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The Journal of Immunology, 2000, 165: 4970-4977.
Copyright © 2000 by The American Association of Immunologists

Stimulatory Function of gp49A, a Murine Ig-Like Receptor, in Rat Basophilic Leukemia Cells1

Kwang Ho Lee2, Masao Ono, Masanori Inui, Takae Yuasa and Toshiyuki Takai3

Department of Experimental Immunology and Core Research for Evolutionary Science and Technology Program, Japan Science and Technology Corp, Institute of Development, Aging and Cancer, Tohoku University, Seiryo, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Murine gp49, a 49-kDa type I transmembrane glycoprotein, is a member of the Ig-like receptors expressed on the surface of cells involved in natural immunity such as mast cells, NK cells, and macrophages. The two major subtypes, gp49A and gp49B, are encoded by two different genes adjacent to each other. gp49B contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic region and is known to function as an inhibitory molecule. In contrast, gp49A does not harbor any specific motif for signal transduction, nor has its physiological role been determined. Here we report on the stimulatory nature of gp49A by analyzing biochemical characteristics of chimeric molecules consisting of an ectodomain of Fc receptor and a C-terminal half of gp49A, namely the pretransmembrane, transmembrane, and cytoplasmic portions, expressed on the rat basophilic leukemia mast cell line. Cross-linking of the chimeric receptors evoked cytoplasmic calcium mobilization, PGD2 release, and transcription of IL-3 and IL-4 genes, but did not elicit degranulation of the cells. The chimeric molecule could be expressed as a singlet and a homodimeric form on the cell surface. A pretransmembrane cysteine residue of gp49A was necessary for dimer formation. Dimerization was be necessary for their incorporation into glycolipid-enriched membrane fraction (GEM) upon cross-linking stimuli. The calcium mobilization response was inhibited by treatment of cells with methyl-ß-cyclodextrin, an inhibitor of GEM formation. Together with these results, it was strongly suggested that gp49A could be expressed as a homodimer and elicit activation signals that lead to calcium mobilization, eicosanoid production, and cytokine gene transcription through its incorporation into GEM.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In addition to well-known immunoreceptors, such as TCR, B cell receptor, and Fc receptor (FcR),4 several new members of the Ig superfamily have been identified recently and suggested to play important roles in cellular functions and in immunity (for review, see Refs. 1, 2, 3, 4). Subfamilies of these include human killer cell Ig-like receptors (KIR) (5, 6, 7), human Ig-like transcript/leukocyte Ig-like receptor/myeloid Ig-like receptor (ILT/LIR/MIR) molecules (8, 9, 10, 11), signal induction receptor proteins (12, 13, 14) in both mouse and man, and murine gp49 molecules (15, 16) and paired Ig-like receptors (PIRs) (17, 18). The common features of members within each of these Ig-like receptor subfamilies are 1) their highly homologous ectodomains and 2) inclusion of one or more inhibitory isoform(s) that harbors an immunoreceptor tyrosine-based inhibitory motif (ITIM) or ITIM-like sequences, as well as 3) the presence of a noninhibitory or activating-type isoform(s), several of which have been shown to be associated with subunit molecules containing an immunoreceptor tyrosine-based activation motif (ITAM) such as FcR {gamma} subunit (FcR{gamma}) or DAP12/KARAP homodimer (19, 20, 21, 22, 23). For example, inhibitory isoforms of KIR, such as KIR2DL, expressed on NK cells and a subset of T cells negatively regulate those cells upon engagement with MHC class I molecules on target cells, so as to prevent these effector cells from damaging normal self cells (24), while activating KIRs such as KIR2DS that associate with a DAP12/KARAP homodimer have been shown to deliver an activation signal upon aggregation (22). Murine ITIM-harboring PIR-B molecule was shown to be inhibitory to cell activation (25, 26, 27), whereas the noninhibitory partner PIR-A was demonstrated to be an activation receptor for cellular responses by associating with FcR{gamma} (19, 20, 28).

gp49 in the murine system is also composed of gp49A and gp49B, noninhibitory and inhibitory isoforms, respectively. Amino acid sequences for ectodomains of gp49A and gp49B are 88% identical and are homologous to other Ig-like receptors, such as mouse PIR, human KIR, ILT/LIR/MIR, and FcR (11, 29). Two separate, but neighboring, genes located on the mouse chromosome 10 B4 region encode the gp49A or gp49B isoform (30). gp49 was first identified as a cell surface molecule that reacts with mAb B23.1, expressed on mononuclear phagocytes, NK cells, and mast cells, but not on neutrophils, thymocytes, fibroblasts, lymph node cells, or splenocytes (31), suggesting a regulatory role for gp49 in the innate immune system, although subsequent flow cytometric analysis of transfectant cells expressing gp49A or gp49B revealed that mAb B23.1 binds only to the gp49B isoform, not to gp49A (32). Several lines of in vivo evidence suggest that gp49B is inhibitory to target cell killing in NK cells (33, 34) and also to the degranulation response of mast cells (35). In contrast, no biochemical or functional study has been reported to date for gp49A, due mainly to the lack of mAb that binds specifically to the isoform. The primary structure of gp49A does not predict any tyrosine-based motifs in its cytoplasmic portion or any positively charged residues in the transmembrane region, which may play an important role in associating with subunit molecules bearing ITAM (20, 36, 37).

