FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044320388> ?p ?o ?g. }

• W2044320388 endingPage "14733" @default.
• W2044320388 startingPage "14726" @default.
• W2044320388 abstract "Post-translational modifications of the extracellular portions of receptors located in the cell membrane can contribute to modulating their biological activity. Using a mutagenesis approach in which single or multiple Tyr-to-Phe, Thr-to-Ala, Ser-to-Ala, and Asn-to-Gln substitutions were made at the appropriate positions, we analyzed the sulfation and glycosylation state of the murine CCR8 chemokine receptor, and the way in which these post-translational modifications affect CCR8 activity. A Y14Y15-to-F14F15 CCR8 mutant was less sulfated than the wild-type receptor. An N8-to-Q8 mutant was less glycosylated than wild-type, and a double T10T12-to-A10A12 mutant showed even less glycosylation. We established a flow cytometric analysis with an Fc-fused form of mouse CCL1 to determine precisely the ligand-binding activity of these mutants. Single mutants at amino acid positions 8, 10 or 12 bound CCL1-Fc similarly to wild-type CCR8, whereas the F14F15 double mutant was essentially inactive and the A10A12 double mutant showed about 65% of wild-type ligand-binding activity. Calcium flux activity assays were performed with these mutants, yielding results consistent with those from the ligand binding assays. These data indicate that sulfation at specific positions of the N-terminal domain of mouse CCR8 is critical for its biological activity, whereas glycosylation has a minor influence. Post-translational modifications of the extracellular portions of receptors located in the cell membrane can contribute to modulating their biological activity. Using a mutagenesis approach in which single or multiple Tyr-to-Phe, Thr-to-Ala, Ser-to-Ala, and Asn-to-Gln substitutions were made at the appropriate positions, we analyzed the sulfation and glycosylation state of the murine CCR8 chemokine receptor, and the way in which these post-translational modifications affect CCR8 activity. A Y14Y15-to-F14F15 CCR8 mutant was less sulfated than the wild-type receptor. An N8-to-Q8 mutant was less glycosylated than wild-type, and a double T10T12-to-A10A12 mutant showed even less glycosylation. We established a flow cytometric analysis with an Fc-fused form of mouse CCL1 to determine precisely the ligand-binding activity of these mutants. Single mutants at amino acid positions 8, 10 or 12 bound CCL1-Fc similarly to wild-type CCR8, whereas the F14F15 double mutant was essentially inactive and the A10A12 double mutant showed about 65% of wild-type ligand-binding activity. Calcium flux activity assays were performed with these mutants, yielding results consistent with those from the ligand binding assays. These data indicate that sulfation at specific positions of the N-terminal domain of mouse CCR8 is critical for its biological activity, whereas glycosylation has a minor influence. Post-translational modifications of amino acid residues located in the extracellular or intracytoplasmic domains of cell membrane receptors can modify the signaling activity of these proteins. In the case of chemokine receptors, a family of seven-transmembrane domain proteins with critical roles in the control of basal and pathological leukocyte trafficking (1Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar), alteration of their activity may modulate the migratory routes of immune cells. Chemokine receptors are known to undergo a variety of post-translational modifications. For instance, phosphorylation of specific intracellular serine residues in the C-terminal region of chemokine receptors is essential for their signal transduction function (2Olbrich H. Proudfoot A.E. Oppermann M. J. Leukoc. Biol. 1999; 65: 281-285Crossref PubMed Scopus (38) Google Scholar). In some chemokine receptors, extracellular regions are also known to be post-translationally modified. Human chemokine receptors CCR2b, CCR5, CX3CR1, and CXCR4 are reported to be sulfated and/or glycosylated at their N-terminal extracellular domains. In vitro, these modifications have diverse consequences for receptor ligand-binding activities, as well as for their function as human immunodeficiency virus coreceptors (3Preobrazhensky A.A. Dragan S. Kawano T. Gavrilin M.A. Gulina I.V. Chakravarty L. Kolattukudy P.E. J. Immunol. 