phosphorylation of carboxyl serines or threonines nor internalization of the receptor is required for robust chemotaxis. Chemokines (chemotactic cytokines) are small, basic peptides that induce directed migration in leukocytes (for reviews, see Refs. 1Oppenheim J.J. Zachariae C.O.C. Mukaida N. Matsushima K. Annu. Rev. Immunol. 1991; 9: 617-648Crossref PubMed Scopus (1831) Google Scholar, 2Rollins B.J. Cancer Cells. 1991; 3: 517-524PubMed Google Scholar, 3Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1130) Google Scholar, 4Schall T.J. Thomson A.W. The Cytokine Handbook. 2nd Ed. Academic Press, London1994: 419-460Google Scholar). The chemokines can be subdivided into two families, based on the positions of the first two cysteines and on the quaternary structure of chemokine homodimers (5Lodi P.J. Garrett D.S. Kuszewski J. Tsang M.L.-S. Weatherbee J.A. Leonard W.J. Gronenborn A.M. Clore G.M. Science. 1994; 263: 1762-1767Crossref PubMed Scopus (211) Google Scholar, 6Clore G.M. Gronenborn A.M. FASEB J. 1995; 9: 57-62Crossref PubMed Scopus (119) Google Scholar). Monocyte chemoattractant 1 (MCP-1) 1The abbreviations used are: MCP-1, monocyte chemoattractant protein 1; CCR, C-C chemokine receptor; HEK, human embryo kidney; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase. is a member of the C-C or β chemokine family, in which the first two cysteines are adjacent, and is a potent chemoattractant for monocytes, basophils, and memory T cells. Chemokine receptors are members of the seven-transmembrane domain family of receptors and mediate leukocyte activation by coupling to G-proteins. We have shown previously that CCR2A and CCR2B, the chemokine receptors for MCP-1, signal through multiple G-proteins, including Gαi, Gαq, and Gα16 (7Arai H. Charo I.F. J. Biol. Chem. 1996; 271: 21814-21819Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In contrast to other seven-transmembrane domain receptors in which an extended third intracellular loop (50–75 amino acids long) interacts directly with G-proteins, the chemokine receptors are characterized by extremely short third loops (12 amino acids in the case of CCR2), and thus other portions of these receptors, such as the carboxyl-terminal domain, may be critically involved in signal transduction (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar). In the case of CCR2, phosphorylation of serine and threonine residues in the carboxyl tail has also been shown to mediate rapid receptor deactivation (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar). Chemotaxis is the prototypic function of the chemokines, but the signal transduction pathways utilized to initiate directed cell migration are not well understood. Moreover, accurate tracking of changing chemokine gradients may require rapid modulation of signaling at the level of the receptor. In the present study, we have created a series of CCR2B mutants to test the hypothesis that receptor deactivation is required for chemotaxis. MCP-1 was obtained from R 22: 2587-2591Crossref PubMed Scopus (216) Google Scholar). Downstream primers were designed to introduce a stop codon as well as an NotI restriction site. An upstream primer was located at ∼10 base pairs 5′ to an internal EcoRI site in CCR2B. Mutation of serine/threonine residues to alanine was also done by polymerase chain reaction as described (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar). All constructs incorporated the Flag epitope and prolactin signal sequence at their 5′ end as described previously (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar), were subcloned into the vector pCMV-1 (10Andersson S. Davis D.L. Dahlbäck H. Jörnvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar), and were sequenced completely before use. Human embryo kidney (HEK)-293 cells were obtained from the American Type Tissue Culture Collection (CRL 1573, Bethesda, MD) and were grown in minimal essential medium with Earle's balanced salt solution supplemented with 10% fetal calf serum, streptomycin (100 μg/ml), and penicillin (100 IU/ml) at 37 °C in 5% CO2. cDNAs were transfected using LipofectAMINE according to the manufacturer's instructions, and stable cell lines were obtained by selection in G418. 300-19 pre-B cells (11Reth M.G. Ammirati P. Jackson S. Alt F.W. Nature. 