In this report we describe stimulatory functions of gp49A analyzed in its chimeric form, in which the ectodomain of a low affinity FcR for IgG, Fc{gamma}RII, was combined with the pretransmembrane, transmembrane, and cytoplasmic portions of gp49A, expressed on the rat basophilic leukemia mast cell line (RBL-2H3). An mAb, 2.4G2 (38), specific for the Fc{gamma}R ectodomain, enables us to cross-link the chimeric receptor altogether or cocross-link the receptor with other cell surface molecules, such as the high affinity FcR for IgE (Fc{epsilon}RI), so as to characterize the nature of signals transduced by this chimeric receptor. We found that cross-linking of the chimeric receptor induced a small, but substantial, cytoplasmic calcium response, PGD2 release, and transcriptional enhancement of IL-3 and IL-4. Although association of the chimeric receptor with FcR{gamma} subunit was not demonstrated, delivery of the activation signal by the receptor was found to be dependent on their entry into the glycolipid-enriched plasma membrane fraction (GEM) upon cross-linking stimuli. Thus, our results strongly suggest that gp49A could be an activating-type receptor for mast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, Abs, and reagents

The rat mast cell line RBL-2H3 was grown in DMEM supplemented with 8% FCS, 2 mM L-glutamine, antibiotics, and 20 µM 2-ME at 37°C in a humidified CO2 incubator. Rat mAb specific for mouse Fc{gamma}RII/III (2.4G2) and biotin-labeled anti-rat IgG2b were obtained from PharMingen (San Diego, CA). Mouse IgG1 Abs specific for trinitrophenyl (TNP) hapten (anti-TNP IgE) (39) were prepared with DEAE-cellulose column chromatography from supernatant of hybridoma. DTT was obtained from Sigma (St. Louis, MO).

Construction of expression vector for FcR-gp49A chimeric receptor and transfection

The integral cDNA fragment coding for mouse gp49A was prepared from mouse bone marrow-derived cultured mast cell RNA by PCR amplification. The primer pairs for this PCR spanned from the codon corresponding to Thr221 (16) to the stop codon and contain ApaI sites at both ends. The PCR products were digested with ApaI and ligated to the ApaI site downstream of cDNA for the mouse Fc{gamma}RII extracellular region that had been inserted into the pcEXV-3 vector (40) as described previously (20) to generate the expression vector for FcR-gp49A chimeric receptor containing an Fc{gamma}RII ectodomain (aa residues 1–175) (40) followed by gp49A pretransmembrane, transmembrane, and cytoplasmic portions (aa residues 221–303) (16). Cys226 (16) in the gp49A portion of the chimeric receptor was replaced by Phe by site-directed mutagenesis using two overlapping PCR primers surrounding the residue to be changed (20). For obtaining stable transfectants, RBL-2H3 cells were cotransfected with 2 µg of linearized pSV2-Neo vector plus 20 µg of each chimeric construct by electroporation at 250 V and 975 µF using the Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA). After selection with 200 µg/ml geneticin (Life Technologies, Grand Island, NY) viable clones were screened for 2.4G2 binding by flow cytometry. Several clones thus obtained were compared for their expression levels of chimeric receptor and chosen for additional experiments.

Calcium mobilization assay

The details of calcium mobilization assay were described previously (20). Cells were incubated with 2 µM fura-2 (Molecular Probes, Eugene, OR) at 35°C for 20 min and for an additional 10 min with biotin-labeled Abs at 20°C. Cells were washed, resuspended in PBS supplemented with 1 mM each of CaCl2 and MgCl2, placed in a fluorometer, and stimulated with 10 µg of avidin while being agitated with a stir bar. Relative free cytoplasmic calcium in the cells was measured by the ratio of fluorescence emission intensities at 510 nm when the samples were exposed to excitation wavelengths of 340 and 360 nm, respectively. Calibration and calculation of calcium concentration were conducted as previously described (41).

Measurement of cytokine production

Cells in a 10-cm dish (1.2 x 106) were plated and cultured overnight. Exponentially growing cells were then sensitized for 30 min in 2 ml of culture medium with biotinylated anti-TNP IgE (4 µg) or biotin-2.4G2 (4 µg). After two washes with medium, cells were stimulated for 2 h with 20 µg of avidin (Wako Pure Chemicals, Osaka, Japan) in 2 ml of culture medium prewarmed to 37°C. After stimulation, cells were processed for RNA extraction using TRIzol (Life Technologies, Grand Island, NY). The RNA sample was resuspended in 20 µl of TE (10 mM Tris-HC1 (pH 7.4), 1 mM EDTA). For each sample, 1 µl of resuspended RNA was reverse transcribed using the first-strand cDNA synthesis kit (Roche, Indianapolis, IN) to a final volume of 20 µl. For the quantification of cytokine synthesis, 1/5 µl, 1/25 µl and 1/125 µl of each cDNA sample were amplified in separate reactions. Rat ß-actin was amplified in parallel for standardization. The annealing temperatures used for amplification for rat IL-3 and IL-4 were 57 and 53°C, respectively. Every cDNA sample was amplified for 30 cycles (30 s at 94°C, 30 s for annealing, and 30 s at 72°C). The primers used for amplification of the corresponding gene transcripts were: IL-3 sense, 5'-AATAGTGACGACAAAGCCAATCTG-3'; IL-3 antisense, 5'-CATTCCACGGTCATAGGGCGAAAG-3';IL-4 sense, 5'-TTTAGGCTTTCCAGGAAGT-3'; IL-4 antisense, 5'-GAGATCATCAACACTTTGAAC-3'; ß-actin sense, 5'-GTGGGGCGCCCCAGGCACCA-3'; and ß-actin antisense, 5'-GTCCTTAATGTCACGCACGATTTC-3'. The predicted PCR products were 317 bp for IL-3, ~300 bp for IL-4, and 526 bp for ß-actin. Referring to the intensity of the ß-actin bands, optimal amounts of PCR products were loaded for agarose gel electrophoretic analysis.