2000; 165: 5295-5303Crossref PubMed Scopus (95) Google Scholar, 4Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 5Farzan M. Babcock G.J. Vasilieva N. Wright P.L. Kiprilov E. Mirzabekov T. Choe H. J. Biol. Chem. 2002; 277: 29484-29489Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6Fong A.M. Alam S.M. Imai T. Haribabu B. Patel D.D. J. Biol. Chem. 2002; 277: 19418-19423Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).CCR8, the receptor for the β-chemokine CCL1 (7Roos R.S. Loetscher M. Legler D.F. Clark L.I. Baggiolini M. Moser B. J. Biol. Chem. 1997; 272: 17251-17254Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 8Goya I. Gutierrez J. Varona R. Kremer L. Zaballos A. Marquez G. J. Immunol. 1998; 160: 1975-1981PubMed Google Scholar, 9Tiffany H.L. Lautens L.L. Gao J.L. Pease J. Locati M. Combadiere C. Modi W. Bonner T.I. Murphy P.M. J. Exp. Med. 1997; 186: 165-170Crossref PubMed Scopus (183) Google Scholar), is expressed in Th2-polarized T cells (10Zingoni A. Soto H. Hedrick J.A. Stoppacciaro A. Storlazzi C.T. Sinigaglia F. D'Ambrosio D. O'Garra A. Robinson D. Rocchi M. Santoni A. Zlotnik A. Napolitano M. J. Immunol. 1998; 161: 547-551PubMed Google Scholar, 11D'Ambrosio D. Iellem A. Bonecchi R. Mazzeo D. Sozzani S. Mantovani A. Sinigaglia F. J. Immunol. 1998; 161: 5111-5115PubMed Google Scholar) and has been implicated in allergic inflammation (12Panina-Bordignon P. Papi A. Mariani M. Di Lucia P. Casoni G. Bellettato C. Buonsanti C. Miotto D. Mapp C. Villa A. Arrigoni G. Fabbri L.M. Sinigaglia F. J. Clin. Invest. 2001; 107: 1357-1364Crossref PubMed Scopus (396) Google Scholar), although whether CCR8 has a critical role in asthma remains controversial (13Chung C.D. Kuo F. Kumer J. Motani A.S. Lawrence C.E. Henderson Jr., W.R. Venkataraman C. J. Immunol. 2003; 170: 581-587Crossref PubMed Scopus (105) Google Scholar, 14Chensue S.W. Lukacs N.W. Yang T.Y. Shang X. Frait K.A. Kunkel S.L. Kung T. Wiekowski M.T. Hedrick J.A. Cook D.N. Zingoni A. Narula S.K. Zlotnik A. Barrat F.J. O'Garra A. Napolitano M. Lira S.A. J. Exp. Med. 2001; 193: 573-584Crossref PubMed Scopus (205) Google Scholar, 15Goya I. Villares R. Zaballos A. Gutierrez J. Kremer L. Gonzalo J.A. Varona R. Carramolino L. Serrano A. Pallares P. Criado L.M. Kolbeck R. Torres M. Coyle A.J. Gutierrez-Ramos J.C. Martinez A.C. Marquez G. J. Immunol. 2003; 170: 2138-2146Crossref PubMed Scopus (96) Google Scholar). CCR8 also has a potential role in atherogenesis (16Harpel P.C. Haque N.S. Isr. J. Med. Assoc. 2002; 4: 1025-1027PubMed Google Scholar), can act as human immunodeficiency virus coreceptor (17Horuk R. Hesselgesser J. Zhou Y. Faulds D. Halks-Miller M. Harvey S. Taub D. Samson M. Parmentier M. Rucker J. Doranz B.J. Doms R.W. J. Biol. Chem. 1998; 273: 386-391Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), and is a target for certain virally encoded chemokines (18Holst P.J. Luttichau H.R. Schwartz T.W. Rosenkilde M.M. Contrib. Microbiol. 2003; 10: 232-252Crossref PubMed Google Scholar). We characterized the mouse version of CCR8, which has only one murine ligand, CCL1 (8Goya I. Gutierrez J. Varona R. Kremer L. Zaballos A. Marquez G. J. Immunol. 1998; 160: 1975-1981PubMed Google Scholar). We developed mouse CCR8-specific antibodies and used them to study the tissue expression of this molecule, which was highest in thymocytes committed to the CD4+ T cell lineage (19Kremer L. Carramolino L. Goya I. Zaballos A. Gutierrez J. del Carmen Moreno-Ortiz M. Martinez A.C. Marquez G. J. Immunol. 