1985; 317: 353-355Crossref PubMed Scopus (108) Google Scholar) were a generous gift from Dr. G. La Rosa (LeukoSite, Cambridge, MA) and were cultured in RPMI 1640 supplemented with 10% fetal calf serum, streptomycin (100 μg/ml), penicillin (100 IU/ml), glutamine (2 mm), and β-mercaptoethanol (55 μm). Transfection of 300-19 cells was done by electroporation (Bio-Rad). Briefly, 2 × 106 cells in 400 μl of medium were mixed with 30 μg of the cDNAs in expression vectors and 1.5 μg of a plasmid that conferred resistance to neomycin (pSV2-neo) and were maintained at 4 °C for 15 min. Electroporation was performed at 250 V and 960 microfarads. After an overnight incubation, positive clones were selected in the presence of 800 μg/ml G418. A pool of G418-resistant cells was analyzed for cell surface expression of receptors by a FACS. Approximately 3 × 106 harvested cells were incubated at room temperature for 1 h with culture medium containing phycoerythrin-conjugated M1 antibody. Unbound antibody was removed by washing with phosphate-buffered saline (PBS), and the cells were resuspended in PBS plus 20 μg/ml propidium iodide. MCP-1 (5 μg) was labeled using the Bolton-Hunter reagent (diiodide, NEN Life Science Products) as described previously (12Monteclaro F.S. Charo I.F. J. Biol. Chem. 1996; 271: 19084-19092Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Unconjugated iodide was separated from labeled protein by elution through a PD-10 column (Pharmacia Biotech Inc.) equilibrated with PBS and bovine serum albumin (BSA) (1% w/v). Specific activity was determined by immunoassay (Quantikine; R 271: 19084-19092Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Briefly, 125I-labeled ligand, with or without a 100-fold excess of unlabeled ligand, was added to 0.5 × 106cells in polypropylene tubes in a total volume of 300 μl (50 mm HEPES, pH 7.4, 1.0 mm CaCl2, 5.0 mm MgCl2, 0.5% BSA) and incubated for 90 min at 27 °C on an orbital shaker set at 150 rpm. The cells were collected on glass-fiber filters presoaked in 0.3% polyethylimine and 0.2% BSA with a Skatron cell harvester (Skatron Instruments, Sterling, VA). Unbound ligand was removed by washing with 4 ml of buffer (10 mm HEPES, pH 7.4, 500 mm NaCl, 0.5% BSA) for 10 s. Competition with unlabeled ligand was determined by incubation of 0.5 × 106 transfected cells (as above) with 1.5 nm radiolabeled ligand in a final volume of 300 μl. The samples were collected, washed, and counted as above. The data were analyzed with the curve-fitting program Prism (GraphPad, San Diego) and the iterative nonlinear regression program LIGAND. Agonist-dependent increases in cytoplasmic Ca2+ were determined in transfected 300-19 cells as described previously (13Myers S.J. Wong L.M. Charo I.F. J. Biol. Chem. 1995; 270: 5786-5792Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). HEK-293 cells stably transfected with wild-type and truncated CCR2B were grown until subconfluent in 12-well dishes and labeled overnight with 2 μCi/ml 3Hadenine (25–30 Ci/mmol) in minimal essential medium with 10% fetal calf serum. The next day, the cells were washed with serum-free medium supplemented with 1 mg/ml BSA and 10 mmHEPES. After removal of the washing medium, the cells were stimulated by the addition of fresh medium containing 50 μmforskolin and the indicated concentrations of MCP-1 in the presence of 1 mm 3-isobutyl-1-methylxanthine (Sigma) for 20 min at room temperature. 3HcAMP and 3HATP levels were quantitated by chromatography on Dowex 50W and neutral alumina columns as described (13Myers S.J. Wong L.M. Charo I.F. J. Biol. Chem. 1995; 270: 5786-5792Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Measurement of ERK activation was performed as described previously (14Arai H. Escobedo J.A. Mol. Pharmacol. 1996; 50: 522-528PubMed Google Scholar). Briefly, 1 × 107 300-19 cells stably transfected with wild-type or mutant CCR2B were maintained in the absence of serum for 24 h and then activated by incubation with 10 nm MCP-1 for 5 min. After two washes with ice-cold PBS containing 1 mm sodium orthovanadate, the cells were lysed in 500 μl of ice-cold buffer (20 mm HEPES, pH 7.4, 50 mm NaCl, 1% Triton X-100, 20 μm leupeptin, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 mm sodium orthovanadate, 50 mm sodium fluoride). After centrifugation at 13,000 × g for 15 min, equal amounts of cell lysates were incubated with an anti-ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibody for 2 h at 4 °C followed by the addition of protein A-agarose beads. Samples were then washed three times with lysis buffer and twice with washing buffer (25 mm Tris-HCl, pH 7.4, 25 mmβ-glycerophosphate, 1 mm sodium orthovanadate) and then incubated in 50 μl of kinase buffer (25 mm HEPES, pH 7.4, 10 mm magnesium acetate, 2 mm dithiothreitol, 2 mm EGTA, 200 μm sodium orthovanadate, 50 μm ATP, 250 μg/ml myelin basic protein (Sigma), 2 μm protein kinase A inhibitor (Sigma), and 2 μCi of γ-32PATP) for 30 min at 25 °C. After incubation, Laemmli sample buffer was added (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), followed by electrophoresis and visualization of the phosphoproteins by autoradiography. A PhosphorImager (Fuji Medical Systems, Stamford, CT) was used to quantitate band intensity. The surface expression of CCR2B was assessed by enzyme-linked immunosorbent assay as described previously (16Wong L.-M. Myers S.J. Tsou C.