FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2163270535> ?p ?o ?g. }

• W2163270535 endingPage "14802" @default.
• W2163270535 startingPage "14792" @default.
• W2163270535 abstract "Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the β-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the β-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function. Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the β-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the β-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function. The functional balance of the immune system is regulated, in part, by inhibitory and activatory receptors found on all leukocytes as well as on many non-immune cells (1Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (843) Google Scholar). Within these activatory and inhibitory receptor families, cytoplasmic consensus motifs have been identified and shown to associate with particular signaling mechanisms. These motifs include the immunoreceptor tyrosine-based activation motifs (ITAMs), 1The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; CTLD, C-type lectin-like domain; LOX-1, lectin-like receptor for oxidized low density lipoprotein-1; BGR, β-glucan receptor; MICL, myeloid inhibitory C-type lectin-like receptor; PMN, polymorphonuclear leukocyte; SHP, Src homology-2 domain-containing tyrosine phosphatase; CHO, Chinese hamster ovary; HA, hemagglutinin; RT, reverse transcription; TNF-α, tumor necrosis factor-α; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; SHIP, Src homology-2 domain-containing inositol phosphatase; contig, group of overlapping clones. 1The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; CTLD, C-type lectin-like domain; LOX-1, lectin-like receptor for oxidized low density lipoprotein-1; BGR, β-glucan receptor; MICL, myeloid inhibitory C-type lectin-like receptor; PMN, polymorphonuclear leukocyte; SHP, Src homology-2 domain-containing tyrosine phosphatase; CHO, Chinese hamster ovary; HA, hemagglutinin; RT, reverse transcription; TNF-α, tumor necrosis factor-α; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; SHIP, Src homology-2 domain-containing inositol phosphatase; contig, group of overlapping clones. which become phosphorylated upon stimulation, sending their activatory signals through a variety of intracellular enzyme cascades and resulting in a wide range of cellular outcomes, and the immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which suppress such activatory signals by recruiting phosphatase enzymes that dephosphorylate ITAMs as well as other components of the activatory pathways. The array of inhibitory and activatory receptors expressed by natural killer cells has recently been the focus of much investigation (2McQueen K.L. Parham P. Curr. Opin. Immunol. 2002; 14: 615-621Crossref PubMed Scopus (164) Google Scholar). In humans, these receptors can be divided into two families: the Ig superfamily, including the killer cell Ig-like receptors (3Vilches C. Parham P. Annu. Rev. Immunol. 2002; 20: 217-251Crossref PubMed Scopus (835) Google Scholar) that bind classical major histocompatibility complex class I or HLA G (non-classical), and the C-type lectin-like family (4Lopez-Botet M. Chambers W.H. Methods (Orlando). 1996; 9: 352-361Google Scholar), including the NKG2 receptors, which bind the non-classical HLA E or other distantly related molecules that are up-regulated on stressed, transformed, or infected cells (5Diefenbach A. Tomasello E. Lucas M. Jamieson A.M. Hsia J.K. Vivier E. Raulet D.H. Nat. Immunol. 2002; 3: 1142-1149Crossref PubMed Scopus (380) Google Scholar, 6Diefenbach A. Hsia J.K. Hsiung M.Y. Raulet D.H. Eur. J. Immunol. 2003; 33: 381-391Crossref PubMed Scopus (131) Google Scholar). The C-type lectin and lectin-like proteins are classified into 14 groups based on the arrangement of their C-type lectin-like domains (CTLDs). 2Available at ctld.glycob.ox.ac.uk/. 