2001; 166: 218-225Crossref PubMed Scopus (36) Google Scholar).The results of that study suggested that modifications in extracellular regions of CCR8 could mask or form part of epitopes recognized by the antibodies. Using a mutagenesis approach and metabolic labeling studies, we have now performed a detailed study of potential post-translational modifications of murine CCR8. Our results indicate that tyrosines at positions 14 and 15 in mouse CCR8 are sulfated amino acid residues, whereas asparagine 8 and threonines 10 and 12 are glycosylated. CCR8 mutants in which those residues are replaced by amino acids subject neither to sulfation nor to glycosylation are affected differently in their ability to bind CCL1, as well as in their signaling. All together, the results show that tyrosine sulfation in the CCR8 N-terminal domain is important for full activity of this chemokine receptor, whereas the influence of asparagine and threonine glycosylation on the receptor activity is less critical.EXPERIMENTAL PROCEDURESCell Culture—Human embryonic kidney (HEK) 1The abbreviations used are: HEK, human embryonic kidney; CM, conditioned medium; PBS, phosphate-buffered saline; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PE, phycoerythrin; FACS, fluorescence-activated cell sorting; Indo 1-AM, Indo 1-acetoxymethyl ester. 293 cells were cultured in Dulbecco's modified Eagle medium (BioWhittaker, Verviers, Belgium) containing 10% fetal calf serum and supplemented with glutamine, penicillin, and streptomycin. Methionine- and cysteine-free medium consisted of RPMI 1640 (BioWhittaker); sulfate-free medium consisted of Eagle's modified minimal essential medium (ICN Biomedicals, Aurora, OH), supplemented with 1 mm CaCl2. In some cases tunicamycin (5 μg/ml) or monensin (5 μm; both from Sigma, St. Louis, MO) was added to the culture. When necessary, G418 (1 mg/ml; Promega, Madison, WI) was used for selection.Mutagenesis and Transfection—The construction of a mammalian expression plasmid containing the coding sequence of mouse CCR8 cDNA has been described previously (8Goya I. Gutierrez J. Varona R. Kremer L. Zaballos A. Marquez G. J. Immunol. 1998; 160: 1975-1981PubMed Google Scholar). A synthetic DNA fragment encoding a c-Myc protein epitope was introduced at the 5′ end of the CCR8 coding sequence, causing the resulting CCR8 protein to have an MEQKLISEEDLLAA N-terminal tag. The myc-CCR8-encoding plasmid was used as template to generate single or double mutants using the QuikChange multisite-directed mutagenesis kit (Stratagene, La Jolla, CA). DNA fragments encoding single mutations were swapped to obtain CCR8 multiple mutants. Sequences of all PCR-produced DNA segments were analyzed to discard clones containing undesired mutations. CCR8-encoding plasmids were prepared using the Jetstar Maxi Kit 50 (Genomed, Bad Oeynhausen, Germany) and transfected into HEK 293 cells with LipofectAMINE Plus (Invitrogen, San Diego, CA) according to the manufacturers' instructions. In some cases, stable transfectants were generated using G418 selection for 2 weeks. Transient transfectants were used unless otherwise indicated.To construct the mouse CCL1-hFc plasmid, the cDNA sequence encoding the entire mouse CCL1 protein was amplified by PCR and fused to the 5′ end of the sequence encoding the Fc portion of human IgG1. Briefly, 5 μg of thymus total RNA extracted with Tri-reagent (Sigma) was reverse-transcribed using random hexamers and 100 units of Superscript II RT (Invitrogen). One-twentieth of the reaction mixture was subjected to 30 PCR cycles (15 s at 94 °C, 15 a at 60 °C, and 60 s at 72 °C) using the Expand High Fidelity DNA polymerase (Roche Applied Science, Mannheim, Germany) and the following primers: forward, 5′-ACGTAAGCTTGCCGCCATGAAACCCACTGCCA-3′; reverse, 5′-ACGTGTCGACGCAGGGGTTCACCTTCT-3′. The amplified product was purified, digested with HindIII and SalI, and fused in-frame to the human IgG1 Fc sequence present in a pCDNA3 derivative. Inserts from individual clones were sequenced, and a plasmid harboring no mutations was selected. The plasmid was transfected into HEK 293T cells with FuGENE (4 μl/μg DNA), as suggested by the manufacturer (Roche Applied Science). Conditioned medium (CM) was produced for 72 h, collected, filtered through 0.22 μm, and stored at 4 °C with 0.1% NaN3.Labeling and Immunoprecipitation—Separate cultures of 5 × 106 transfected HEK 293 cells expressing different murine CCR8 variants were washed in phosphate-buffered saline (PBS) and depleted (2 h) of either methionine and cysteine, or sulfate. Culture medium was then added containing either [35S]methionine and [35S]cysteine, or sodium [35S]sulfate (Amersham Biosciences; Buckinghamshire, UK), and