The cytoplasmic tail of FcgammaRIIIAalpha is involved in signaling by the low affinity receptor for immunoglobulin G.

The low affinity receptor for IgG, FcgammaRIIIA, is a multimeric receptor composed of the ligand binding subunit FcgammaRIIIAalpha (CD16) in association with the signal-transducing subunits zeta or gamma. Previous studies suggested that the cytoplasmic tail of FcgammaRIIIAalpha was not required for FcgammaRIIIAalpha-zeta association or signaling by FcgammaRIIIA. However, in these studies, the truncated FcgammaRIIIAalpha chains still expressed the four most membrane-proximal amino acids of the cytoplasmic tail (amino acids 230-233). By successive truncations from the C terminus of FcgammaRIIIAalpha, we have studied the role played by the membrane-proximal amino acids of the cytoplasmic tail of FcgammaRIIIAalpha in (i) FcgammaRIIIA expression, (ii) FcgammaRIIIAalpha-zeta association, and (iii) signal transduction. We provide evidence that this region is not required for FcgammaRIIIA expression or FcgammaRIIIAalpha-zeta association. However, signaling by FcgammaRIIIA is strictly dependent on the membrane-proximal amino acids in the cytoplasmic tail of FcgammaRIIIAalpha. Thus, total deletion of the cytoplasmic tail of FcgammaRIIIAalpha results in a severely impaired tyrosine phosphorylation of phospholipase C-gamma1, zap, and syk and rise in intracellular free Ca2+ following receptor ligation with specific anti-CD16 monoclonal antibody or Ig-anti-Ig complexes, suggesting that FcgammaRIIIAalpha-zeta association per se is not sufficient to establish the signal function of FcgammaRIIIA. In conclusion, the present findings demonstrate that the most membrane-proximal amino acids of the FcgammaRIIIAalpha cytoplasmic tail play a critical role in ligand-induced signal transduction by the FcgammaRIIIAalpha-zeta complex.

cells are cytotoxic for target cells coated with IgG antibodies. This property is referred to as antibody-dependent cellular cytotoxicity and is mediated through binding to target-bound IgG by the low affinity FcR for IgG expressed at the NK cell surface. Two distinct forms of the human low affinity FcR for IgG, Fc␥RIIIA and Fc␥RIIIB, have been identified (1). Fc␥RIIIA, which is found on NK cells and macrophages, is a transmembrane protein complex, whereas the form expressed on neutrophils, Fc␥RIIIB, is anchored to the outer plasma membrane by a glycosylphosphatidylinositol moiety (1). Fc␥RIIIA, the T cell receptor (TCR), and the B cell receptor (BCR) have many structural and functional features in common. Like TCR and BCR complexes, Fc␥RIIIA exist as multimeric receptor complexes composed of the ligand-binding Fc␥RIIIA␣ (CD16) chain noncovalently associated with various disulfide-linked subunits (2)(3)(4)(5). The associated chains are defined as a family of disulfide-linked dimers (6), which are closely related and can substitute for each other (7,8). They can be found as a homodimer (2, 3), a ␥␥ homodimer, originally described as a component of the high affinity FcR for IgE (4), or as a ␥ heterodimer (9). In addition, all of these dimers can form part of the TCR complex (8,10) and cell surface expression of both Fc␥RIIIA␣ and the TCR is dependent on coexpression of the or the ␥ chains (4,(11)(12)(13). It is not entirely clear how the Fc␥RIIIA␣-association takes place.