2Available at ctld.glycob.ox.ac.uk/. In particular, group V receptors include the lectin-like NKG2 family mentioned above, but also a distinct subgroup of these receptors that are expressed predominantly on myeloid and endothelial cells (7Sobanov Y. Bernreiter A. Derdak S. Mechtcheriakova D. Schweighofer B. Duchler M. Kalthoff F. Hofer E. Eur. J. Immunol. 2001; 31: 3493-3503Crossref PubMed Scopus (97) Google Scholar). The members of this subgroup appear to bind diverse ligands, as exemplified by LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) (8Mehta J.L. Li D. J. Am. Coll. Cardiol. 2002; 39: 1429-1435Crossref PubMed Scopus (143) Google Scholar) and the β-glucan receptor (BGR), which is an activatory phagocytic receptor for β-glucans (9Brown G.D. Gordon S. Nature. 2001; 413: 36-37Crossref PubMed Scopus (1293) Google Scholar). 3J. Herre, A. S. J. Marshall, E. Caron, A. D. Edwards, D. L. Williams, E. Schweighoffer, V. L. Tybulewicz, C. Reis e Sousa, S. Gordon, and G. D. Brown, manuscript in preparation. 3J. Herre, A. S. J. Marshall, E. Caron, A. D. Edwards, D. L. Williams, E. Schweighoffer, V. L. Tybulewicz, C. Reis e Sousa, S. Gordon, and G. D. Brown, manuscript in preparation. Although they share structuralhomologywithclassicalcarbohydrate-bindingC-type(Ca2+-dependent) lectins (10Drickamer K. Biochem. Soc. Trans. 1993; 21: 456-459Crossref PubMed Scopus (44) Google Scholar), the group V molecules are termed C-type lectin-like because they lack the residues involved in Ca2+ coordination (11Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (884) Google Scholar). Here, we describe the identification and characterization of a myeloid inhibitory C-type lectin-like receptor (MICL), a novel group V C-type lectin-like receptor that contains an ITIM in its cytoplasmic tail and is most homologous to the LOX-1/BGR subgroup. We demonstrate that it is expressed at the cell surface and is highly N-glycosylated and that its expression is restricted to polymorphonuclear leukocytes (PMNs) and monocytes. We also show that it associates with the signaling phosphatases Src homology-2 domain-containing tyrosine phosphatase (SHP)-1 and SHP-2 and that it can inhibit cellular activation. Cell Lines and Growth Conditions—NIH3T3 fibroblasts, RAW264.7 monocytes, and the PT67 and HEK293T-based Phoenix ecotropic retroviral packaging cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mm l-glutamine. CHO-K1 cells were grown in Ham's F-12 nutrient mixture, and CHO-Lec1 cells were grown in α-minimal essential medium with ribonucleosides and deoxyribonucleosides, both with the supplements described above. All cell lines were obtained from the cell bank of the Sir William Dunn School of Pathology, for except CHO-Lec1 (American Type Culture Collection CRL-1735) (12Stanley P. Caillibot V. Siminovitch L. Cell. 1975; 6: 121-128Abstract Full Text PDF PubMed Scopus (183) Google Scholar, 13Stanley P. Narasimhan S. Siminovitch L. Schachter H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3323-3327Crossref PubMed Scopus (160) Google Scholar), Phoenix ecotropic (a gift from Dr. Gary Nolan, Stanford University), and PT67 (Clontech). In Silico Analysis—Sequence analyses were performed using several on-line tools. 4Available at ca.expasy.org/, www.justbio.com/tools.php, and www.ncbi.nlm.nih.gov/BLAST/. Sequences were aligned with ClustalX (14Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35414) Google Scholar); dendrogram analysis and percentage identities were calculated by DNAMAN Version 4.0 (Lynnon BioSoft). Cloning and Generation of Stable Cell Lines—All routine nucleic acid manipulation techniques were performed essentially as described by Sambrook et al. (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The complete MICL open reading frame was originally cloned into the pBluescript SK II+ vector (Stratagene) from human peripheral blood mononucleocyte cDNA using the Advantage HF2-PCR kit (Clontech) with primers 5′-AAAGGATCCTCTTTACATATTCATCAATG-3′ and 5′-AAACTCGAGACACTCCTTAAATGTATTTG-3′. Hemagglutinin (HA) and V5 epitope tags (used as described below) were generated using adaptor duplex oligonucleotides as described previously (16Brown G.D. Herre J. Williams D.L. Willment J.A. Marshall A.S. Gordon S. J. Exp. Med. 2003; 197: 1119-1124Crossref PubMed Scopus (990) Google Scholar). 