incubation continued overnight. Labeling media contained 200 μCi of [35S]methionine/cysteine or 500 μCi of [35S]sulfate. Labeled cells were harvested, washed in PBS, and lysed in Tris-buffered saline containing 0.4% n-dodecylmaltoside, 10% glycerol, and a protease inhibitor mixture. A culture supernatant from the anti-c-Myc monoclonal antibody (mAb)-producing 9E10 hybridoma was used to immunoprecipitate myc-tagged CCR8; the immunoprecipitate was analyzed by SDS-PAGE, as described (20Carramolino L. Kremer L. Goya I. Varona R. Buesa J.M. Gutierrez J. Zaballos A. Martinez A.C. Marquez G. J. Leukoc. Biol. 1999; 66: 837-844Crossref PubMed Scopus (48) Google Scholar).Immunoprecipitates of unlabeled cells were analyzed by SDS-PAGE and Western blot using rabbit anti-c-Myc antibody (Upstate, Charlottesville, VA) and peroxidase-labeled goat anti-rabbit immunoglobulin, essentially as described previously (20Carramolino L. Kremer L. Goya I. Varona R. Buesa J.M. Gutierrez J. Zaballos A. Martinez A.C. Marquez G. J. Leukoc. Biol. 1999; 66: 837-844Crossref PubMed Scopus (48) Google Scholar).Neuraminidase Digestions—Transfected HEK 293 cells were harvested, washed, and incubated alone or with 0.25 unit of neuraminidase from Arthrobacter ureafaciens (Roche Applied Science) in HEPES-buffered Dulbecco's modified Eagle medium containing a protease inhibitor mixture (37 °C, 2 h). Cells were then diluted and washed in PBS, samples were taken for functional assays, and the remainder was stored frozen until Western blot analysis.Ligand Binding Assay—Transfected HEK 293 cells were harvested, washed, and incubated (30 min) with 10% (v/v) of a 9E10 hybridoma supernatant containing anti-myc mAb and 10% (v/v) of HEK 293T CM containing CCL1-Fc; other concentrations of CCL1-Fc CM were used in some cases, as indicated. Purified mouse CCL1 (BD Pharmingen, San Diego, CA) was also used in some experiments to compete for CCL1-Fc binding. Cells were then washed twice and incubated (20 min) with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (Fc) (Beckman Coulter, Marseille, France) and phycoerythrin (PE)-conjugated goat anti-mouse IgG (BD Pharmingen). After two final washes, stained cells were fixed in PBS with 4% formaldehyde. All washes and incubations were performed at 0 °C in PBS with 0.5% bovine serum albumin and 0.01% NaN3. FITC cell fluorescence (525 nm) was analyzed by flow cytometry on an Epics XL (Coulter, Miami, FL), gating a fixed medium to high level of PE fluorescence emission (575 nm).Calcium Flux Assay—Transfected HEK 293 cells were harvested, washed, and incubated (30 min) with 3 μm Indo 1-AM (Molecular Probes, Leiden, The Netherlands). Cells were washed twice, resuspended at 3 × 106 cell/ml, and stored on ice until use. All incubations and washes were carried out in Hanks' balanced salt solution with 0.5% bovine serum albumin, 10 mm HEPES, 0.8 mm CaCl2, and 250 μm sulfinpyrazone. Aliquots of the cell suspension were heated for 5 min at 37 °C, excited with light at 335 nm, and stimulated with various concentrations of purified mouse CCL1; intracellular Ca2+ levels were assessed as Indo 1 fluorescence at 390 nm and recorded as a time trace in an F2500 fluorometer (Hitachi; Tokyo, Japan).Confocal Microscopy Analysis—Transfected HEK 293 cells were cultured in poly-l-Lys-coated glass Lab-Tek II chamber slides (Nalge Nunc, Naperville, IL). Culture medium was withdrawn, and the cells were washed, fixed in 2% paraformaldehyde (20 min, room temperature), and permeabilized with 0.1% saponin in PBS for 15 min. After thorough washing, slides were incubated with a culture supernatant from the anti-CCR8 mAb-producing hybridoma 3H9, which recognizes a fixation-resistant epitope at the C-terminal end of CCR8. 