In addition to structural similarities, there are significant similarities between the signaling pathways coupled to Fc␥RIIIA, the TCR, and the BCR, respectively. Thus, ligation of each of these receptors induces tyrosine phosphorylation of several substrates including phospholipase C-␥1 (PLC-␥1) (14 -17), and a rise in intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) (18,19). The precise pathway by which receptor engagement initiates these events is not fully known, although src family kinases, the zap/syk kinases, and the tyrosine-containing activation motif (TAM) in the cytoplasmic tail of the associated subunits, all play important roles (reviewed in Ref. 20). Previous studies suggested that the cytoplasmic tail of Fc␥RIIIA␣ might not be required for signaling by Fc␥RIIIA (21)(22)(23). In all of these studies, however, the truncated Fc␥RIIIA␣ chains still expressed the four most membrane-proximal amino acids of the cytoplasmic tail. Thus, it remained unknown whether the membrane-proximal amino acids in the cytoplasmic tail of Fc␥RIIIA␣ also play a role in signal transduction following Fc␥RIIIA ligation. Although chimeric receptor molecules containing the extracellular part of Fc␥RIIIA␣ and the cytoplasmic domain of subunits with TAM are effective signal transducers when cross-linked by extracellular ligands (24,25), it is possible that the cytoplasmic tail of the ligand binding subunit in intact receptors plays a role in signaling, as recently demonstrated for the BCR (26).
In this study, we show that signaling by Fc␥RIIIA following stimulation with specific anti-CD16 mAb or immune complexes is strictly dependent on the membrane-proximal amino acids in the cytoplasmic tail of Fc␥RIIIA␣. In contrast, this region is not required for Fc␥RIIIA␣association and cell surface expression.
Constructs and Transfection-All mutations were constructed as described previously (29 -31) by the polymerase chain reaction using Vent DNA polymerase containing 3Ј 3 5Ј proofreading exonuclease activity (New England Biolabs, Inc., Beverly, MA) and as template Fc␥RIII The polymerase chain reaction products were cut with XbaI and StyI and ligated into the 4.1 kilobase XbaI-StyI fragment of pBluescript-␤WT (29). From here the constructs were cut out with XbaI-BamHI and ligated into the expression vector pT␤Fneo (32). Mutations were confirmed by complete DNA sequencing. Sequencing demonstrated one nucleotide mismatch at base position 559 as compared to the previously published sequence (1,33). In all constructs, a guanosine instead of a thymidine was found at position 559 (resulting in a valine instead of a phenylalanine at amino acid position 176 in the extracellular domain). Accordingly, two independent samples of the original Fc␥RIII-2 plasmid (kindly donated by Dr. J. V. Ravetch) were sequenced and in both samples the base at position 559 was found to be a guanosine and not a thymidine as previously published. These results indicated that position 559 in Fc␥RIIIA␣ might be polymorphic in the population or that a variation had been introduced into position 559 as a result of propagation of the plasmids. 2 The last possibility seems less likely as identical sequencing results were obtained from two independent plasmids. The constructs were transfected into JGN cells using the Bio-Rad Gene Pulser at a setting of 270 V and 960 F with 40 g of plasmid per 2 ϫ 10 7 cells. After 3-4 weeks of selection, G418-resistant clones were expanded and maintained in medium without G418.
Flow Cytometric Analyses and Intracellular Calcium-FACS analyses were performed as described previously on a FACScan (Becton Dickinson) (31). [Ca 2ϩ ] i of cells was measured with the intracellular fluorescent indicator fura-2/AM (Sigma). Fura-2/AM was added to a 1-ml cell suspension (4 ϫ 10 6 cells) in complete medium to a final concentration of 2.5 M. After a 20-min incubation at room temperature, cells were washed twice and resuspended in 2 ml of assay buffer (115 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl 2 , 1.5 mM KH 2 PO 4 , 1.5 mM MgSO 4 , 24 mM NaHCO 3 , and 10 mM HEPES, pH 7.4). Fluorescence measurements were performed using FLDM software in a Perkin-Elmer LS-50B luminescence spectrometer (Emeryville, CA) equipped with a thermostatic cuvette holder maintained at room temperature with continuous stirring. The ratio of fura-2/AM fluorescence at 340 nm to that at 380 nm was recorded in real time and expressed in arbitrary units. To initiate calcium mobilization, anti-CD16 or F101.01 mAb was added at time 50 s. At time 300 s, F(ab) 2 fragments of RAM Ig or intact RAM Ig was added. Ionophore-Ca 2ϩ salt (Serva, Heidelberg, Germany) was added at time 500 s as a receptor independent control. The background values of the samples were determined first and subtracted from the values obtained in the stimulation assay. At least two independent clones of each transfectant were analyzed.