5Available at biochem.boehringer-mannheim.com/prod_inf/manuals/epitope/p20.pdf. All tags were inserted at the 3′-end of each sequence, corresponding to the C-terminal extracellular domain of each receptor. The MICL/BGR chimera was generated using overlap extension PCR (17Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6825) Google Scholar) with primers 5′-CTACAACTGATGAGTAACTTCCTATCAAGAAATAAAG-3′ and 5′-TTTATTTCTTGATAGGAAGTTACTCATCAGTTGTAG-3′ such that the MICL 5′-sequence ran into the murine BGR sequence after 95 amino acids, translating as MICL88NISLQLMSMICL95/BGR83NFLSRNKENBGR91. PCR was used to generate cytoplasmic mutant constructs from the V5-tagged full-length MICL/BGR chimera, including the truncated mutant, in which the 5′-end of the gene encoding the cytoplasmic tail (amino acids 1–39) was replaced with a start codon (AUG) and a Kozak sequence, and the ITIM mutant, in which the ITIM (VTYADL, amino acids 5–10) was mutated to VTFADL to inactivate the signaling domain. A schematic of all constructs (without epitope tags) that were used in these experiments is presented in Fig. 1. The fidelity of all clones was confirmed by sequencing. To obtain stable cell lines, constructs were subcloned into the pFBneo (Stratagene) or pMXs-IP (a gift from Professor Toshio Kitamura, University of Tokyo) (18Kitamura T. Koshino Y. Shibata F. Oki T. Nakajima H. Nosaka T. Kumagai H. Exp. Hematol. 2003; 31: 1007-1014Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar) retroviral vector; packaged into virions using HEK293T-based Phoenix ecotropic or PT67 cells as described previously (19Willment J.A. Gordon S. Brown G.D. J. Biol. Chem. 2001; 276: 43818-43823Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar); and transduced into NIH3T3, CHO-K1, CHO-Lec1, or RAW264.7 cells. RAW264.7 and CHO-K1 cells were pretreated with 0.2 μg/ml tunicamycin to increase transduction efficiency. All cell lines were used as non-clonal populations to reduce any founder effects and were generated and tested at least twice to confirm their phenotype. Stable cell lines were selected and maintained in 0.6 mg/ml Geneticin or 3 μg/ml puromycin. RNA Blot Analysis and Reverse Transcription (RT)-PCR—Northern blotting was performed using a full-length MICL cDNA probe as described previously (19Willment J.A. Gordon S. Brown G.D. J. Biol. Chem. 2001; 276: 43818-43823Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 20Stacey M. Lin H.-H. Hilyard K.L. Gordon S. McKnight A.J. J. Biol. Chem. 2001; 276: 18863-18870Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). RNA isolation and RT-PCR were also performed as described previously (19Willment J.A. Gordon S. Brown G.D. J. Biol. Chem. 2001; 276: 43818-43823Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), except that random hexamer primers were used for first-strand cDNA synthesis. Leukocytes were isolated from buffy coats (Bristol Blood Donor Services) and separated into adherent and non-adherent mononuclear cells using Ficoll-Paque™ Plus (Amersham Biosciences) and from fresh peripheral blood and separated into PMNs and total mononuclear leukocytes using Polymorphprep™ (Axis-Shield, Oslo, Sweden) according to the manufacturers' protocols. Fluorescent Zymosan Binding and Internalization and Tumor Necrosis Factor-α (TNF-α) Assays—Fluorescence-based binding assays using fluorescein isothiocyanate (FITC)-labeled zymosan (Molecular Probes, Inc.) were performed as follows. NIH3T3 transfectants were plated at 2 × 105 cells/well in 24-well plates the day prior to each experiment. The cells were washed three times; 100 μg/ml laminarin (Sigma) was added when appropriate; and the cells were incubated for 20 min at 37 °C to allow inhibition of BGR (21Brown G.D. Taylor P.R. Reid D.M. Willment J.A. Williams D.L. Martinez-Pomares L. Wong S.Y. Gordon S. J. Exp. Med. 2002; 196: 407-412Crossref PubMed Scopus (812) Google Scholar). Following the addition of FITC-labeled zymosan (25 particles/cell), the cells were incubated at 37 °C in 5% CO2 for 60 min. After washes to remove unbound particles, the cells were lysed in 3% Triton X-100, and the amount of FITC-labeled zymosan bound by the cells was quantified using a Titertek Fluoroskan II (Labsystems Group Ltd.) as described (21Brown G.D. Taylor P.R. Reid D.M. Willment J.A. Williams D.L. Martinez-Pomares L. Wong S.Y. Gordon S. J. Exp. Med. 2002; 196: 407-412Crossref PubMed Scopus (812) Google Scholar). For the internalization assays, NIH3T3 transfectants were plated at 2 × 105 cells/well in 12-well plates the day prior to each experiment. At the start of the assay, the cells were washed three times with culture medium, and when necessary, cytochalasin D (1 μm; Sigma) was added to the cells 40 min prior to the start of the assay and then maintained throughout the experiment. Following the addition of FITC-labeled zymosan (5 particles/cell), the cells were incubated at 37 °C in 5% CO2 for 30 min, washed to remove unbound particles, and then incubated for a further 90 min at 37 °C. External zymosan was stained with rabbit anti-zymosan antibody (Molecular Probes, Inc.) after blocking with phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 5% heat-inactivated goat serum and then detected with allophycocyanin-conjugated goat anti-rabbit antibody (Molecular Probes, Inc.). The cells were lifted and fixed in 1% formaldehyde, and flow cytometry analysis (carried out according to conventional protocols) was performed by gating on the FITC-positive cell populations, which had bound zymosan. The percentage of phagocytosis was determined by comparing the allophycocyanin-negative (internalized particles) and allophycocyanin-positive (non-internalized particles) cell populations. TNF-α production in RAW264.7 transfectants stimulated with zymosan was determined as described previously (16Brown G.D. Herre J. Williams D.L. Willment J.A. Marshall A.S. Gordon S. J. Exp. Med. 2003; 197: 1119-1124Crossref PubMed Scopus (990) Google Scholar). After taking samples for anti-TNF-α enzyme-linked immunosorbent assay, the amount of FITC-labeled zymosan bound by the cells was quantified as described above. All graphical data are presented as the means ± S.D. from a representative experiment. Immunoprecipitation—To identify MICL-associated phosphatases in RAW264.7 transfectants, cells were washed twice with PBS and resuspended in PBS containing 0.5% bovine serum albumin and 2 mm NaN3. Aliquots containing 5 × 107 cells were incubated for 2 h at 4 °C with 5 μg of anti-HA antibody. Excess antibody was then removed by washing, and the cells were resuspended in 2.25 ml of PBS and prewarmed to 37 °C for 3 min before stimulation with 250 μl of pervanadate (or PBS-only control), followed by incubation at 37 °C for 10 min. Pervanadate (22Swarup G. Cohen S. Garbers D.L. Biochem. Biophys. Res. Commun. 1982; 107: 1104-1109Crossref PubMed Scopus (571) Google Scholar) was prepared from 1 mm activated Na3 VO4 6Available at www.upstate.com/misc/protocols.q.prot.e.activation/Activation+of+Sodium+Orthovanadate. and 10 mm H2O2 (final concentrations). Cells were lysed in Nonidet P-40 buffer (1% Nonidet P-40, 150 mm NaCl, 10 mm EDTA, 10 mm NaN3, 10 mm Tris-HCl (pH 8), 2 mm Na3VO4, 10 mm NaF, and complete EDTA-free protease inhibitors (Roche Applied Science)) and incubated on ice for 45 min. Nuclei were pelleted at 12,000 × g for 20 min at 4 °C. Supernatants were added to 5 μl of anti-mouse Dynabeads® (precoated with 2% bovine serum albumin, Dynal Biotech) and incubated overnight at 4 °C. Beads were then washed four times with Nonidet P-40 buffer, and precipitated proteins were denatured by boiling for 5 min in reducing sample buffer. Aliquots were subjected to SDS-10% PAGE, and proteins were transferred to Hybond-C Extra membranes (Amersham Biosciences) for 90 min at 100 V. Proteins were probed with primary antibody in PBS containing 2% skimmed milk powder for 2 h at room temperature, followed