FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2097908478> ?p ?o ?g. }

• W2097908478 endingPage "33165" @default.
• W2097908478 startingPage "33157" @default.
• W2097908478 abstract "To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9–76]MCP-1 (MCP-1 lacking residues 2–8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9–76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9–76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9–76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists. To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9–76]MCP-1 (MCP-1 lacking residues 2–8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9–76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9–76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9–76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists. Chemokines are small secreted proteins that function as intercellular messengers to control migration and activation of specific subsets of leukocytes (1Miller M.D. Krangel M.S. Crit. Rev. Immunol. 1992; 12: 17-46PubMed Google Scholar, 2Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 3Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1984) Google Scholar). This process is mediated by the interaction of chemokines with seven transmembrane G-protein-coupled receptors on the surface of target cells. Interest in these proteins was first stimulated by the observation of elevated levels in a number of inflammatory diseases (4Proost P. Wuyts A. van Damme J. Int. J. Clin. Lab. Res. 1996; 26: 211-223Crossref PubMed Scopus (172) Google Scholar, 5Taub D.D. Cytokine Growth Factor Rev. 1996; 7: 355-376Crossref PubMed Scopus (152) Google Scholar) including rheumatoid arthritis (6Hosaka S. Akahoshi T. Wada C. Kondo H. Clin. Exp. Immunol. 1994; 97: 451-457Crossref PubMed Scopus (149) Google Scholar, 7Robinson E. Keystone E.C. Schall T.J. Gillett N. Fish E.N. Clin. Exp. Immunol. 1995; 101: 398-407Crossref PubMed Scopus (163) Google Scholar), arteriosclerosis (8Nelken N.A. Coughlin S.R. Gordon D. Wilcox J.N. J. Clin. Invest. 1991; 88: 1121-1127Crossref PubMed Google Scholar, 9Yla-Herttuala S. Lipton B.A. Rosenfeld M.E. Sarkioja T. Yoshimura T. Leonard E.J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5252-5256Crossref PubMed Scopus (805) Google Scholar), and asthma (10Rothenberg M.E. MacLean J.A. Pearlman E. Luster A.D. Leder P. J. Exp. Med. 1997; 185: 785-790Crossref PubMed Scopus (473) Google Scholar). Although it is not clear whether excessive production is the cause or consequence of these diseases, the demonstration that neutralizing antibodies (11Harada A. Sekido N. Akahoshi T. Wada T. Mukaida N. Matsushima K. J. Leukocyte Biol. 1994; 56: 559-564Crossref PubMed Scopus (785) Google Scholar, 12Wenzel U. Schneider A. Valente A.J. Abboud H.E. Thaiss F. Helmchen U.M. Stahl R.A. Kidney Int. 1997; 51: 770-776Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 13Feng L. Xia Y. Yoshimura T. Wilson C.B. J. Clin. Invest. 1995; 95: 1009-1017Crossref PubMed Scopus (167) Google Scholar) reduced symptoms in a number of animal models generated optimism that receptor antagonists may have therapeutic benefit (14Gong J.H. Ratkay L.G. Waterfield J.D. Clark-Lewis I. J. Exp. Med. 1997; 186: 131-137Crossref PubMed Scopus (349) Google Scholar). Recently, it has been shown that certain chemokine receptors also serve as obligate coreceptors for entry of the human immunodeficiency virus into CD4+ cells (15Choe H. Farzan M. Sun Y. Sullivan N. Rollins B. Ponath P.D. Wu L. Mackay C.R. LaRosa G. Newman W. Gerard N. Gerard C. Sodroski J. Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2090) Google Scholar, 16Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3629) Google Scholar, 17Deng H. Liu R. Ellmeier W. Choe S. Unutmaz D. Burkhart M. Di Marzio P. Marmon S. Sutton R.E. Hill C.M. Davis C.B. Peiper S.C. Schall T.J. Littman D.R. Landau N.R. Nature. 