Iodination and Western Blotting-Cells were surface-iodinated as described previously (28,34) and solubilized for 45 min at 4°C in lysis buffer (20 mM Tris-HCl, pH 8.0, 1 mM MgCl 2 , 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 8 mM iodoacetamide, and 1% digitonin (Sigma)). For immunoprecipitation, precleared lysate was incubated with 1.0 g of mAb for 2 h at 4°C. Protein A-Sepharose (PA) was added and the incubation continued for 2 h. The immunoprecipitates were washed five times in lysis buffer, boiled for 5 min with sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromphenol), and subjected to SDS-PAGE on 10% acrylamide gels under nonreducing conditions. Autoradiography of the dried gels was performed by using Hyperfilm-MP (Amersham Laboratories, Amersham, UK). 14 C-Labeled proteins from Amersham were used as molecular weight markers. For Western blotting, 5 ϫ 10 6 cells in 1 ml of RPMI medium were stimulated with the anti-CD16 mAb 3G8 plus F(ab) 2 fragments of RAM Ig, F101.01 plus intact RAM Ig, or PBS at 37°C for 2 min. The pelleted cells were lysed in modified RIPA buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5) containing phosphatase and protease inhibitors (1 mM vanadate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride). The postnuclear fractions were precleared by incubation with PA at 4°C for 30 min and precipitated with the indicated mAb for 2 h at 4°C. PA beads were added and the incubation continued for 2 h. Beads were washed four times in modified RIPA buffer without phenylmethylsulfonyl fluoride. The immunoprecipitates were eluted with sample buffer plus 2% 2-mercaptoethanol by boiling for 5 min, separated by 8 -10% SDS-PAGE, and electrotransferred to nitrocellulose membrane (Hybond-ECL, Amersham) as described previously (35). The membranes were blocked in 3% skim milk, 1% bovine serum albumin, and 0.5% Tween 20 in PBS and incubated with the indicated primary mAb. Washing, incubation with peroxidase-conjugated secondary antibodies, and visualization of the proteins by the enhanced chemiluminescence technique were performed as described previously (35). At least two independent clones of each transfectant were analyzed.

Cell Surface Expression of Truncated Fc␥RIIIA␣-Conflict-
ing results have been presented concerning the role of the cytoplasmic tail of Fc␥RIIIA␣ in the assembly of Fc␥RIIIA␣ and /␥. One report suggested that both the transmembrane region and the cytoplasmic tail of Fc␥RIIIA␣ were involved in the interaction with ␥ (11), whereas another report found that the cytoplasmic tail of Fc␥RIIIA␣ was dispensable for Fc␥RIIIA␣-association (4). However, in both reports, the truncated Fc␥RIIIA␣ chains studied contained the four most membrane-proximal amino acids of the cytoplasmic tail. To our knowledge, the role of these amino acids for Fc␥RIIIA␣-association and function has not been described previously. To analyze the role of the cytoplasmic tail of Fc␥RIIIA␣ for Fc␥RIIIA␣-complex formation and function in stable transfectants, stop codons were inserted in Fc␥RIIIA␣ cDNA corresponding to residues 242, 234, 233, 232, 231, and 230 resulting in truncated Fc␥RIIIA␣ chains designated Fc␥t242, -t234, -t233, -t232, -t231, and -t230, respectively (Fig. 1A) (Fc␥RIIIA␣ residue numbering according to Ref. 1). To avoid any possible interference in signaling by the TCR, these constructs were transfected into the TCR cell surface negative Jurkat variant JGN (27). G418-resistant clones were isolated and analyzed for cell surface expression of Fc␥RIIIA␣. All of the truncated Fc␥RIIIA␣ chains were expressed at the surface of JGN with comparable intensity as the wild-type Fc␥RIIIA␣ as determined by FACS analyses using CLB/FcRgran1, 3G8, or Leu-11c mAb (Fig. 1B).