by peroxidase-conjugated secondary antibody for 60 min at room temperature and detection using ECL substrate (Amersham Biosciences). The primary antibodies used were mouse anti-HA (HA.11, Covance), rabbit anti-SHP-1 (Upstate Biotechnology, Inc.), mouse anti-SHP-2 (clone 79, BD Biosciences) and rabbit anti-Src homology-2 domain-containing inositol phosphatase (SHIP) (a gift from Dr. Gerry Krystal). MICL Is a C-type Lectin-like Transmembrane Receptor with a Cytoplasmic ITIM—We identified a novel human immunoreceptor that we named MICL (Fig. 2A) through data base searches for molecules homologous to BGR. The MICL gene consists of six exons and is located on the (+)-strand of chromosome 12 (locus 12p13.31) within the natural killer gene complex (23Yokoyama W.M. Plougastel B.F. Nat. Rev. Immunol. 2003; 3: 304-316Crossref PubMed Scopus (476) Google Scholar), spanning ∼14 kb on GenBank™/EBI genomic contig NT_009714 (Fig. 2B). It is positioned at the telomeric end of a cluster of homologous genes that include LOX-1, BGR, CLEC-1, and CLEC-2 (Fig. 2C) (7Sobanov Y. Bernreiter A. Derdak S. Mechtcheriakova D. Schweighofer B. Duchler M. Kalthoff F. Hofer E. Eur. J. Immunol. 2001; 31: 3493-3503Crossref PubMed Scopus (97) Google Scholar). The predicted open reading frame encodes a 265-amino acid type II transmembrane polypeptide with an expected mass of ∼31 kDa and comprises one CTLD, a stalk/neck region, a single transmembrane domain, and a cytoplasmic tail containing an archetypal ITIM (Figs. 1A and 2A). The CTLD contains the six canonical cysteines, present in almost all C-type lectins; and in the stalk, there are two extra cysteines that may allow homo- or heterodimerization (24Lazetic S. Chang C. Houchins J.P. Lanier L.L. Phillips J.H. J. Immunol. 1996; 157: 4741-4745PubMed Google Scholar). The ectodomain also contains several putative N-glycosylation sites and one predicted O-glycosylation site. 7Available at www.cbs.dtu.dk/services/NetNGlyc/ and www.cbs.dtu.dk/services/NetOGlyc/. Two single nucleotide polymorphisms in the coding region have been identified (at 255 and 790 bp in Fig. 2A), although only the latter one results in an amino acid substitution (Q244K). The Lys244 allele was previously deposited in the GenBank™/EBI Data Bank under accession number NM_138337, but its functional significance is not yet known. MICL Shares Homology with LOX-1, BGR, CLEC-1, and CLEC-2 as Well as Other Inhibitory Receptors—MICL is most homologous to other human C-type lectin-like molecules on chromosome 12 that are found in the same cluster (Fig. 2C), including two novel transcripts with GenBank™/EBI accession numbers AY358265 and AY358810 (Fig. 3A). The predicted MICL CTLD sequence is most similar to those of AY358265, LOX-1, and BGR (Fig. 3B). Alignments of the CTLDs of MICL and related proteins show that they all contain the six conserved cysteines, but lack the Ca2+-binding residues that are present in classical C-type lectins such as rat MBP-A (Fig. 3A). Furthermore, none of the known sugar-binding motifs present in classical C-type lectins, e.g. EPN/QPD (11Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (884) Google Scholar), are found in MICL. Analysis of the MICL cytoplasmic tail revealed homology to other ITIM-containing group V C-type lectin-like molecules (Fig. 3C) such as AY358810, CD72, and murine Ly49C. The ITIM is also similar to those of certain better characterized type I transmembrane receptors, viz. signal-regulatory protein-α (SIRP-α), ILT5, KIR3DL1, and KIR2DL4 (data not shown). Recently, the murine ortholog of MICL was identified (Gen-Bank™/EBI accession number XM_149798). This gene is located within the equivalent gene cluster on the syntenic region of mouse chromosome 6. The encoded protein has 49% overall identity (∼70% similarity) to human MICL and shares many of the features mentioned above, e.g. exon structure, N-glycosylation sites, and extra cysteines in the stalk. Similar MICL-related sequences are also present in other vertebrate genomes, including Rattus