Measurement of PGD2 release

Cells (3 x 105/well) were cultured overnight in a six-well plate. Exponentially growing cells were then sensitized by adding biotin-labeled 2.4G2 (5 µg/ml) or IgE (2 µg/ml) in fresh medium, followed by incubation at room temperature for 30 min. Avidin (5 µg/ml) in 1 ml of medium was added after washing twice with medium. Culture supernatants (100 µl) were collected at various intervals and diluted for ELISA. The Prostaglandin D2-MOX Enzyme Immunoasay Kit (Cayman Chemical, Ann Arbor, MI) was commercially purchased.

Serotonin release assay

RBL-2H3 cells plated into the culture medium in six-well plates (~6 x 105 cells/well) were grown overnight. Precultured cells were trypsinized, plated again into 96-well plates (5 x 103/well), and cultured for 2 h. The adhered cells were loaded with [1,2-3H]hydroxytryptamine creatine sulfate (4 µCi/ml; Amersham Pharmacia Biotech, Aylesbury, U.K.) for 6 h. After washing, the cells were sensitized at the indicated Abs for 30 min. The secretion was measured for the release of this preloaded mediator, and the percent serotonin release (percent degranulation) was calculated as described previously (20).

Immunoprecipitation and Western blot analysis

Semiconfluent RBL-2H3 transfectants in a 10-cm dish were washed three times with Dulbecco’s PBS without calcium and magnesium (PBS(-); pH 8.0), and biotinylated for 5 min by adding the same volume of PBS(-) plus 1 mg/ml EZ-link sulfo-NHS-biotin (Pierce, Rockford, IL) in PBS(-). After rinsing the cells twice with cold PBS(-), cells were lysed in 450 µl of digitonin lysis solution (20). The lysates were scraped from the dish with a rubber policeman and shaken on ice for 15 min. After centrifugation, the supernatants of the lysates were immunoprecipitated with 50 µg of 2.4G2 conjugated to Sepharose 4B beads (Amersham Pharmacia Biotech). Immunoadsorbed beads were rinsed four times with lysis solution and heat denatured at 95°C for 5 min in the presence or the absence of 5% 2-ME. Samples were resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Membranes were incubated with the Abs indicated, followed by probing with peroxidase-linked streptavidin (Amersham Pharmacia Biotech) or peroxidase-linked donkey anti-rabbit Ig (Amersham Pharmacia Biotech).

N-glycosidase digestion, SDS-PAGE, and silver staining

Monolayers of transfectants in a 15-cm dish were lysated by RIPA buffer (20) and immunoprecipitated with 2.4G2-Sepharose 4B. The Sepharose gel was then resuspended in 20 µl of digestion buffer containing 0.5% SDS, 50 mM 2-ME, and 0.55 M sodium phosphate. A 10-µl aliquot of the resuspended sample was digested in 5 µl of 7.5% Nonidet P-40, 1 U of glycopeptidase F (Sigma), and 13.8 µl of H2O overnight at 37°C. Digested samples were resuspended in 4x SDS-PAGE sample buffer, heated for 5 min at 95°C, and electrophoresed on 12% polyacrylamide gels. Protein bands were stained with a silver staining kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

Transient transfection and assay for association of chimeric receptor with FcR{gamma} subunit

COS-7 cells (1.5 x 105/well) were cultured for 2 days in a 60-mm dish. Five micrograms of each construct was transfected into semiconfluent COS-7 cells by use of DEAE-dextran. Briefly, cells were rinsed three times with DMEM buffered with 50 mM Tris-HCl (pH 7.4; DMEM-Tris) and then incubated with transfection mixture and 0.1 mM chloroquine (Sigma) for 3–4 h at 37°C. For the transfection mixture, DNA together with 400 µg/ml of DEAE-dextran and 1.5 ml of DMEM-Tris were mixed and incubated for 15 min. Cells were rinsed twice with DMEM-Tris to remove the transfection mixture and returned to DMEM containing 5% FCS. After 48-h incubation, protein samples were prepared with digitonin lysis solution. The procedures for immunoprecipitation with 2.4G2 and Western blot were described above. SDS-PAGE was conducted in a reduced condition. To detect FcR{gamma}, membranes were incubated with anti-FcR{gamma} (polyclonal rabbit IgG) followed by peroxidase-linked donkey anti-rabbit Ig (Amersham Pharmacia Biotech). Fc{gamma}RIII, Fc{gamma}RII, and FcR{gamma} in pcEXV-3 were described in detail previously (20).