2L. Kremer, unpublished results. Cells were further washed and incubated with an Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes). Saponin was maintained throughout the process to this step. After two final washes, slides were mounted and visualized on a Leica DM IRB fluorescence microscope. Serial confocal images along the z-axis were acquired using an Ar-Kr laser scanning TCS-NT Leica system; images were selected corresponding to thin optical cell sections at the nuclear level to assess CCR8 distribution. Samples were in duplicate, and at least two independent transfections were performed for each CCR8 variant.RESULTSMyc-tagged Mouse CCR8 Is Expressed Functionally at the Cell Surface—The study of potential extracellular post-translational modifications of mouse CCR8 required a tool to measure precisely cell surface expression levels of the protein independently of such modifications. We therefore produced a plasmid encoding a version of CCR8 with a c-Myc epitope tagged at its N-terminal end. This CCR8 variant was tested for expression and function in transfected HEK 293 cells. Tagged CCR8 was recognized by an anti-myc mAb (Fig. 1A), yielding a FACS staining pattern that closely matched that of the CCR8-specific mAb 8F4 (19Kremer L. Carramolino L. Goya I. Zaballos A. Gutierrez J. del Carmen Moreno-Ortiz M. Martinez A.C. Marquez G. J. Immunol. 2001; 166: 218-225Crossref PubMed Scopus (36) Google Scholar). HEK 293 stable transfectant cells expressing myc-tagged or untagged CCR8 were obtained and tested for ligand-induced Ca2+ flux. The ligand sensitivity of cells transfected with the tagged version was similar to that of cells expressing the untagged version; they responded readily to 0.1 and 1 nm CCL1 and showed a typical desensitization pattern following addition of 10 nm CCL1 (Fig. 1B). The myc-tagged CCR8 was thus expressed efficiently and functioned similarly to the untagged version.CCL1-Fc and CCL1 Have Similar CCR8-binding Activity— To analyze the ligand-binding activity of CCR8 and its variants, we produced a human Fc-fused version of CCL1. Conditioned medium (CM) containing CCL1-Fc and secondary FITC-conjugated reagents recognizing the Fc portion were used to stain cells transfected with a plasmid encoding CCR8 or the void vector. At high CCL1-Fc concentrations, CCR8-expressing cells stained intensely; staining faded with dilution and was no longer detectable at CCL1-Fc CM concentrations below 0.01% (v/v) (Fig. 1C). The vector-transfected cells did not stain above background level at any CCL1-Fc CM concentration; in addition, mock CM was produced and tested similarly on CCR8-expressing cells; as predicted, no staining was detected above background levels (not shown). To verify that Fc-linked cell staining was a reliable measure of CCL1 binding to its receptor, CCR8-expressing cells were incubated with 10% CCL1-Fc CM in the presence of purified mouse CCL1 at various concentrations. At 0.25 nm, purified CCL1 competed slightly for CCL1-Fc staining; competition increased gradually by augmenting CCL1 concentration up to 200 nm, above which CCL1-Fc staining was negligible (Fig. 1D). The CCL1 concentration yielding half-maximal competition was ∼2 nm, which matches the dissociation constant reported for the CCL1-CCR8 interaction (8Goya I. Gutierrez J. Varona R. Kremer L. Zaballos A. Marquez G. J. Immunol. 1998; 160: 1975-1981PubMed Google Scholar). CCL1-Fc CM is thus a reliable tool for measuring ligand-binding activity of CCR8-bearing cells by flow cytometry.Flow Cytometric Analysis of CCL1 Binding to CCR8 —Simultaneous measurement of cell surface CCR8 expression and CCL1 binding in a single flow cytometry assay would facilitate testing the activity of receptor variants. This was analyzed by incubating myc-tagged CCR8-transfected cells with CCL1-Fc CM, with anti-myc 9E10 mAb, or with both simultaneously. Secondary FITC- or PE-conjugated reagents were used to develop bound Fc or myc epitopes, respectively. Cells stained with CCL1-Fc alone were recorded as typical FITC fluorescent events (Fig. 2A), and cells stained with the anti-myc 9E10 mAb were essentially PE events (Fig. 2B); cells stained with both reagents displayed a linear distribution along the FL1/FL2 diagonal (Fig. 2C). Anti-myc 9E10 mAb binding was inhibited weakly by CCL1-Fc binding (74.2% PE+ cells in plot B versus 60.7% PE+ cells in plot C), although strong overall correlation was observed between CCR8 levels and CCL1-Fc binding. In addition, CCL1-Fc binding to myc-CCR8 was estimated to have a Kd of 1–2 nm, concurring with previously published results for the untagged CCL1:CCR8 pair (8Goya I. Gutierrez J. Varona R. Kremer L. Zaballos A. Marquez G. J. Immunol. 