norvegicus, Bos tauros, and Danio rerio (data not shown). MICL Has at Least Three Alternatively Spliced Transcripts, and Expression Is Restricted to PMNs and Monocytes—We examined the expression of human MICL by northern blotting and RT-PCR analysis. By northern blotting using a full-length cDNA probe, we were able to detect MICL transcripts in several tissues, especially those rich in leukocytes. One predominant transcript of 1.5 kb was observed in positive tissues, as were several transcripts of greater length. High levels of MICL transcripts were detected in peripheral blood leukocytes and bone marrow, with lower levels in spleen, fetal liver, heart, colon, placenta, and lung (Fig. 4A) as well as testis (data not shown). Blotting with an alternative MICL cDNA probe and increased hybridization stringency gave identical results, thus confirming the specificity of the hybridizing bands (data not shown). We then determined the peripheral blood cell populations expressing MICL by RT-PCR and detected high levels of MICL transcripts in both PMNs and total mononuclear leukocytes (Fig. 4B). We separated the mononuclear leukocyte populations into non-adherent cells (predominantly lymphocytes) and adherent cells (predominantly monocytes), and the latter were also allowed to mature (from 1 to 7 days) into macrophages. MICL was detected in immature monocytes, but expression was gradually lost upon maturation toward macrophages; lymphocytes were MICL-negative. This experiment was repeated using blood from three separate donors, and comparable results were obtained, although the degree of MICL down-regulation concomitant with monocyte maturation was variable. Several human cell lines from a range of sources were also analyzed for MICL expression. Consistent with previous data, only the monocytic/promyelocytic cells tested (U937, HL-60, and MonoMac6) were clearly MICL-positive (Fig. 4C). Moreover, maturation of the monocytic/promyelocytic cell lines THP-1 and HL-60 by phorbol 12-myristate 13-acetate, which induces a macrophage-like phenotype, down-regulated MICL expression as shown by northern blotting (Fig. 4D). In contrast, when HL-60 cells were treated with all-trans-retinoic acid and Me2SO, which induce a PMN-like phenotype (25Collins S.J. Bodner A. Ting R. Gallo R.C. Int. J. Cancer. 1980; 25: 213-218Crossref PubMed Scopus (328) Google Scholar), MICL expression was maintained. This PMN/monocyte-restricted mRNA expression was further corroborated by SAGE, 8E. J. Evans, personal communication. which identified MICL transcripts in only myeloid cells, viz. granulocytes, monocytes, and immature dendritic cells (26Evans E.J. Hene L. Sparks L.M. Dong T. Retiere C. Fennelly J.A. Manso-Sancho R. Powell J. Braud V.M. Rowland-Jones S.L. Mc-Michael A.J. Davis S.J. Immunity. 2003; 19: 213-223Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 27Hashimoto S. Nagai S. Sese J. Suzuki T. Obata A. Sato T. Toyoda N. Dong H.Y. Kurachi M. Nagahata T. Shizuno K. Morishita S. Matsushima K. Blood. 2003; 101: 3509-3513Crossref PubMed Scopus (48) Google Scholar). 9Available at bloodsage.gi.k.u-tokyo.ac.jp/. MICL was not detected in any SAGE library of lymphoid origin or in mature dendritic cells. All non-immune tissue libraries, except lung and liver, were also MICL-negative (28Lash A.E. Tolstoshev C.M. Wagner L. Schuler G.D. Strausberg R.L. Riggins G.J. Altschul S.F. Genome Res. 2000; 10: 1051-1060Crossref PubMed Scopus (353) Google Scholar). 10Available at www.ncbi.nlm.nih.gov/SAGE. Thus, MICL appears to be expressed preferentially by granulocytes and monocytes. RT-PCR analysis of peripheral blood leukocytes (Fig. 4B) identified three alternatively spliced isoforms of ∼800, 700, and 1100 bp (MICL-α, -β, and -γ, respectively). These were cloned and sequenced, and their predicted structures are depicted in Fig. 2B. MICL-α is the isoform characterized in this work; MICL-β lacks exon 2 (encoding the transmembrane region); and in the MICL-γ transcript, the second intron is un-spliced, resulting in the introduction of a stop codon after 18 codons. Transcripts similar to the MICL-γ isoform were also found in several human expressed sequence tags. MICL Is Highly Glycosylated and Is Expressed at the Cell Surface—To investigate MICL protein expression and function, we generated HA epitope-tagged MICL transfectants in a variety of cell lines. Because several similar C-type lectin and lectin-like proteins are not expressed at the cell surface when transfected into certain cell lines (29Colonna M. Samaridis J. Angman L. Eur. J. Immunol. 