Detergent solubilization and sucrose gradient centrifugation

Cells (~7 x 106) were plated and grown overnight in a 15-cm dish. All the following steps were performed on ice. Immune complex was prepared by mixing anti-TNP IgG1 and TNP7-OVA to 100 and 5 µg/ml, respectively, for the final concentration. The cells were rinsed with PBS(-) and stimulated with ice-cold immune complex for 30 s. After rinsing cells with PBS(-), pH 8.0, cells were labeled with biotin for 10 min as described above. After thorough rinsing, the cells were lysed in 2 ml of ice-cold MES-buffered saline (MBS) lysis buffer (42), followed by scraping the lysates from the dish with a rubber policeman. After 30-min incubation on ice, the lysates were homogenized with 10 strokes of a loose-fitting Dounce homogenizer. The lysates were then gently mixed with an equal volume of 85% sucrose (w/v) in MBS and placed in the bottom of an SW40 centrifuge tube (Beckman, Palo Alto, CA). Six milliliters of 35% sucrose and 2.5 ml of 5% sucrose in MBS were then layered over the lysate. The gradients were centrifuged for 12 h at 200,000 x g at 4°C in a Beckman SW41 rotor. Fractions were harvested by collecting 1.5 ml from the bottom. Fractions 1–3 were diluted with the same volume of RIPA buffer. Insoluble phases of the fractions (fraction 4) were solubilized by adding Nonidet P-40 to 1% and deoxycholate to 0.4%. Immunoprecipitation of FcR-gp49A chimeric molecules was then performed by adding 2.4G2 Sepharose. Immunoprecipitated samples were rinsed four times with RIPA buffer and resuspended in SDS-PAGE sample buffer. Chimeric molecules were detected by Western blot analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aggregation of FcR-gp49A chimeric receptor-induced calcium response

We transfected RBL-2H3 cells with a chimeric receptor construct for gp49A linked to the extracellular region of mouse Fc{gamma}RII (FcR-gp49A) and isolated the stable transfectants expressing the chimeric receptor to define its function (Fig. 1GoA). In addition we prepared another chimeric receptor, which harbored a point mutation of Cys226 to Phe, so as to investigate a possible association of the chimeric receptor with other cell surface molecules. We chose several stable clones highly expressing the chimeric receptors among transfectants by flow cytometric analysis with 2.4G2, which binds to the ectodomain of Fc{gamma}RII (Fig. 1GoB). RBL-2H3 cells were stained by neither 2.4G2 nor isotype-matched control Ab, rat IgG2b, whereas the transfectants were weakly stained with rat IgG2b. These results indicate the absence of intrinsic receptor recognized by 2.4G2 on RBL-2H3 cells and the presence of nonspecific Fc binding to chimeric receptors. Cross-linking of FcR-gp49A chimeric receptor or its mutant on the cell surface was induced with biotin-2.4G2 and avidin. The effects of aggregation of chimeric receptors on cellular activation were analyzed by cytoplasmic calcium mobilization (Fig. 1Go, C and D). We found that the aggregation of wild-type receptors, which preserved the cysteine residue in its pretransmembrane region, triggered cytoplasmic calcium mobilization, albeit the signal was delayed and weak compared with the stimulation of Fc{epsilon}RI as shown in Fig. 1GoC. Addition of control rat IgG2b followed by anti-rat IgG2b to the transfectant culture did not elicit calcium mobilization (Fig. 1Go, C and D), although the results obtained with the combination of rat IgG2b and anti-rat IgG2b may not be best suited for comparison to those obtained with biotin-2.4G2 and avidin. Untransfected RBL-2H3 (data not shown) and, importantly, Cys-mutated transfectant did not show any calcium mobilization upon addition of biotin-2.4G2 and avidin (Fig. 1GoD). These observations rule out any contribution of intrinsic FcR to the effect of aggregation of wild-type chimeric receptors on calcium mobilization. The cytoplasmic calcium mobilization triggered by cross-linking of FcR-gp49A was also observed under the calcium-depleted condition in the extracellular environment achieved by the presence of EGTA (data not shown), suggesting that calcium mobilization is initiated by calcium release from endoplasmic reticulum. Taken together, the C-terminal half of gp49A, namely pretransmembrane, transmembrane, and cytoplasmic portions, is capable of generating a cellular activation signal in terms of cytoplasmic calcium mobilization upon aggregation. In addition, a pretransmembrane cysteine residue may be essential for this activation.



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  		FIGURE 1. Schematic structure and stimulatory function of FcR-gp49A chimeric receptor in RBL-2H3 cells. A, The schematic structure of FcR-gp49A chimeric receptor. The extracellular region containing two Ig domains of Fc{gamma}RII was ligated to Thr of gp49A. The predicted transmembrane (TM) is boxed. Cys226 was replaced by Phe for mutant transfectant, and each prototype and mutated chimeric receptor was transfected into RBL-2H3 cells. B, Surface expression of transfected receptors on RBL-2H3 cells. Prototype and Cys mutant of gp49A chimeric receptors as well as nontransfected RBL-2H3 cells were stained by PE-2.4G2 or PE-rat IgG2b and analyzed by flow cytometry. Nonstained RBL-2H3 cells were used as a negative control (-). C and D, Intracellular calcium mobilization of transfectants. Prototype (C) and Cys-mutated (D) transfectants were labeled by fura-2 and stimulated by avidin (arrow) after sensitization with indicated biotin-Abs. Stimulation of transfectants with rat IgG2b followed by anti-rat IgG2b was performed.