1996; 381: 661-666Crossref PubMed Scopus (3194) Google Scholar) and that viral replication can be inhibited by the ligands of the coreceptors (18Simmons G. Clapham P.R. Picard L. Offord R.E. Rosenkilde M.M. Schwartz T.W. Buser R. Wells T.N.C. Proudfoot A.E. Science. 1997; 276: 276-279Crossref PubMed Scopus (592) Google Scholar, 19Cocchi F. DeVico A.L. Garzino-Demo A. Arya S.K. Gallo R.C. Lusso P. Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2626) Google Scholar, 20Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.L. Arenzana-Seisdedos F. Schwartz O. Heard J.M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1481) Google Scholar, 21Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar). Thus a wide range of clinically important diseases are associated with chemokines and their receptors, motivating many studies to understand the molecular details of chemokine function. Chemokines have been classified into two major families based on their pattern of cysteine residues, their chromosomal location, and their cell specificities (22Schall T.J. Bacon K.B. Curr. Opin. Immunol. 1994; 6: 865-873Crossref PubMed Scopus (610) Google Scholar). α-Chemokines such as IL-8 1The abbreviations used are: IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; [1+9–76]MCP-1, MCP-1 lacking residues 2–8; WT, wild type MCP-1; WT*, WT MCP-1 with a mutation of M64I; WT*(C77), WT* with a cysteine added to the C terminus; [1+9–76](C77), [1+9–76]MCP-1 with a cysteine added to the C terminus; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single quantum correlation. 1The abbreviations used are: IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; [1+9–76]MCP-1, MCP-1 lacking residues 2–8; WT, wild type MCP-1; WT*, WT MCP-1 with a mutation of M64I; WT*(C77), WT* with a cysteine added to the C terminus; [1+9–76](C77), [1+9–76]MCP-1 with a cysteine added to the C terminus; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single quantum correlation. have a conserved CXC cysteine motif and act predominantly on neutrophils, whereas β-chemokines have a CC signature and attract monocytes and T-cells. The recently discovered chemokines lymphotactin (23Kelner G.S. Kennedy J. Bacon K.B. Kleyensteuber S. Largaespada D.A. Jenkins N.A. Copeland N.G. Bazan J.F. Moore K.W. Schall T.J. Zlotnik A. Science. 1994; 266: 1395-1399Crossref PubMed Scopus (627) Google Scholar) and fractalkine/neurotactin (24Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Greaves D.R. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1697) Google Scholar, 25Pan Y. Lloyd C. Zhou H. Dolich S. Deeds J. Gonzalo J.A. Vath J. Gosselin M. Ma J. Dussault B. Woolf E. Alperin G. Culpepper J. Gutierrez-Ramos J.C. Gearing D. Nature. 1997; 387: 611-617Crossref PubMed Scopus (573) Google Scholar) are characterized by C and CX 3C motifs, respectively, and chemoattract T-cells and NK cells. Mutagenesis studies, particularly of α- and β-chemokines, have provided some insight into the structural determinants of receptor binding and the specificity of these proteins, but many details have yet to emerge. Considerable effort has been devoted to characterizing the stochiometry of chemokine-receptor complexes because most chemokines oligomerize to an extent that depends on concentration and pH (26Lowman H.B. Fairbrother W.J. Slagle P.H. Kabakoff R. Liu J. Shire S. Hebert C.A. Protein Sci. 1997; 6: 598-608Crossref PubMed Scopus (79) Google Scholar, 27Paolini J.F. Willard D. Consler T. Luther M. Krangel M.S. J. Immunol. 1994; 153: 2704-2717PubMed Google Scholar, 28Leong S.R. Lowman H.B. Liu J. Shire S. Deforge L.E. Gillece-Castro B.L. McDowell R. Hebert C.A. Protein Sci. 1997; 6: 609-617Crossref PubMed Scopus (48) Google Scholar, 29Zhang Y. Rollins B.J. Mol. Cell. Biol. 1995; 15: 4851-4855Crossref PubMed Scopus (144) Google Scholar, 30Rajarathnam K. Sykes B.D. Kay C.M. Dewald B. Geiser T. Baggiolini M. Clark-Lewis I. Science. 