2000; 30: 697-704Crossref PubMed Scopus (187) Google Scholar, 30Bellon T. Heredia A.B. Llano M. Minguela A. Rodriguez A. Lopez-Botet M. Aparicio P. J. Immunol. 1999; 162: 3996-4002PubMed Google Scholar), 11E. P. McGreal, G. D. Brown, S. Heinsbroek, S. Zamze, S. Y. Wong, S. Gordon, L. Martinez-Pomares, and P. R. Taylor, manuscript in preparation. presumably due to the lack of accessory molecules, we examined the surface expression of MICL transfected into NIH3T3 fibroblasts (Fig. 5A). Analysis by flow cytometry of live transfected cells demonstrated high levels of MICL expression at the cell surface, indicating that accessory molecules were not required. We also examined HA-tagged MICL expression by western blotting and observed bands between 40 and 75 kDa, which are much higher molecular masses than predicted from the polypeptide sequence alone (Fig. 5B). These bands were detected similarly under both reducing and nonreducing conditions in NIH3T3 and RAW264.7 lysates (data not all shown), suggesting they were due to glycosylation of MICL rather than dimerization. To confirm that MICL was glycosylated, we expressed MICL-HA in CHO-Lec1 cells, which are N-glycosylation-deficient (12Stanley P. Caillibot V. Siminovitch L. Cell. 1975; 6: 121-128Abstract Full Text PDF PubMed Scopus (183) Google Scholar, 13Stanley P. Narasimhan S. Siminovitch L. Schachter H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3323-3327Crossref PubMed Scopus (160) Google Scholar), as well as in wild-type CHO-K1 cells. CHO-K1 cells expressed glycosylated MICL in a similar fashion to NIH3T3 cells, but in CHO-Lec1 cells, the molecular mass of MICL was reduced to near its predicted unglycosylated mass (∼31 kDa). Therefore, MICL is expressed at the cell surface and is highly N-glycosylated. MICL Is Associated with SHP-1 and SHP-2, but Not with SHIP—ITIM-containing receptors are known to exact their inhibitory functions through the recruitment of phosphatase enzymes, including SHP-1, SHP-2, and SHIP (1Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (843) Google Scholar). We therefore wanted to determine which, if any, of these phosphatases are recruited by the ITIM of MICL. To this end, we expressed HA-tagged MICL in RAW264.7 cells because this cell line is known to express all three phosphatases (31Angata T. Kerr S.C. Greaves D.R. Varki N.M. Crocker P.R. Varki A. J. Biol. Chem. 2002; 277: 24466-24474Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 32Lucas D.M. Rohrschneider L.R. Blood. 1999; 93: 1922-1933Crossref PubMed Google Scholar) and isolated MICL-positive signaling complexes from these transfectants by anti-HA immunoprecipitation following treatment with and without pervanadate (Fig. 6), which stimulates recruitment of signaling molecules (22Swarup G. Cohen S. Garbers" @default.
• W2163270535 created "2016-06-24" @default.
• W2163270535 creator A5004410792 @default.
• W2163270535 creator A5007773455 @default.
• W2163270535 creator A5033677559 @default.
• W2163270535 creator A5059582877 @default.
• W2163270535 creator A5060388462 @default.
• W2163270535 creator A5070609764 @default.
• W2163270535 date "2004-04-01" @default.
• W2163270535 modified "2023-10-16" @default.
• W2163270535 title "Identification and Characterization of a Novel Human Myeloid Inhibitory C-type Lectin-like Receptor (MICL) That Is Predominantly Expressed on Granulocytes and Monocytes" @default.
• W2163270535 cites W1484816825 @default.
• W2163270535 cites W1511482735 @default.
• W2163270535 cites W1529522766 @default.