 
Stimulation of FcR-gp49A chimeric receptor enhanced transcription of cytokine mRNAs and PGD2 release, but did not elicit degranulation

Mast cells perform a significant role in host defense against parasitic and some bacterial infections. A signaling cascade initiated by aggregation of the Fc{epsilon}RI leads to rapid responses, including enhancement of gene expression and release of multiple cytokines and activation of enzymes for proinflammatory mediators such as leukotriene C4 and PGD2, and release of preformed, granule-associated mediators, such as histamine, serotonin, and proteases (43). To test the involvement of gp49A in mast cell functions, we stimulated the chimeric receptor through aggregation with 2.4G2 and examined the amount of cytokine mRNAs by RT-PCR, PGD2 release, and serotonin release. As shown in Fig. 2GoA, stimulation of FcR-gp49A by either 2.4G2 or IgE enhanced the expression of both IL-3 and IL-4 mRNAs about 5- and 25-fold, respectively. However, stimulation of the Cys mutant receptor did not induce any cytokine mRNA tested. Significant cytokine transcription was not observed in cells stimulated with avidin alone. These results indicate that the chimeric receptor, but not the Cys mutant receptor, can activate the signal transducers, which lead to the transcriptional activation of cytokines such as IL-3 or IL-4.



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  		FIGURE 2. Cytokine mRNA expression, PGD2 release and degranulation response of FcR-gp49A transfectants. A, Cytokine mRNA induction of transfectants. Prototype (wild) and Cys-mutated (cys-) transfectants were sensitized with the indicated biotin-Abs and stimulated with avidin. The stimulation with avidin alone was also used as a negative control (-). RNA was prepared from stimulated cells, and cDNA was synthesized by RT-PCR as shown in Materials and Methods. RT-PCR amplification of indicated cytokine messages (arrowed) was performed with ß-actin as a positive control. B, PGD2 release from transfectants after cross-linking of 2.4G2 or IgE. Prototype (wild) and Cys-mutated (cys-) transfectants were sensitized with biotinylated Abs for 30 min and stimulated with avidin. Culture supernatants from the stimulated cells were harvested at the indicated intervals. The amount of released PGD2 was determined by ELISA. The assay was conducted in duplicate. Each sample was assayed at two points of serial dilution. C, Degranulation response of transfectants upon receptor aggregation. The percentage of degranulation denotes the percentage of serotonin released into medium. Receptors on RBL-2H3 transfectants were aggregated with the indicated Abs. {square}, Negative control without Ab; and , biotinylated 2.4G2 alone and avidin alone. Nontransfected RBL-2H3 cells were used as a negative control. The RBL-2H3 transfectant harboring Fc{gamma}RIII (20 ) was used as a positive control. Each column represents the mean of triplicate determinations ± SEM.

 
PGD2 is a proinflammatory eicosanoid synthesized upon activation of enzymes in arachidonic acid cascades. Fig. 2GoB shows a marked elevation of PGD2 release by RBL-2H3 transfectant with the prototype FcR-gp49A receptors after stimulation with 2.4G2. Again, stimulation of Cys mutant receptor did not evoke PGD2 release (Fig. 2GoB), indicating that the prototype FcR-gp49A chimeric receptor, but not Cys mutant receptor, is capable of activating arachidonic acid cascades upon aggregation.

Release of preformed vasoactive amines such as histamine and serotonin is a hallmark of mast cell degranulation. We evaluated degranulative responses of RBL-2H3 cells after aggregation of transfected FcR-gp49A chimeric receptors by measuring serotonin release as shown in Fig. 2GoC. Unexpectedly, none of the wild-type or Cys-mutated receptor transfectants showed any detectable degranulation after stimulation with 2.4G2 (Fig. 2GoC). A marked degranulation upon Fc{gamma}RIII aggregation on RBL-2H3 cells (Fig. 2GoC) indicates that the experimental design for cross-linking the receptors with biotin-2.4G2 and avidin is sufficient for inducing degranulative response. These results suggest that the signal pathway from calcium mobilization to degranulative response might be defective or insufficient upon aggregation of FcR-gp49A chimeric receptors.

FcR-gp49A chimeric receptor functions by a homodimeric structure linked via the pretransmembrane cysteine residues

A large number of receptors mediating intracellular activation signals tend to be expressed as multimeric forms. In this report we found that FcR-gp49A functions as an activation-type receptor in terms of calcium mobilization, cytokine mRNA transcription, and proinflammatory mediator production. Thus, we questioned whether the chimeric receptor is also expressed as a multimeric form on the cell surface as well. To answer this question, we analyzed mutant receptor as well as prototype FcR-gp49A by SDS-PAGE as shown in Fig. 3Go. Both the transfectants were labeled with biotin, lysed, immunoprecipitated with 2.4G2, and detected by Western blot analysis. As shown in Fig. 3GoA, the prototype FcR-gp49A, which has a calculated Mr of 29.5 kDa, was detected as both a high Mr form (~100 kDa) and a lower Mr form (~48 kDa). However, replacement of the Cys226 residue into Phe abolished the formation of the high Mr form. When the samples were reduced with 2-ME and analyzed by SDS-PAGE, only the lower Mr species was detected in the mutant as well as the prototype transfectant (Fig. 3GoA). To investigate whether the high Mr form of the chimeric receptor was composed of single polypeptide species, the total lysates were treated with N-glycosidase followed by reduction with 2-ME, and detected by silver staining after SDS-PAGE. As shown in Fig. 3GoB, only a single band with a Mr of 30 kDa corresponding to the monomeric FcR-gp49A protein was detected for both the prototype and Cys mutant transfectants. Taken together, it is strongly suggested that the prototype FcR-gp49A molecule can be expressed as glycosylated forms of homodimer protein as well as a singular receptor on RBL-2H3 cells.



