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Mediate Endocytosis and Phagocytosis Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5030-5034, June 1992 Immunology Two forms of the low-affinity Fc receptor for IgE differentially mediate endocytosis and phagocytosis: Identification of the critical cytoplasmic domains (CD23/endocytosis/phagocytosis) AKIRA YOKOTA*, KAzUNORI YUKAWA*, AKITSUGU YAMAMOTOt, KENJI SUGIYAMA*, MASAKI SUEMURAt, YUTAKA TASHIROt, TADAMITSU KISHIMOTO*t, AND HITOSHI KIKUTANI* *Institute for Molecular and Cellular Biology, Osaka University, 1-3, Yamada-oka, Suita, Osaka 565, Japan; *Department of Medicine III, Osaka University Medical School, 1-1-50, Fukushima, Fukushima-ku, Osaka 553, Japan; and tDepartment of Physiology, Kansai Medical University, Moriguchi-shi, Osaka 570, Japan Contributed by Tadamitsu Kishimoto, February 21, 1992 ABSTRACT We have previously identified two species of FceRIIb may be involved in B-cell function and IgE-mediated the low-affinity human Fc receptor for IgE, FceRIIa and immunity, respectively. FceRIIb, which differ only in a short stretch of amino acids at In this paper, we have attempted to elucidate the molecular the N-terminal cytoplasmic end. Their differential expressions basis ofthe functional difference between these two forms of on B cells and monocytes suggest that FceRlla and FceRIIb are receptor molecules by using stable transfectants expressing involved in B-cell function and IgE-mediated immunity, re- either human wild-type or mutated FceRII and have demon- spectively. Here we show that FceRII-mediated endocytosis is strated that endocytosis is mediated only through FceRIIa observed only in FceRlia-expressing cells, whereas IgE- and that phagocytosis is mediated only through FceRIIb. dependent phagocytosis isobserved only in FceRIEb-expressing Furthermore, the minimum amino acid residues necessary cells, demonstrating the functional difference between FceRIIa for endocytosis and phagocytosis have been determined. The and FceR1Ib. Furthermore, site-directed mutagenesis revealed results indicate that endocytosis and phagocytosis of FceRII that the tyrosine residue in the FceRlla-speciflc region is are independent phenomena mediated through distinct amino important for endocytosis, and the Asn-Pro residues in the acid residues. FceRllb-specific region are required for phagocytosis. These fidings suggest that endocytosis and phagocytosis are func- tionally separable phenomena involving distinct amino acid MATERIALS AND METHODS residues. Establishment ofWild-Type and Mutant FceRII-Expressing Transfectants. The FceRIIb cDNA (16) was cloned into the Subpopulations of leukocytes express the low-affinity Fc M13 phage vector. Each ofthe mutations was introduced into receptor for IgE (FceRII), which is believed to have multiple the region of FceRIIb encoding the cytoplasmic domain by functions in B lymphocytes and in effector cells of IgE- using the Amersham in vitro mutagenesis kit according to the mediated immunity (1-3). FceRII has been known as a B-cell manufacturer's instruction. The FceRIIa (15), FcERIIb, and differentiation antigen, CD23 (4-7), and has been proposed to mutant FcERIIb cDNAs were cloned into the pZip-neo vector be involved in several B-cell functions such as the growth- and transfected into qk2 cells by calcium phosphate copre- promoting effect (8, 9), cell adhesion (10), and IgE-mediated cipitation. After G418 selection, cells that strongly expressed antigen presentation.(11). On the other hand, FceRII has also FceRII epitopes were selected by using a fluorescence- been reported to be expressed on macrophages, eosinophils, activated cell sorter J774 cells were and platelets and to be an effector molecule of IgE-mediated (FACS). macrophage immunity, including immediate-type allergy and cytotoxicity infected with retroviruses carrying wild-type or mutant against certain parasites (2). IgE can mediate release of FceRII sequences by coculturing with the above 4f2 trans- varieties of chemical mediators from eosinophils, mono- fectants. J774 cells strongly expressing FcERII determinants cytes, and platelets (12-14). were sorted by a FACS. We have previously identified two species of human Endocytosis Assay. Cells were preincubated with 1251- FceRII, FceRIIa and FceRIIb (15, 16). They are generated by labeled 3-5 anti-FceRII monoclonal antibody at 4°C for 30 utilizing different transcriptional initiation sites and 5' exons min, washed, and incubated at 37°C in RPMI 1640 medium of the single genomic gene, and they differed only in a short supplemented with 20 mM Hepes (pH 7.4) and bovine serum amino acid stretch in the N-terminal cytoplasmic end. albumin (5 mg/ml) for the time indicated. Then, surface- FceRIIa is constitutively, but cell-type-specifically, ex- bound 1251-labeled 3-5 antibodies were acid-stripped with pressed on B cells. On the other hand, monocytes and citrate buffer [10 mM sodium citrate/0.14 M NaCl/bovine eosinophils express only FceRIlb. FceRIIb is expressed on serum albumin (50 ,ug/ml), pH 2.0], and acid-inaccessible normal peripheral blood B cells and monocytes only after internalized