1998; 160: 1975-1981PubMed Google Scholar). These results allowed specific gating for myc-associated fluorescence, i.e. CCR8 levels, and measuring the amount of Fc-associated fluorescence, i.e. CCL1 binding, only for the specifically gated cell population. By gating on myc, it was thus possible to compare the ligand-binding activity of CCR8 variants with different cell surface expression efficiency.Fig. 2CCL1-Fc binding by myc-tagged CCR8. Wild-type mycCCR8-expressing cells were tested in the FACS ligand binding assay. Cells were incubated with CCL1-Fc-conditioned medium (10% v/v) (A), anti-myc mAb 9E10 (B), or with both together (C). FITC fluorescence associates to Fc and measures CCL1 binding; PE fluorescence associates to myc and measures surface CCR8 expression. The percentage of events is presented in each quadrant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Targeting of Potential Post-translational Modification Sites in the CCR8 Amino Acid Sequence—Post-translational modifications relevant to chemokine receptor function have been described mainly at the N-terminal domain. In particular, protein sulfation at tyrosine residues, as well as both N- and O-glycosylation have been reported for other CC chemokine receptors. There is no strict consensus site for tyrosine sulfation, but sulfated tyrosines in CCR5 and CCR2b are adjacent to acidic amino acids (3Preobrazhensky A.A. Dragan S. Kawano T. Gavrilin M.A. Gulina I.V. Chakravarty L. Kolattukudy P.E. J. Immunol. 2000; 165: 5295-5303Crossref PubMed Scopus (95) Google Scholar, 4Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). We detected four such potential sulfation sites in two segments of the mouse CCR8 sequence predicted to be extracellular, three at the N-terminal domain and an additional site in the second extracellular loop (Fig. 3A). For N-linked glycosylation, the asparagine residue at CCR8 position 8 conforms to requirements of the well defined consensus site for this type of modification (asparagine-X-serine/threonine) (8Goya I. Gutierrez J. Varona R. Kremer L. Zaballos A. Marquez G. J. Immunol. 1998; 160: 1975-1981PubMed Google Scholar). Finally, O-linked glycosylation is the least characterized modification in terms of consensus sites, but this modification often occurs within threonine and/or serine residue clusters. We identified two such sites, an N-terminal domain threonine pair that partially overlaps the N-glycosylation consensus site, and a threonine/serine pair at the third extracellular loop (Fig. 3A).Fig. 3Targeted positions in the CCR8 amino acid sequence and nomenclature of all mutants used. Targeted positions are boxed and labeled in A; the specific mutations designed at each position, their combinations, and mutant names are presented in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To study potential post-translational modifications of mouse CCR8, we generated a set of myc-tagged CCR8 mutants at these nine positions. Conservative changes were designed, consisting of tyrosine-to-phenylalanine, which is not subject to sulfation, asparagine-to-glutamine, not subject to N-glycosylation, and threonine- or serine-to-alanine, not subject to O-glycosylation (Fig. 3B).CCR8 Is Modified by Sulfate—Myc-tagged CCR8-expressing cells were incubated with either [35S]methionine/cysteine or sodium [35S]sulfate, followed by myc immunoprecipitation, electrophoresis, and autoradiography. Three bands were observed in autoradiographs corresponding to wild-type CCR8-expressing cells cultured with [35S]methionine/cysteine; the upper band migrated as host c-Myc and was present in all immunoprecipitates analyzed, including a mock-transfected control (Fig. 4A, lane 8). Two bands were seen corresponding to CCR8; the upper band was broad and migrated at 41 kDa; the lower, sharper band migrated as 35 kDa (Fig. 4A, lane 1). Similar results were obtained in Western blot analysis of unlabeled cells (see below). The autoradiograph of wild-type CCR8-expressing cell cultures treated with [35S]sulfate showed one band corresponding to the 41-kDa form (Fig. 4B, lane 1). This showed that CCR8 is indeed a sulfated protein and