1994; 264: 90-92Crossref PubMed Scopus (283) Google Scholar). Above micromolar concentrations many form homodimers, whereas at nanomolar concentrations the monomeric form predominates in solution. High resolution structures of IL-8 (31Clore G.M. Appella E. Yamada M. Matsushima K. Gronenborn A.M. Biochemistry. 1990; 29: 1689-1696Crossref PubMed Scopus (416) Google Scholar), MGSA/GRO (32Fairbrother W.J. Reilly D. Colby T.J. Hesselgesser J. Horuk R. J. Mol. Biol. 1994; 242: 252-270Crossref PubMed Scopus (81) Google Scholar), MCP-1 (33Handel T.M. Domaille P.J. Biochemistry. 1996; 35: 6569-6584Crossref PubMed Scopus (148) Google Scholar, 34Lubkowski J. Bujacz G. Boque L. Domaille P.J. Handel T.M. Wlodawer A. Nat. Struct. Biol. 1997; 4: 64-69Crossref PubMed Scopus (140) Google Scholar), RANTES (35Skelton N.J. Aspiras F. Ogez J. Schall T.J. Biochemistry. 1995; 34: 5329-5342Crossref PubMed Scopus (139) Google Scholar, 36Chung C.W. Cooke R.M. Proudfoot A.E. Wells T.N. Biochemistry. 1995; 34: 9307-9314Crossref PubMed Scopus (111) Google Scholar), and MIP-1β (37Lodi P.J. Garrett D.S. Kuszewski J. Tsang M.L. Weatherbee J.A. Leonard W.J. Gronenborn A.M. Clore G.M. Science. 1994; 263: 1762-1767Crossref PubMed Scopus (211) Google Scholar) have also revealed a striking correlation between chemokine class and mode of dimerization (38Clore G.M. Gronenborn A.M. FASEB J. 1995; 9: 57-62Crossref PubMed Scopus (118) Google Scholar), suggesting that dimerization may play an important role in chemokine function. A key question, however, is whether dimerization is required for receptor binding and activation or whether it plays a more subtle role in protein stability, regulation, surface presentation and retention, formation of the chemotactic gradient, or other processes unrelated to chemotaxis. In the case of the CXC chemokine IL-8, monomeric mutants were shown to recruit and activate neutrophils in vitro as efficiently as wild type (26Lowman H.B. Fairbrother W.J. Slagle P.H. Kabakoff R. Liu J. Shire S. Hebert C.A. Protein Sci. 1997; 6: 598-608Crossref PubMed Scopus (79) Google Scholar, 30Rajarathnam K. Sykes B.D. Kay C.M. Dewald B. Geiser T. Baggiolini M. Clark-Lewis I. Science. 1994; 264: 90-92Crossref PubMed Scopus (283) Google Scholar), consistent with the view that it interacts with its receptor as a monomer. For the CC chemokine, MCP-1, some data suggest that a dimer may be the receptor-bound form of the protein (29Zhang Y. Rollins B.J. Mol. Cell. Biol. 1995; 15: 4851-4855Crossref PubMed Scopus (144) Google Scholar). In these studies, a heterogeneous mixture of chemically cross-linked MCP-1 was shown to be active in chemotaxis assays. In addition, a deletion mutant lacking residues 2–8 ([1+9–76]MCP-1), which acts as a receptor antagonist, was also shown to inhibit chemotaxis by wild type but not by chemically cross-linked MCP-1. Finally, [1+9–76]MCP-1 containing a C-terminal FLAG epitope tag coprecipitated iodinated MCP-1 in an immunoprecipitation assay. Based on these results, it was postulated that MCP-1 interacts as a dimer and that [1+9–76]MCP-1 acts as an dominant negative antagonist by formation of inactive heterodimers with the wild type protein. Although this conclusion is consistent with the data, the heterogeneous nature of the cross-linked MCP-1 species allows alternative interpretations. Other data including the function of IL-8 obligate monomers, geometric considerations based on the three-dimensional structure of MCP-1, and the best data and models regarding G-protein-coupled receptor structure and function (39Unger V.M. Hargrave P.A. Baldwin J.M. Schertler G.F. Nature. 1997; 389: 203-206Crossref PubMed Scopus (481) Google Scholar, 40Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R.A. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (995) Google Scholar, 41DeMartino J.A. Van Riper G. Siciliano S.J. Molineaux C.J. Konteatis Z.D. Rosen H. Springer M.S. J. Biol. Chem. 1994; 269: 14446-14450Abstract Full Text PDF PubMed Google Scholar, 42Siciliano S.J. Rollins T.E. DeMartino J. Konteatis Z. Malkowitz L. Van Riper G. Bondy S. Rosen H. Springer M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1214-1218Crossref PubMed Scopus (218) Google Scholar) also seem difficult to reconcile with a heterodimer model. Because resolving this issue is necessary for a complete understanding of the molecular mechanisms of leukocyte migration and for the modeling and utilization of structural data for the design of receptor antagonists, we felt compelled to investigate further whether MCP-1 can bind and activate its receptor as a monomer. To explore the requirement of dimerization in the interaction of MCP-1 with its receptor, we mutated residues that contribute significantly to the dimer interface and assessed the effect of these mutations on receptor binding, on activation, and on dimerization. We also characterized the aggregation state of [1+9–76]MCP-1 at concentrations up to the millimolar range to assess whether it can form a homodimer. Finally, to elucidate the mechanism by which [1+9–76]MCP-1 acts as a receptor antagonist, we carried out binding competition experiments to address whether it functions as a dominant negative or classic competitive inhibitor. With the exception of [1+9–76]MCP-1, all mutants were made in the context of MCP-1 M64I, referred to as WT*, and expressed in E. coli. We have demonstrated that WT* behaves identically to WT in binding and activity assays (see Table I). This alteration in the primary structure improves the purity and homogeneity of the mutants by eliminating the formation of species containing methionine-sulfoxide at position 64.Table ISummary of binding and activity data for WT MCP-1 and all mutantsMutationBinding to THP-1 cellsBinding to CCR2-CHL cellsInhibition of cAMP synthesisStimulation Ca+2influxStimulation of chemotaxisFoldK dFoldK dFoldIC50FoldEC50FoldEC50Mut/wtnmMut/wtnmMut/wtnmMut/wtnmMut/wtnm[1–76] (Wild type)1.00.035 ± 0.021 (441)1.00.030 ± 0.017 (46)1.00.048 ± 0.017 (21)1.03.39 ± 2.3 (19)1.00.360 ± 0.315 (16)[1–76, M64I] (Wild Type*)0.90.031 ± 0.018 (5)0.90.027 ± 0.016 (6)1.10.054 ± 0.022 (4)0.50.170 ± 0.085 (2)[P8A]*1.00.035 ± 0.015 (4)0.70.020 ± 0.012 (4)0.5bp < 0.01, significant.0.024 ± 0.009 (4)0.41.49 ± 0.80 (3)0.20.089 ± 0.003 (2)[V9A]*1.30.046 ± 0.016 (5)1.40.041 ± 0.012 (5)2.10.098 ± 0.068 (3)0.50.175 ± 0.021 (2)[V9E]*6.2ap < 0.001, highly significant.0.217 ± 0.015 (4)7.6ap < 0.001, highly significant.0.229 ± 0.009 (2)13.4ap < 0.001, highly significant.0.641 ± 0.339 (4)6.3ap < 0.001, highly significant.2.27 ± 0.55 (3)[V9A + T10A]*2.30.080 ± 0.027 (2)2.8bp < 0.01, significant.0.083 ± 0.020 (2)8.3ap < 0.001, highly significant.0.398 ± 0.018 (2)[T10E]*20ap < 0.001, highly significant.0.704 ± 0.298 (7)27ap < 0.001, highly significant.0.801 ± 0.510 (8)118ap < 0.001, highly significant.5.654 ± 3.936 (5)64ap < 0.001, highly significant.23.0 ± 14.1 (2)[Y13A]*95ap < 0.001, highly significant.3.319 ± 1.433 (20)160ap < 0.001, highly significant.4.768 ± 2.973 (6)1065ap < 0.001, highly significant.50.82 ± 36.48 (11)45ap < 0.001, highly significant.153 ± 109 (2)Antagonist>100,000dNo chemotaxis was observed at 100 μm, the highest concentration tested.(4)[1 + 9–76]6.8ap < 0.001, highly significant.0.240 ± 0.153 (5)7.2ap < 0.001, highly significant.0.215 ± 0.033 (3)109ap < 0.001, highly significant.5.198 ± 4.34 (5)Antagonist>200,000cNo calcium influx was observed at 200 μm, the highest concentration tested.