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  		FIGURE 3. SDS-PAGE analysis of FcR-gp49A. A, Biotin-labeled prototype (wild) and Cys-mutated transfectants (cys-) were lysed with digitonin. Cell extracts were subsequently incubated with 2.4G2-Sepharose 4B. Sepharose 4B was separated from the extracts by centrifugation, followed by SDS-PAGE in reducing condition (+2-ME) or nonreducing condition (-2-ME). Eight and 12% SDS-polyacrylamide gels were used for nonreducing and reducing conditions, respectively. Peroxidase-linked streptavidin was used for the detection. Nontransfected RBL-2H3 cells were used for negative control. B, SDS-PAGE of N-glycosidase-treated FcR-gp49A. RBL-2H3 cells or transfectants (wild and cys-) were lysed with RIPA buffer, followed by immunoprecipitation with 2.4G2-Seharose 4B. The proteins were then digested with N-glycosidase (+). Digested samples were separated on 12% SDS-PAGE and subjected to silver staining. The band corresponding to FcR-gp49A is marked by an arrow. The samples from prototype transfectant cells and RBL-2H3 without treatment of N-glycosidase were used as the negative control (-). SM, size marker.

 
As shown in the preceding sections, activation of transfected RBL-2H3 cells could be observed only in those expressing the prototype FcR-gp49A (Figs. 1Go and 2Go), suggesting the importance of the dimeric form for elicitation of stimulatory signals. Therefore, we next treated the wild-type transfectant with DTT to disrupt disulfide bonds. By treatment of the transfectant with the indicated concentrations of DTT for 1 h, a dimeric form of FcR-gp49A disappeared from SDS-PAGE and Western blot (Fig. 4GoA). By these treatments with DTT, aggregation of the chimeric receptors with 2.4G2 could no longer activate calcium mobilization (Fig. 4GoB). Flow cytometric analysis showed that DTT treatment up to 10 mM did not affect 2.4G2 binding to transfected receptors (data not shown). In contrast, similar treatments with DTT did not affect calcium mobilization after cross-linking Fc{epsilon}RI complex, in which three different subunits, Fc{epsilon}RI{alpha}, Fc{epsilon}RIß, and dimeric FcR{gamma}, are known to be assembled with nondisulfide bonds (Fig. 4GoC). Thus, it is suggested that gp49A can form a homodimer through Cys226, and the dimeric form is essential for mediating stimulatory signals of gp49A.



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  		FIGURE 4. DTT treatment of RBL-2H3 transfectants. A, The prototype transfectant cells (1 x 106) were treated with DTT at the indicated concentrations for 1 h. Cells were then labeled with biotin and subsequently lysed with RIPA buffer. The cell extracts were immunoprecipitated with 2.4G2-Sepharose 4B, and separated by 10% SDS-PAGE under the nonreducing condition. The gel was subjected for immunoblot with peroxidase-linked streptavidin. The bands corresponding to chimeric FcR-gp49A are marked by an arrow. B and C, The prototype transfectants (1 x 106) were similarly treated with DTT and labeled with fura-2. The labeled cells were then stimulated by avidin (arrow) after sensitization with biotin-labeled 2.4G2 (B) or anti-TNP IgE (C).

 
The FcR-gp49A chimeric receptor does not associate with FcR {gamma} subunit

Possible mechanism for activation signaling of gp49A could be an association of the receptor with ITAM-bearing subunit molecules such as an FcR common {gamma} subunit as shown for PIR-A signaling in RBL-2H3 cells described previously by us (20) and in B cells and mast cells by others (19, 28). We tried to detect possible association of FcR{gamma} with FcR-gp49A chimeric receptor on COS-7 cells by transiently transfecting FcR-gp49A and FcR{gamma} construct. However, we could not demonstrate any association of the chimeric receptor with FcR{gamma} as analyzed by immunoprecipitation and Western blot (Fig. 5Go).



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  		FIGURE 5. FcR {gamma} subunit does not associate with FcR-gp49A chimeric molecule. COS-7 cells were cotransfected with FcR{gamma} and the indicated constructs. Fc{gamma}RIII was used for positive control (Fc{gamma}RIII). Single transfection of FcR{gamma} (-) or cotransfection of Fc{gamma}RII (Fc{gamma}RII) was used as the negative control. Digitonin lysates from these cotransfectants were used for immunoprecipitation with 2.4G2 (IP with 2.4G2) followed by immunoblot with anti-FcR{gamma} and peroxidase-linked donkey anti-rabbit Ig. Whole cell lysates (total lysate) were immunoblotted as well. The bands corresponding to FcR{gamma} are marked with an arrow.