that sulfate is incorporated into the low mobility protein form.Fig. 4CCR8 is a sulfated receptor whose undersulfation affects its ligand binding ability. Cells expressing myc-tagged versions of wild-type or tyrosine-to-phenylalanine-substituted CCR8 proteins, as indicated, were labeled with [35S]methionine/cysteine (A) or sodium [35S]sulfate (B), immunoprecipitated with an anti-myc antibody, electrophoresed, and autoradiographed. [35S]sulfate to [35S]methionine/cysteine ratios: 0.25, 0.17, 0.18, 0.16, 0.24, 0.07, and 0.06 in lanes 1–7, respectively. C and D, FACS ligand binding assay of wild-type CCR8-expressing cells cultured with (C) or without (D) sulfate. In D, sulfation was further inhibited by addition of 10 mm sodium chlorate to cultures. CCL1-Fc-conditioned medium was used at 10% (v/v). FITC fluorescence associates to Fc and measures CCL1 binding; PE fluorescence associates to myc and measures surface CCR8 expression. A gate was established for medium to high myc expression levels; the mean Fc-associated fluorescence of gated events is indicated above each gate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To analyze whether the targeted tyrosine residues were involved in CCR8 sulfation, cells transfected with plasmids encoding the appropriate mutants were labeled, immunoprecipitated, and autoradiographed. For correct comparison of the sulfation level of CCR8 variants, autoradiographs were densitometered, and the amount of [35S]methionine/cysteine label was used to normalize the amount of [35S]sulfate label for protein mass. All the single mutants incorporated [35S]sulfate label in amounts similar to wild-type (Fig. 4B, lanes 2–5); double or quadruple mutants involving tyrosines 14 and 15 significantly reduced the [35S]sulfate label (Fig. 4B, lanes 6 and 7), whereas all mutants were similarly labeled by [35S]methionine and [35S]cysteine (Fig. 4A, lanes 2–7). These results suggest that tyrosines 14 and 15 are indeed subject to sulfation and that there is additional sulfate incorporation throughout the molecule not involving the targeted tyrosines. A slight decrease in CCR8 apparent molecular weight was seen in the [35S]methionine/cysteine autoradiograph in samples from single mutants at positions 14 or 15, particularly at the lower band; this mobility shift was even more marked in the double or quadruple mutants involving these positions (Fig. 4A, lanes 4–7). Similar small electrophoretic mobility shifts were also reported for sulfatable tyrosine mutants of C5a receptor (21Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Kuhn J. Gerard C. Gerard N.P. Choe H. J. Exp. Med. 2001; 193: 1059-1066Crossref PubMed Scopus (74) Google Scholar). Cells transfected with void vector were treated similarly and yielded only the band corresponding to host c-Myc (Fig. 4, A and B, lanes 8).Inhibition of Sulfation Impairs CCR8 Ligand Binding— Cells transfected with myc-tagged CCR8 were depleted of sulfate and incubated with 10 mm sodium chlorate, a concentration reported to inhibit sulfation (22Mintz K.P. Fisher L.W. Grzesik W.J. Hascall V.C. Midura R.J. J. Biol. Chem. 1994; 269: 4845-4852Abstract Full Text PDF PubMed Google Scholar). Control cells were incubated in the presence of sulfate. Cells were harvested and analyzed in the CCL1-Fc binding FACS assay. The results indicated that even cells with low surface CCR8 expression, cultured in the presence of sulfate, bound CCL1 as predicted (Fig. 4C). To the contrary, sulfate-depleted cells cultured in sodium chlorate bound small amounts of CCL1 only when expressing high CCR8 levels (Fig. 4D). This result indicated that sulfation is necessary for the full ligand-binding activity of CCR8.Mutations at Tyrosines 14 or 15 Decrease CCR8 Ligand-binding Activity—Cells transfected with wild-type myc-tagged CCR8 or selected tyrosine-to-phenylalanine mutants were analyzed in the CCL1-Fc binding FACS assay. A gate was establish" @default.
• W2044320388 created "2016-06-24" @default.
• W2044320388 creator A5017974200 @default.
• W2044320388 creator A5030269913 @default.
• W2044320388 creator A5034123077 @default.
• W2044320388 creator A5046575837 @default.
• W2044320388 creator A5051411928 @default.