-L. Gosling J. H. Charo I.F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Briefly, × 106 were washed twice with PBS and then incubated with 1 μg/ml M1 antibody in RPMI 1640 containing BSA for 1 h at 37 °C. The cells were then incubated with or without 50 nm MCP-1 for times at 37 °C and with at 4 °C for 15 min. After washing with PBS, the cells were incubated with in RPMI 1640, BSA) for 30 min at room temperature and washed with was by the addition of 1 mg/ml (Sigma) in pH with at nm was with an enzyme-linked immunosorbent assay The of internalization was expressed as a of receptor expression in the presence or absence of The migration of 300-19 cell lines was determined using a of the of J.J. S. J. Biol. 1996; PubMed Scopus Google Scholar). Briefly, 300-19 cells stably transfected with or wild-type CCR2B were resuspended in RPMI 1640 plus BSA was to 5 × 106 and 5 × cells were added to the of a Cambridge, MA) and incubated for 3 h at 37 °C in an containing 5% CO2. that through the were collected from the well and counted in a San Chemotaxis was from by as described by and J. PubMed Scopus Google Scholar). determined with a were in with the of the To the role of the carboxyl-terminal domain of CCR2B in chemotaxis and we created a series of mutants in which this region of 52 amino acids was truncated progressively to and 8 amino acids. constructs were and phosphorylation of carboxyl tail serine and threonine residues is to to the of (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar), we created two the carboxyl tail in which all of the serine and threonine residues total of were to In the serine and threonine in were to transfected cell lines were in a murine pre-B lymphocyte cell (11Reth M.G. Ammirati P. Jackson S. Alt F.W. Nature. 1985; 317: 353-355Crossref PubMed Scopus (108) Google Scholar), and by cell surface expression of the to that of wild-type of wild-type and truncated receptors in cells. transfected 300-19 cells were for expression of the wild-type and truncated or receptors by incubation with the Flag antibody M1 with 300-19 cells are by the wild-type receptor; with serine and threonine to all 10 serine and threonine residues in the CCR2B carboxyl tail to We determined previously that the region of CCR2B is the binding site for MCP-1 (12Monteclaro F.S. Charo I.F. J. Biol. Chem. 1996; 271: 19084-19092Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In binding of the carboxyl tail truncation mutants 125I-labeled MCP-1 with an to that of wild-type CCR2B An cytoplasmic carboxyl is not required for binding of MCP-1 to of MCP-1 of Ca2+ and of binding data shown are the of at three not in a The data shown are the of at three not the CCR2B truncation mutants MCP-1 with we next assessed their to initiate signal We first of intracellular Ca2+ in the 300-19 cell In to a of MCP-1, a robust was in cells and the two alanine mutants 3 was not in the truncated was to which carboxyl tail had a of these constructs a of MCP-1 concentrations that had to that of the wild-type receptor 3 and with (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar), of carboxyl tail serines and or of these residues as a of receptor to a of the in intracellular 3 of extracellular with did not affect the or the of the in the truncation mutants not CCR2B to to levels (7Arai H. Charo I.F. J. Biol. Chem. 1996; 271: 21814-21819Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, S.J. Wong L.M. Charo I.F. J. Biol. Chem. 1995; 270: 5786-5792Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and as a assay of signal transduction, we determined the of mutant to for the truncation mutants were as as wild-type CCR2B in this assay in the for was which were performed in a cell the of the in 300-19 cells. In contrast to the of the and was as potent as wild-type CCR2B or the truncation mutants in ERK data that the carboxyl tail of CCR2B not a role in activation of the protein kinase In to MCP-1, CCR2B was with of the wild-type receptor removed from the cell surface by 15 min Truncation of the carboxyl tail resulted in a of internalization, such that of and on the cell surface at 15 min. The internalization of the truncation mutants be into three was the and had an level of and and were as well as the wild-type receptor. data that multiple of the carboxyl tail in the amino acids and in receptor The data further that neither the serine nor the threonine in a role in receptor internalization, of to alanine did not the of internalization with we of the mutants for to induce chemotaxis stably expressed in 300-19 cells. shown in cells transfected with CCR2B a to The MCP-1 for chemotaxis was 1 and the of cells in the the absence of was in excess of that all of this was by chemotaxis and not not for a was with of the truncation the to be In the was to that in the wild-type receptor but was to In cells the was the was also and the at 10 that this was not by not We have shown previously that the cytoplasmic carboxyl of CCR2B an role in receptor deactivation and internalization (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar). To this region of the receptor is critically involved in chemotaxis, we created a series of of the carboxyl domain and also all serine and threonine residues to are two of this the amino acids and are critically involved in signal transduction, internalization, and chemotaxis. rapid deactivation of the by phosphorylation of carboxyl domain serine and threonine residues or by receptor internalization, is not required for chemotaxis. To this is the first of a of receptor and chemotaxis. Chemotaxis is the prototypic function of the chemokine receptors, little is of the signaling pathways and receptor involved in this transfected cells not robust A. A. J.J. J. A. J.