antibodies were measured by a gamma counter. stimulation with interleukin 4, which is known to be the Phagocytosis Assay. Cells were preincubated with or with- cytokine responsible for the isotype switching of B cells to out interferon y (IFN-y) at 200 units/ml for 30 h and were IgE (17-19). Moreover, peripheral blood lymphocytes from incubated with human IgE conjugated to sheep erythrocytes atopic individuals expressed FceRIIb without stimulation (SRBCs) by using a chromium chloride method. After a with interleukin 4 (16). The differential manner of gene 60-min incubation at 37°C, cells were washed with phosphate- expression for these receptors suggests that FceRIIa and buffered saline and then with 0.16 M NH4Cl to lyse extra- The publication costs of this article were defrayed in part by page charge Abbreviations: FcERII, low-affinity Fc receptor for IgE; SRBC, payment. This article must therefore be hereby marked "advertisement" sheep erythrocyte; FACS, fluorescence-activated cell sorter; IFN-y, in accordance with 18 U.S.C. §1734 solely to indicate this fact. interferon y. 5030 Downloaded by guest on September 27, 2021 Immunology: Yokota et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5031 A 1 2 3 4 5 6 7 8 9 10 amino acid residues at the N-terminal cytoplasmic domain as shown in Fig. 1A. If these receptors are also functionally - - - - -4erGl - - FccRIIa Met Glu Glu Gly Gln -_Tyr Ile Glu different, these regions must contain critical sequences. We FccRIIb Met-Asn-Pro- Pro --Ser-GI Glu - Le - Glu first examined the ability of these two forms of FceRII to 1 2 3 4 5 6 7 8 9 mediate endocytosis. A mouse fibroblast cell line if2 was B transfected with human FceRIIa or FcERIIb cDNA. Result- ing transfectants were found to express comparable amounts b-Phe5 Met - Asn - Pro - Pro - Phe - Gln - ci - ne - GIu of FceRII (Fig. 2A). They were incubated with 1251-labeled 3-5 anti-FceRII monoclonal antibody (3) and examined for b-Arg4 Met - An - Pm -Arg - ser - Gin - Giu - Ie - Glu their ability to internalize labeled antibody. As shown in Fig. 2B, the amount of internalized antibody (acid inaccessible) b-Arg3 Met - Asn - Arg -Pro - ser - Ghi - Glu - Ile - Glu rose rapidly and reached a plateau within 60 min in cells expressing FcERIIa, whereas the internalization was se- b-Lys2 Met -Lys - Pro * Pro Ser - Gin - Glu - Ie - Glu verely impaired in cells expressing FceRIIb. The same results were obtained in a murine myeloid leukemia cell line Ml and b-A2-6 Met - Glu - lle - Glu a murine macrophage cell line J774 transfected with either FIG. 1. Amino acid sequences of wild-type FceRIIa and FceRIIb human FceRIIa or FcERIIb cDNA (data not shown). These and FcERIIb mutants. (A) The amino acid sequences of N-terminal findings show that FcERIIa but not FcERIIb mediates effi- cytoplasmic ends of FcERIIa and FcERIIb. Sequences downstream cient endocytosis. of the vertical line are identical. The 5-amino acid pattern reported Restoration of Endocytosis in the Mutant FceRllb with an by Vega and Strominger (20) is found in FceRIIa and is double- underlined (see Discussion). The incomplete 5-amino acid pattern Amino Acid Substitution in the N-Terminal Cytoplasmic Do- found in FceRIIb is underlined. (B) The N-terminal sequences of main. It has been suggested that aromatic amino acids in the mutant FceRIIb. The receptors named b-Phe5, b-Arg4, b-Arg3, cytoplasmic region are required for efficient endocytosis of b-Lys2, and b-A2-6 were mutated, respectively, so that Ser-5 was various receptors (21-23). FceRIIa contains a tyrosine resi- changed to Phe, Pro-4 was changed to Arg, Pro-3 was changed to due at position 6 whereas FcERIIb has a serine residue at its Arg, Asn-2 was changed to Lys, and amino acids from positions 2 to equivalent position as shown in Fig. 1A. Thus, we substituted were 6 deleted. phenylalanine for serine at position 5 of FcERIIb (Fig. 1B). This mutant receptor was also expressed on 4i2 cells (Fig. cellular SRBCs. Cells were then fixed with 100% ethanol, 2C). As shown in Fig. 2D, the ability of this mutant receptor stained with hematoxylin/eosin, and photographed. to mediate endocytosis was significantly restored. Therefore, Immunoelectron Microscopic Observation. Cells were in- impaired endocytosis of FcERIIb can be explained by a lack cubated with the 3-5 at for 30 and at antibody 4°C min 37°C of aromatic amino acid at position 5 in the cytoplasmic for 2 min, as described in the endocytosis assay, and fixed for domain. 10 min with 2% (wt/vol) paraformaldehyde/0.1% glutaralde- The distribution of FcERII on transfectants was also ex- hyde in '0.1 M sodium phosphate buffer (pH 7.4). After amined by immunogold electron microscopy. As shown in incubation with rabbit anti-mouse IgG (0.1 mg/ml) for 60 min Fig. 3, gold particles were found in more than two-thirds of and then with 4-nm protein A-gold complex (OD525 = 0.020) the coated pits of cells expressing FcERIIa, whereas most of for 30 min, cells were fixed in 2% glutaraldehyde and then in coated pits did not contain gold particles in FcERIIb- 1% OS04 and embedded in Epon.
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