• W2044320388 creator A5055503370 @default.
• W2044320388 date "2004-04-01" @default.
• W2044320388 modified "2023-10-18" @default.
• W2044320388 title "Analysis of Post-translational CCR8 Modifications and Their Influence on Receptor Activity" @default.
• W2044320388 cites W1503000778 @default.
• W2044320388 cites W1517863220 @default.
• W2044320388 cites W1530393280 @default.
• W2044320388 cites W1539535477 @default.
• W2044320388 cites W1607416604 @default.
• W2044320388 cites W1893012437 @default.
• W2044320388 cites W1994015218 @default.
• W2044320388 cites W2012730930 @default.
• W2044320388 cites W2036821049 @default.
• W2044320388 cites W2037373268 @default.
• W2044320388 cites W2056967947 @default.
• W2044320388 cites W2062250702 @default.
• W2044320388 cites W2086232654 @default.
• W2044320388 cites W2092664889 @default.
• W2044320388 cites W2101436464 @default.
• W2044320388 cites W2130364297 @default.
• W2044320388 cites W2139705683 @default.
• W2044320388 cites W2149864650 @default.
• W2044320388 cites W2155691564 @default.
• W2044320388 cites W2159839943 @default.
• W2044320388 cites W2165494030 @default.
• W2044320388 cites W2271289211 @default.
• W2044320388 cites W2398616419 @default.
• W2044320388 cites W4313308187 @default.
• W2044320388 cites W4313331406 @default.
• W2044320388 cites W4313333788 @default.
• W2044320388 cites W97158386 @default.
• W2044320388 doi "https://doi.org/10.1074/jbc.m309689200" @default.
• W2044320388 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14736884" @default.
• W2044320388 hasPublicationYear "2004" @default.
• W2044320388 type Work @default.
• W2044320388 sameAs 2044320388 @default.
• W2044320388 citedByCount "38" @default.
• W2044320388 countsByYear W20443203882012 @default.
• W2044320388 countsByYear W20443203882013 @default.
• W2044320388 countsByYear W20443203882014 @default.
• W2044320388 countsByYear W20443203882015 @default.
• W2044320388 countsByYear W20443203882017 @default.
• W2044320388 countsByYear W20443203882018 @default.
• W2044320388 countsByYear W20443203882021 @default.
• W2044320388 countsByYear W20443203882023 @default.
• W2044320388 crossrefType "journal-article" @default.
• W2044320388 hasAuthorship W2044320388A5017974200 @default.
• W2044320388 hasAuthorship W2044320388A5030269913 @default.
• W2044320388 hasAuthorship W2044320388A5034123077 @default.
• W2044320388 hasAuthorship W2044320388A5046575837 @default.
• W2044320388 hasAuthorship W2044320388A5051411928 @default.
• W2044320388 hasAuthorship W2044320388A5055503370 @default.
• W2044320388 hasBestOaLocation W20443203881 @default.
• W2044320388 hasConcept C185592680 @default.
• W2044320388 hasConcept C55493867 @default.
• W2044320388 hasConcept C86803240 @default.
• W2044320388 hasConceptScore W2044320388C185592680 @default.
• W2044320388 hasConceptScore W2044320388C55493867 @default.
• W2044320388 hasConceptScore W2044320388C86803240 @default.
• W2044320388 hasIssue "15" @default.
• W2044320388 hasLocation W20443203881 @default.
• W2044320388 hasOpenAccess W2044320388 @default.
• W2044320388 hasPrimaryLocation W20443203881 @default.
• W2044320388 hasRelatedWork W1531601525 @default.
• W2044320388 hasRelatedWork W2319480705 @default.
• W2044320388 hasRelatedWork W2384464875 @default.
• W2044320388 hasRelatedWork W2398689458 @default.
• W2044320388 hasRelatedWork W2606230654 @default.
• W2044320388 hasRelatedWork W2607424097 @default.
• W2044320388 hasRelatedWork W2748952813 @default.
• W2044320388 hasRelatedWork W2899084033 @default.
• W2044320388 hasRelatedWork W2948807893 @default.
• W2044320388 hasRelatedWork W2778153218 @default.
• W2044320388 hasVolume "279" @default.
• W2044320388 isParatext "false" @default.
• W2044320388 isRetracted "false" @default.
• W2044320388 magId "2044320388" @default.
• W2044320388 workType "article" @default.