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), and this has been a in the chemokine have utilized a of the chemotaxis assay described by J.J. S. J. Biol. 1996; PubMed Scopus Google Scholar). lines of wild-type and CCR2B were in a pre-B lymphocyte cell and a (for the wild-type in excess of as to 15 for transfected HEK-293 cells A. A. J.J. J. A. J.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). this we have that truncation of CCR2B to 8 amino acids of the not chemotaxis but in a as well as a to MCP-1 concentrations for cell The in chemotaxis well with signal transduction in the and and is thus as a of coupling of the truncated receptor to G-proteins. the truncated mutant well in the protein kinase kinase assay the multiple of signal ligand binding and ERK the wild-type and to mediate chemotaxis was also markedly the of receptor is for robust chemotaxis. To this we created a in which all 10 of the carboxyl tail serine and threonine residues were to Agonist-dependent internalization of seven-transmembrane domain receptors is in phosphorylation of these serine and threonine residues (for see M.G. FASEB J. PubMed Scopus Google Scholar), and as The was as robust as that of the wild-type receptor. In a of other mutants were to the wild-type CCR2B in their We conclude that receptor internalization is not an of the at as in this rapid (8Franci C. Gosling J. Tsou C.-L. Coughlin S.R. Charo I.F. J. Immunol. 1996; 157: 5606-5612PubMed Google Scholar), we further conclude that neither of the these two well described of receptor deactivation is critically involved in chemotaxis. these that the cell in a that not require modulation at the level of the receptor. receptor internalization and are for a cell to a changing to be Truncation of the carboxyl-terminal domain of CCR2B to 8 amino acids markedly impaired signal The cytoplasmic carboxyl of seven-transmembrane domain receptors have been in M.G. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) that the carboxyl portion of the third intracellular loop to a the membrane-proximal region of the carboxyl tail in the coupling of the receptor to S. S. M.G. S. A. PubMed Scopus Google Scholar) that of the membrane-proximal amino acids of the carboxyl tail of the receptor with the region of the receptor resulted in a marked in activation of C. A. A. S. A. S. Nature. PubMed Scopus Google Scholar) that of the carboxyl-terminal domain of the receptor to G-proteins. In the study, coupling was assessed by of in transfected HEK-293 as well as by intracellular in the 300-19 cells. In cell data an role for the membrane-proximal amino acids of the carboxyl tail amino acids in coupling to G-proteins. Truncation of the cytoplasmic carboxyl tail also to a in the of the mutants to was impaired in to also that carboxyl tail residues other the 10 serines and threonines a role in receptor for this is from the that and had in of receptor residues in the carboxyl of the receptors are for and may to a intracellular loop S. S. M.G. S. A. PubMed Scopus Google Scholar, M.G. S. A. 1994; PubMed Scopus Google Scholar). is not of the two cysteines in this region of CCR2B and is of these and was to the wild-type receptor in signaling and chemotaxis The residues in the carboxyl tail of CCR2B, not to be for receptor To other has the role of the carboxyl-terminal domain of a chemokine receptor in chemotaxis A. A. J.J. J. A. J.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In that study, truncation of the carboxyl tail of to amino acids completely and the a role for this receptor domain in chemotaxis A. A. J.J. J. A. J.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). other of signal transduction were not was that the truncated receptor was from G-proteins. in the this and further that of rapid receptor such as and are not involved in chemotaxis. In we have shown that the amino acids and of the CCR2B carboxyl domain an role in chemotaxis, signal transduction, and receptor receptor internalization nor phosphorylation of carboxyl tail serine/threonine residues to be critically involved in of these including chemotaxis. of this may for in the of We and for of the for and for of the and and for