(2)Antagonist>10,000eNo chemotaxis was observed at 10 μm, the highest concentration tested. (4)The columns labeled Fold (Mut/Wt) are the ratio of the measured parameter for the mutant MCP-1 to WT MCP-1. The error statistic is the standard deviation for the number of replicate experiments shown in parentheses. Blank cells represent unmeasured parameters.a p < 0.001, highly significant.b p < 0.01, significant.c No calcium influx was observed at 200 μm, the highest concentration tested.d No chemotaxis was observed at 100 μm, the highest concentration tested.e No chemotaxis was observed at 10 μm, the highest concentration tested. Open table in a new tab The columns labeled Fold (Mut/Wt) are the ratio of the measured parameter for the mutant MCP-1 to WT MCP-1. The error statistic is the standard deviation for the number of replicate experiments shown in parentheses. Blank cells represent unmeasured parameters. The gene for WT* MCP-1 was constructed by standard gene synthesis techniques with optimal codon usage for expression in E. coli (43Kane J.F. Curr. Opin. Biotechnol. 1995; 6: 494-500Crossref PubMed Scopus (598) Google Scholar). Mutant constructs were made by polymerase chain reaction mutagenesis of the WT* template and cloned into a pET3 based plasmid, pAED-4 (44Doerring D.S. Functional and Structural Studies of a Small f-actin Binding Domain. MIT Press, Cambridge, MA1992Google Scholar). All sequences were confirmed by double-stranded DNA sequencing. Plasmids were then transformed into TAP302 cells, 2S. Lichter, T. M. Handel, H. J. George, and T. Patterson, unpublished results. which are BL21 pLys S cells engineered with a thioredoxin reductase knockout to make the intracellular redox potential more conducive to disulfide bond formation. Using this strain, disulfide bonds appear to be formed in the cell, eliminating the need for a refolding step. Cells were grown in Luria broth containing 100 μg/ml of ampicillan and 34 μg/ml chloramphenicol at 30 °C in standard 2.8-liter shaker flasks. When the cell density reached 0.6 A 600, protein expression was induced by adding isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.5 mm. After 1 h, rifampicin was added to a final concentration of 24 μm. Cells were incubated with shaking for an additional 3–4 h and harvested by centrifugation. 15N-Labeled proteins were expressed in the same manner except with MOPS (45Neidardt F. Bloch P. Smith D. J. Bacteriol. 1974; 119: 736-747Crossref PubMed Google Scholar) minimal medium containing15N-ammonium sulfate (Cambridge Isotope Laboratories, Andover, MA) in place of Luria broth. Post-induction times varied from 3 to 18 h. Typically, proteins were purified from 1.5-liter E. colicells, harvested by centrifugation for 20 min at 6000 ×g. Cells were lysed in 200 ml of buffer by sonication on ice for three 5-min cycles with 5 min of cooling between cycles, using a 1.25-cm horn type sonicator at 80–90% power. The lysis buffer contained 10 mm K2PO4, pH 7.5, 100 mm NaCl, 5 mm MgCl2, and 3000 units DNase I. Cell lysate was cleared by centrifugation at 13,800 ×g for 1 h at 4 °C and then loaded on a 40-ml SP-Sepharose column equilibrated with lysis buffer. Protein was eluted with a gradient of NaCl in 10 mmK2PO4, pH 7.5; typically MCP-1 mutants elute at 0.4 ± 0.1 m NaCl. Peak fractions were pooled and further purified by reversed-phase HPLC using a 4.6 × 250 mm C18 column with a 5-μm particle size and 300-Å pore size. Proteins were eluted using a gradient of increasing acetonitrile containing 0.1% trifluoroacetic acid; typically, proteins eluted at 34 ± 5% acetonitrile. They were then lyophilized, dissolved at 1 mg/ml in 35 mm Tris, pH 8, reacted with 15 μg of aminopeptidase (Peprotech, Rock Hill, NJ)/1 mg of protein for 36 h at room temperature, and repurified by reversed-phase HPLC. Aminopeptidase treatment removes only the N-terminal methionine when proline is the second residue of the mature protein. The protein was then lyophilized, redissolved in water at 1–5 mg/ml, and stored in small aliquots at −80 °C. These methods typically yielded 0.5–5 mg of purified protein/1.5 liter of bacteria. The N-terminal truncation mutant, [1+9–76]MCP-1, was