 
GEM is involved in activation signals from FcR-gp49A

Some microdomains that are enriched in glycosphingolipids, cholesterol, and specific membrane proteins have been found on plasma membrane (44, 45). Often denoted as GEM or rafts, these domains can be distinguished from the rest of the plasma membrane by their relative insolubility in detergents and low buoyant density (46). Phosphatidylinositol-anchored membrane proteins on lymphocytes are enriched in the detergent-insoluble fractions. These fractions have been implicated in many cellular processes, especially in endocytosis (47, 48) and signal transduction from cell surface receptors (49). Because gp49A does not have any predictive motif for cellular signal events, it was assumed that GEM could be involved in signal transmission from gp49A. The prototype or Cys-mutated chimeric receptors were aggregated with immune complex (anti-TNP IgG1 bound to TNP7-OVA). The cell surface proteins including stimulated constructs on each transfectant were then labeled with biotin and lysed in a buffer containing nonionic detergent. The lysates were then fractionated by isopycnic sucrose gradient centrifugation (42, 46), followed by immunoprecipitation with 2.4G2. The 2.4G2-immunoprecipitated protein from each fraction was fractionated on an acrylamide gel and detected by Western blot. As shown in Fig. 6GoA, incorporation of FcR-gp49A into the seventh fraction could be detected only after stimulation of the chimeric gp49A with immune complex. In our experimental condition, the seventh fraction reproducibly corresponded to the interface between two discontinuous sucrose density layers, where detergent-insoluble substances were visible as an opaque disk, suggesting GEM for this fraction. In contrast, the chimeric receptor in the seventh fraction was little or not detected in either unstimulated wild-type FcR-gp49A (Fig. 6GoB) or stimulated Cys-mutated construct (Fig. 6GoC).



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  		FIGURE 6. SDS-PAGE of GEM fraction. Prototype (A and B) or Cys mutant (C) transfectants were either stimulated (+) or unstimulated (-) with immune complex (anti-TNP IgG1 bound to TNP7-OVA) for 30 s. Cells were then labeled with biotin and lysed with MBS lysis buffer. Lysates were mixed with the same volume of 85% sucrose, and 35 and 5% sucrose were sequentially overlaid on mixed lysate. FcR-gp49A chimeric proteins were immunoprecipitated from 1.5-ml fractions collected from the bottom of the tubes after centrifugation. Individual fractions were analyzed by SDS-PAGE and Western blot for the presence of FcR-gp49A chimeric protein using peroxidase-linked streptavidin (arrow).

 
To determine the possible role of GEM for activation signals from gp49A, we employed methyl-ß-cyclodextrin (MBCD), which is a specific cholesterol-binding agent that neither binds to nor is inserted into plasma membrane but preferentially extracts cholesterol from plasma membrane, resulting in the inhibition of GEM formation. A reduction in calcium response by MBCD treatment was observed after IgE stimulation (Fig. 7GoC), because the Fc{epsilon}RI-mediated response is dependent on its incorporation into GEM (50). Interestingly, cholesterol depletion of membranes with MBCD in 2.4G2-stimulated transfectants resulted in diminished intracellular calcium mobilization, albeit the response was higher in 5 mM MBCD than without treatment (Fig. 7GoB). These results support the hypothesis that gp49A chimeric receptor moves to GEM after stimulation with ligand and functions at least for activation of intracellular calcium mobilization, although the possibility cannot be ruled out that a signaling event other than GEM may be involved during the transduction of signals from gp49A.



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  		FIGURE 7. Calcium mobilization after MBCD treatment of FcR-gp49A transfectants. A, Surface expression of FcR-gp49A after MBCD treatment. Prototype transfectant was treated with the indicated amount of MBCD for 30 min at 37°C, and the surface density of FcR-gp49A was determined using PE-2,4G2. Staining without Ab was used as a negative control ({blacksquare}). MBCD treatment does not change the surface density of FcR-gp49A. Viability of cells was determined by staining with trypan blue. The viability decreased after treatment with >50 mM MBCD. B and C, Calcium mobilization of RBL-2H3 transfectants after MBCD treatment. The same transfectants treated in the same way were stimulated with avidin after sensitization with the indicated Abs (arrow), and their calcium mobilization was determined ([Ca2+]i; nanomolar concentrations).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several novel groups of receptors belonging to the Ig superfamily, collectively termed Ig-like receptors, are shown to be expressed on lymphocytes, myeloid cells, or cells involved in innate immunity. Many of these receptors, including human KIR and ILT/LIR/MIR and murine PIR and gp49, are suggested to play important roles in regulating cellular effector functions (51). Our present report focused on the elucidation of the biochemical and functional nature of gp49A, which has a highly homologous structure to the gp49B isoform, but has not been identified for any speculative motifs relevant to intracellular signaling, or a positively charged amino acid residue in its transmembrane region that is important for association with any ITAM-bearing subunit such as FcR{gamma}. FcR {gamma}-chain binds to activating receptors such as PIR-A (19, 20, 28), activating subsets of ILT (21), and FcRs (2, 3, 4, 52, 53). In contrast, DAP12 associates with noninhibitory KIRs (22), CD94/NKG2, and Ly49D (54). Recently, a novel activating-type subunit called DAP10 has been found to be associated with NKG2D (55). Each subunit bears a negative charge in their transmembrane region that is important for associating with the receptor molecules harboring a positive charge in its transmembrane domain and has been known to be involved in activating signals. Because gp49A lacks a charged amino acid in the transmembrane domain, it is unlikely that gp49A associates with these activation subunits. In support of this idea, attempts to detect any association of FcR-gp49A chimeric molecule with FcR{gamma} have not been successful in transient cotransfection experiments in COS-7 cells (Fig. 5Go). In addition, immunoprecipitation and Western blot analyses of the prototype RBL-2H3 transfectant did not reveal any association of FcR{gamma} with FcR-gp49A (data not shown).