• W2163270535 cites W1538902372 @default.
• W2163270535 cites W1550634531 @default.
• W2163270535 cites W1588352485 @default.
• W2163270535 cites W1676290296 @default.
• W2163270535 cites W1918312395 @default.
• W2163270535 cites W1963962536 @default.
• W2163270535 cites W1964297277 @default.
• W2163270535 cites W1971899067 @default.
• W2163270535 cites W1979707641 @default.
• W2163270535 cites W1980138952 @default.
• W2163270535 cites W1985106225 @default.
• W2163270535 cites W1985789843 @default.
• W2163270535 cites W1988621302 @default.
• W2163270535 cites W1988940275 @default.
• W2163270535 cites W1991197142 @default.
• W2163270535 cites W1992120836 @default.
• W2163270535 cites W1994603967 @default.
• W2163270535 cites W1995245022 @default.
• W2163270535 cites W1999472502 @default.
• W2163270535 cites W1999841276 @default.
• W2163270535 cites W2013394935 @default.
• W2163270535 cites W2019062483 @default.
• W2163270535 cites W2020745942 @default.
• W2163270535 cites W2021716242 @default.
• W2163270535 cites W2028407058 @default.
• W2163270535 cites W2042728577 @default.
• W2163270535 cites W2058771802 @default.
• W2163270535 cites W2059688685 @default.
• W2163270535 cites W2060913542 @default.
• W2163270535 cites W2066967399 @default.
• W2163270535 cites W2071548379 @default.
• W2163270535 cites W2076227586 @default.
• W2163270535 cites W2077527031 @default.
• W2163270535 cites W2078958514 @default.
• W2163270535 cites W2079581923 @default.
• W2163270535 cites W2080683076 @default.
• W2163270535 cites W2083920899 @default.
• W2163270535 cites W2090487140 @default.
• W2163270535 cites W2092736520 @default.
• W2163270535 cites W2095331504 @default.
• W2163270535 cites W2096294540 @default.
• W2163270535 cites W2097382368 @default.
• W2163270535 cites W2101994976 @default.
• W2163270535 cites W2107531246 @default.
• W2163270535 cites W2110882632 @default.
• W2163270535 cites W2114970533 @default.
• W2163270535 cites W2120017010 @default.
• W2163270535 cites W2122200639 @default.
• W2163270535 cites W2125558666 @default.
• W2163270535 cites W2126924404 @default.
• W2163270535 cites W2128850053 @default.
• W2163270535 cites W2131384515 @default.
• W2163270535 cites W2132268212 @default.
• W2163270535 cites W2135364324 @default.
• W2163270535 cites W2141181488 @default.
• W2163270535 cites W2150784028 @default.
• W2163270535 cites W2150943213 @default.
• W2163270535 cites W2169261909 @default.
• W2163270535 cites W2170533080 @default.
• W2163270535 cites W2171332001 @default.
• W2163270535 cites W2172180689 @default.
• W2163270535 cites W2212341047 @default.
• W2163270535 cites W2334505016 @default.
• W2163270535 cites W2346213975 @default.
• W2163270535 cites W2409887359 @default.
• W2163270535 cites W4237747603 @default.
• W2163270535 cites W4255256360 @default.
• W2163270535 cites W4313331951 @default.
• W2163270535 doi "https://doi.org/10.1074/jbc.m313127200" @default.
• W2163270535 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14739280" @default.
• W2163270535 hasPublicationYear "2004" @default.
• W2163270535 type Work @default.
• W2163270535 sameAs 2163270535 @default.
• W2163270535 citedByCount "129" @default.
• W2163270535 countsByYear W21632705352012 @default.
• W2163270535 countsByYear W21632705352013 @default.
• W2163270535 countsByYear W21632705352014 @default.
• W2163270535 countsByYear W21632705352015 @default.
• W2163270535 countsByYear W21632705352016 @default.
• W2163270535 countsByYear W21632705352017 @default.
• W2163270535 countsByYear W21632705352018 @default.
• W2163270535 countsByYear W21632705352019 @default.
• W2163270535 countsByYear W21632705352020 @default.
• W2163270535 countsByYear W21632705352021 @default.

• next