expressed as a secreted protein in Pichia strain GS115 (Invitrogen, San Diego, CA) using expression vectors, pPIC9 (Invitrogen). Expression cassettes consist of the AOX1 promoter, a secretion sequence, [1+9–76]MCP-1 cDNA derivative, and a transcription terminator. Vector pPIC9 uses the Saccharomyces α-mating factor prepro-secretion sequence, and we inserted the [1+9–76]MCP-1 gene immediately after the Lys-Arg codons of the α-mating factor sequence. Thus the sequence of the junction between these two proteins is GVSLEKR↓QVTCCY, where the downward facing arrow represents the cleavage site of the fusion protein, pSRF224. Expression cassettes were integrated into the AOX1 genes. This was done by first linearizing the expression plasmids at the edges of the 5′AOX1 and 3′ AOX1 homology region usingBglII and subsequently transforming into yeast. Successful integration was determined by histidine prototropy, mut−phenotype, and MCP-1 expression. Pichia strains containing MCP-1 expression cassettes were grown to saturation by shaking for up to 2 days at 30 °C in noninducing medium. Noninducing medium is BMGY (Invitrogen), containing buffered complex medium and 1% glycerol. Two liters of cells could be grown in this manner. Expression of MCP-1 was induced by resuspending the cells in 400 ml of medium containing 0.5% methanol and either BMMY (Invitrogen) containing buffered complex medium or synthetic medium. The cultures were shaken at 30 °C for an additional 2–6 days. Buffered complex medium contains: 1% yeast extract, 2% peptone, 100 mmK2PO4, pH 6.0, 1.34% yeast nitrogen base without amino acids, and 0.4 μg/ml biotin. Synthetic medium contains: 1% casamino acids, 58 mm H3PO4, 3.4 mm CaSO4, 53 mmK2SO4, 31 mm MgSO4, 37 mm KOH, 1.0 mm FeSO4, 0.10 mm CuSO4, 0.30 mmZnSO4, 0.08 mm MnSO4, 0.43 μg/ml biotin, adjusted with NH4OH to pH 5.8 (46Clare J.J. Romanos M.A. Rayment F.B. Rowedder J.E. Smith M.A. Payne M.M. Sreekrishna K. Henwood C.A. Gene (Amst .). 1991; 105: 205-212Crossref PubMed Scopus (300) Google Scholar, 47Brierley R.A. Bussineau C. Kosson R. Melton A. Siegel R.S. Ann. N. Y. Acad. Sci. 1990; 589: 350-362Crossref PubMed Scopus (140) Google Scholar). These methods typically yielded 30 mg/liter of crude secreted MCP-1 derivative and often 20 mg/liter of purified protein. Proteins were purified and characterized as described above for E. colimaterial, starting with the cell growth medium but without the aminopeptidase step. The molecular masses of all proteins were characterized by electrospray mass spectrometry and differed by no more than 1 Da from the expected value. Protein purity was analyzed using reversed-phase HPLC; the average purity was 95 ± 5%. All protein concentrations were determined using an ε280 extinction coefficient calculated from the amino acid composition (48Gill S. von Hippel P. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5048) Google Scholar). A complete description of our binding assay can be found elsewhere. 3S. Hemmerich, C. Paavola, A. Bloom, S. Bhakta, R. Freedman, D. Grunberger, J. Krstenansky, S. Lee, D. McCarley, M. Mulkins, B. Wong, J. Pease, T. Mirzadegan, I. Polsky, K. Thompson, L. S. Mizoue, T. M. Handel, and K. Jarnagin, manuscript in preparation. Briefly, binding was measured using membranes prepared from two cell lines, THP-1 and CCR2-CHL. Each assay was composed of membranes, 50 pm125I-MCP, MCP buffer, protease inhibitors, and test protein. Equilibrium was achieved by incubation at 28 °C for 90 min. Membrane-bound 125I-MCP was collected by filtration through GF/B filters presoaked in polyethyleneimine and bovine serum albumin, followed by four rapid washes with approximately 0.5 ml of ice-cold buffer containing 0.5 m NaCl and 10 mm HEPES, pH 7.4. MCP buffer consists of 50 mm HEPES, pH 7.2, 1 mm CaCl2, 5 mm MgCl2, and 0.1% bovine serum albumin. Protease inhibitors include 0.1 mm phenylmethylsulfonyl