Mast cells play their role by releasing proinflammatory mediators, which are granule associated or newly synthesized upon activation, and by secreting cytokines or chemokines. These effector functions are regulated by cell surface receptors and their signal transducing events. One of the prominent features during cellular activation is intracellular calcium mobilization. FcR-gp49A chimeric receptors cross-linked by 2.4G2 induced the release of intracellular calcium mobilization, albeit it was weaker than that after Fc{epsilon}RI stimulation. Transcription for cytokine mRNAs such as those for IL-3 and IL-4 after stimulation with 2.4G2 were promoted at comparable levels as those by Fc{epsilon}RI stimulation, suggesting that the signals triggered by a low amount of calcium release might be sufficient to induce the activation of IL-3 and IL-4 genes. In addition, we observed PGD2 release upon activation. It is interesting to clarify whether the induction of cytokine transcripts and PGD2 generation is truly calcium dependent. In this context, the possible involvement of inducible cyclo-oxygenase-2 in PGD2 generation remains to be determined. In any case, IL-4 is known to be the most important cytokine that mediates IgE synthesis (56) and to be involved in eosinophil recruitment to the airway (57). IL-3 is involved in the growth of mast cells and basophils, especially in eosinophil survival (58). PGD2 act as a vasodilator and as a bronchoconstrictor. Therefore, we speculate that gp49A is closely involved in hypersensitive responses in mice.

Sphingolipid microdomains are thought to be the result of the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the GPI-anchored proteins are enriched. Cholesterol associates with sphingolipids in the Golgi complex and stabilizes the microdomains to which GPI-anchored proteins become associated by way of lipid-lipid interaction (44, 46). Lowering the cellular cholesterol levels markedly affects the properties of GPI-anchored proteins and leads to their dispersion on the cell surface (59), a decrease in their cell surface expression (60), their release as membrane vesicles (61), and an increase in solubility in nonionic detergents (60). MBCD was known to preferentially extract cholesterol from the outside, rather than within, the sphingolipid microdomains, and this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and nonvesicular forms (62). In our experiments, cholesterol extraction by MBCD generally led to a decrease in intracellular calcium mobilization through FcR-gp49A signals in a dose-dependent manner (Fig. 7GoB). Repeated experiments, however, indicated that a small amount of MBCD (5 mM) conversely augmented the intracellular calcium mobilization more than without MBCD treatment. We suggest that signal transmission from gp49A might not be entirely dependent on sphingolipid microdomains, and other events can be involved in complete signal transduction from gp49A.

Based on our observation shown in Fig. 6Go, we speculated that dimer formation of gp49A could be a prerequisite for incorporation into GEM. The wild-type monomeric receptor was also observed in GEM in concert with incorporation of dimeric receptor into GEM. As the Cys-mutated monomeric receptor by itself was not found incorporated into GEM, it is suggested that the wild-type monomeric receptor may be involved in the receptor assembly including dimeric receptor upon 2.4G2 cross-linking and is passively incorporated into GEM. Mechanisms for compartmentation of the dimeric receptor represented here should be of interest as a novel paradigm of GEM-associated signal transduction.

Our knowledge on Ig-like receptors was accumulated during the last several years in terms of the structure, expression, and mode of functions. Many, but not all, inhibitory-type and noninhibitory-type Ig-like receptors within a subfamily tend to be expressed on cell surfaces in a pairwise fashion. For example, stimulating-type PIR-A and inhibitory PIR-B are coexpressed on many cell types (18, 28, 63). Like other Ig-like receptors, gp49A and gp49B are also coexpressed on mast cells and other cells, as shown at least in RT-PCR analysis (K. H. Lee and T. Takai, unpublished observation). Stimulating-type Ig-like receptors, such as ILT-1 (21), PIR-A (19, 20, 28), and KIR2DS (22), are demonstrated to be associated with ITAM-harboring FcR{gamma} or DAP12. Our present report strongly suggests that gp49A functions as a unique stimulating-type receptor that does not associate with an activating subunit, but is incorporated into GEM so as to stimulate cellular function.


   Footnotes
 
1 This work was supported by Core Research for Evolutionary Science and Technology, Japan Science and Technology Corp, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan. Back

2 Current address: Faculty of Life Science, College of Natural Science, Konkuk University, Danwol Dong, Chung Ju, Chung Buk 380-701, South Korea. Back

3 Address correspondence and reprint requests to Dr. Toshiyuki Takai, Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo 4-1, Sendai 980-8575, Japan. Back

4 Abbreviations used in this paper: FcR, Fc receptor; KIR, killer cell Ig-like receptor; ILT/LIR/MIR, Ig-like transcript/leukocyte Ig-like receptor/myeloid Ig-like receptor; PIR, paired Ig-like receptor; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; FcR{gamma}, FcR common {gamma} subunit; Fc{epsilon}RI, high affinity FcR for IgE; Fc{gamma}R, FcR for IgG; Fc{gamma}RII and Fc{gamma}RIII, type II and type III FcR for IgG; GEM, glycolipid-enriched membrane fraction; MBCD, methyl-ß-cyclodextrin; MBS, MES-buffered saline; PBS(-), Dulbecco’s PBS without calcium and magnesium; TNP, trinitrophenyl; RBL-2H3, rat basophilic leukemia cells. Back

Received for publication March 27, 2000. Accepted for publication August 8, 2000.


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