fluoride, 1 μmleupeptin, and 0.35 μg/ml pepstatin. THP-1 cells are from a human monocyte cell line (ATCC TIP-202) that expresses both CCR1 and CCR2. CCR2-CHL cells are Chinese hamster lung cells (ATCC CRL-1657) that have been stably transformed with an expression vector, pSW104, bearing the human CCR2b receptor and a neomycin resistance marker plasmid as described previously (49Jarnagin K. Bhakta S. Zuppan P. Yee C. Ho T. Phan T. Tahilramani R. Pease J.H. Miller A. Freedman R. J. Biol. Chem. 1996; 271: 28277-28286Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The CCR2-CHL and the THP-1–4X cells express approximately 10,000 CCR2 receptors per cell, whereas THP-1, HEK-293-CCR2b, and CHO-K1-CCR2b-cAMP-Luc-neo-22 cells express approximately 5000 CCR2 receptors/cell (see below). The methods for measurement of intracellular signaling and chemotaxis have been described in a separate paper. 4K. Jarnagin, D. Grunberger, M. Mulkins, B. Wong, S. Hemmerich, C. Paavola, A. Bloom, S. Bhakta, R. Freedman, D. McCarley, I. Polsky, A. Ping-Tsou, and T. M. Handel, manuscript in preparation. Briefly, adenylate cyclase inhibition was measured by direct cAMP measurement using stable HEK293-CCR2b-transfected cells that were labeled with [H3]adenine, stimulated with forskolin, and inhibited by exposure to the appropriate mutant for 15 min. cAMP was isolated with ion exchange chromatography and quantified by radioactivity determination. Adenylate cyclase inhibition was also measured using the firefly-luciferase reporter gene attached 3′ to a cAMP response enhancer, CREB; these elements were contained within the cell clone, CHO-K1-CCR2b-cAMP-Luc-neo-22. The assay measured adenylate cyclase inhibition after 6 h of exposure to the appropriate mutant in the presence of forskolin. Luciferase expression was quantified using the Luclite substrate (Packard Inst, Downers Grove, IL). Cytosolic calcium influx was measured in THP-1–4X cells loaded with the fluorescent dye Fura-2-AM (50Blue Jr., D.R. Craig D.A. Ransom J.T. Camacho J.A. Insel P.A. Clarke D.E. J. Pharmacol. Exp. Ther. 1994; 268: 1588-1596PubMed Google Scholar). Quantitation of fluorescence intensity was done by integrating the signal for 82 s following the addition of mutant protein. Conversion of signal intensity to calcium molarity was done by standard methods (51Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar).4 Chemotaxis was measured over 1 h using THP-1–5X cells in a 96-well Boyden chamber apparatus. Cell migration through the polycarbonate filter was quantified by fluorescent staining using propidium iodide in 0.1% Triton X-100. These assays typically gave a stimulated to unstimulated migration of 4–10-fold (average 6-fold) using 3 nm MCP-1, a maximally effective concentration for this protein. All 441 determinations of the K d for the WT protein were tested for statistical normality; the data are log-normally distributed as would be expected from the ratio definition ofK d. Similarly, the WT tests of CCR2-CHL binding, inhibition of cAMP synthesis, calcium flux, and chemotaxis were found to be log-normally distributed. Thus all of the statistical tests on the K d values and IC50 values were performed as pK d values and pIC50values. Significance is noted in Table I as explai" @default.
• W2097908478 created "2016-06-24" @default.
• W2097908478 creator A5004526431 @default.
• W2097908478 creator A5006306961 @default.
• W2097908478 creator A5007852420 @default.
• W2097908478 creator A5023746719 @default.
• W2097908478 creator A5025004675 @default.
• W2097908478 creator A5050290780 @default.
• W2097908478 creator A5055302959 @default.
• W2097908478 creator A5058776594 @default.
• W2097908478 creator A5061322167 @default.
• W2097908478 creator A5071000489 @default.
• W2097908478 creator A5076222574 @default.
• W2097908478 creator A5082059757 @default.
• W2097908478 creator A5090401657 @default.
• W2097908478 date "1998-12-01" @default.
• W2097908478 modified "2023-10-16" @default.
• W2097908478 title "Monomeric Monocyte Chemoattractant Protein-1 (MCP-1) Binds and Activates the MCP-1 Receptor CCR2B" @default.
• W2097908478 cites W1486554974 @default.
• W2097908478 cites W1506918918 @default.
• W2097908478 cites W1526864759 @default.
• W2097908478 cites W1544899066 @default.
• W2097908478 cites W1607234954 @default.
• W2097908478 cites W1652900453 @default.
• W2097908478 cites W1781531090 @default.
• W2097908478 cites W1809178030 @default.
• W2097908478 cites W1963479874 @default.
• W2097908478 cites W1963984954 @default.
• W2097908478 cites W1964120116 @default.
• W2097908478 cites W1965019797 @default.
• W2097908478 cites W1966010345 @default.
• W2097908478 cites W1974602509 @default.
• W2097908478 cites W1977920448 @default.
• W2097908478 cites W1978958492 @default.
• W2097908478 cites W1980198584 @default.
• W2097908478 cites W1981440980 @default.
• W2097908478 cites W1984061807 @default.
• W2097908478 cites W1984115097 @default.
• W2097908478 cites W1984811742 @default.
• W2097908478 cites W1987712445 @default.
• W2097908478 cites W1989035987 @default.
• W2097908478 cites W1991720779 @default.
• W2097908478 cites W1995905351 @default.
• W2097908478 cites W1998819077 @default.
• W2097908478 cites W2005986251 @default.
• W2097908478 cites W2008386999 @default.
• W2097908478 cites W2008669330 @default.
• W2097908478 cites W2011548322 @default.
• W2097908478 cites W2014462918 @default.
• W2097908478 cites W2021873664 @default.
• W2097908478 cites W2022481460 @default.
• W2097908478 cites W2023853843 @default.
• W2097908478 cites W2024510500 @default.
• W2097908478 cites W2028231353 @default.
• W2097908478 cites W2028304773 @default.
• W2097908478 cites W2029262446 @default.
• W2097908478 cites W2031311732 @default.
• W2097908478 cites W2038182929 @default.
• W2097908478 cites W2039707307 @default.
• W2097908478 cites W2053315646 @default.
• W2097908478 cites W2056258550 @default.
• W2097908478 cites W2057350048 @default.
• W2097908478 cites W2058720223 @default.
• W2097908478 cites W2062148688 @default.
• W2097908478 cites W2067403399 @default.
• W2097908478 cites W2067964854 @default.
• W2097908478 cites W2069602336 @default.
• W2097908478 cites W2071148453 @default.
• W2097908478 cites W2075309835 @default.
• W2097908478 cites W2076321944 @default.
• W2097908478 cites W2086495450 @default.
• W2097908478 cites W2094210266 @default.
• W2097908478 cites W2111650420 @default.
• W2097908478 cites W2114144487 @default.
• W2097908478 cites W2116210034 @default.
• W2097908478 cites W2117135532 @default.
• W2097908478 cites W2141260882 @default.
• W2097908478 cites W2150396060 @default.
• W2097908478 cites W2153748057 @default.
• W2097908478 cites W2160272188 @default.
• W2097908478 cites W2160914594 @default.
• W2097908478 cites W2163865126 @default.
• W2097908478 cites W2166858952 @default.
• W2097908478 cites W2395981476 @default.
• W2097908478 cites W4211264883 @default.
• W2097908478 cites W4232488115 @default.
• W2097908478 doi "https://doi.org/10.1074/jbc.273.50.33157" @default.
• W2097908478 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9837883" @default.
• W2097908478 hasPublicationYear "1998" @default.
• W2097908478 type Work @default.
• W2097908478 sameAs 2097908478 @default.
• W2097908478 citedByCount "182" @default.
• W2097908478 countsByYear W20979084782012 @default.
• W2097908478 countsByYear W20979084782013 @default.
• W2097908478 countsByYear W20979084782014 @default.
• W2097908478 countsByYear W20979084782015 @default.
• W2097908478 countsByYear W20979084782016 @default.
• W2097908478 countsByYear W20979084782017 @default.

• next