Association of the Truncated Fc␥RIIIA␣ with the Subunit-Several studies have demonstrated the requirement of the /␥ subunits for signaling through Fc␥RIIIA␣ (21)(22)(23). It was therefore important to analyze whether the truncated Fc␥RIIIA␣ chains expressed at the cell surface were associated with the chain. The chain is poorly labeled by cell surface iodination; however, it should be possible to detect Fc␥RIIIA␣-association by coprecipitation-labeled Fc␥RIIIA␣ with anti-mAb. Accordingly, cells were surface radioiodinated, lysed in 1% digitonin lysis buffer, and immunoprecipitated with anti-CD16 mAb CLB/FcRgran1, anti-mAb, or PBS. The precipitates were analyzed by SDS-PAGE and autoradiography. Precipitates obtained with the anti-CD16 mAb clearly demonstrated the presence of the Fc␥RIIIA␣ in all of the transfectants (Fig. 2). The Fc␥RIIIA␣ chains moved as a broad band with a molecular mass between 50 and 70 kDa for Fc␥WT and a molecular mass between 40 and 60 kDa for the truncated Fc␥RIIIA␣ chains. As expected, the band was only weakly seen in the precipitates from all transfectants. However, most importantly, anti-mAb clearly coprecipitated the Fc␥RIIIA␣ chain from all transfectants with comparable intensities (Fig. 2).

Receptor-induced Mobilization of Intracellular Calcium-
The ability of Fc␥RIIIA containing truncated Fc␥RIIIA␣ chains to mediate a rise in [Ca 2ϩ ] i following stimulation with the specific anti-CD16 mAb 3G8, was next studied. As shown in Fig. 3B, 3G8 induced a rapid but weak rise in [Ca 2ϩ ] i in JGN cells transfected with Fc␥WT. This response was further potentiated following cross-linking with F(ab) 2 fragments of RAM Ig (Fig. 3B). As expected, no detectable rise in [Ca 2ϩ ] i was observed in the Fc␥RIIIA negative parent cell line, JGN (Fig.  3A). In contrast to JGN-Fc␥WT cells, none of the transfectants expressing truncated Fc␥RIIIA␣ chains showed a rise in [Ca 2ϩ ] i following stimulation with 3G8 alone. Following optimal stimulation by cross-linking with RAM Ig, a rise in [Ca 2ϩ ] i in JGN-Fc␥t242 and JGN-Fc␥t234 cells was seen equal to the one observed for JGN-Fc␥WT cells. However, further truncation resulted in an impaired rise in [Ca 2ϩ ] i (Fig. 3) suggesting that the most membrane-proximal amino acids were required for an efficient rise in [Ca 2ϩ ] i . Indeed, cells expressing Fc␥RIIIA␣ lacking all of the amino acids of the cytoplasmic tail were nearly unable to respond to optimal stimulation (Fig. 3H). Similar results were obtained following stimulation of the transfectants with Ig-anti-Ig complexes. Whereas the rise in [Ca 2ϩ ] i was comparable for JGN-Fc␥WT, JGN-Fc␥t242, and JGN-Fc␥t234 cells, truncations from residue 233 to 230 resulted in a progressive weakening of the response following treatment with Ig-anti-Ig complexes (Fig. 4).
Receptor-induced Protein Tyrosine Phosphorylation-One of the earliest signaling events following Fc␥RIIIA ligation is tyrosine phosphorylation of several cytoplasmic substrates including the subunit (14). To analyze the role of the Fc␥RIIIA␣ tail in receptor-mediated protein tyrosine phosphorylation, JGN-Fc␥WT and JGN-Fc␥t230 cells were treated with either cross-linked anti-CD16 mAb (3G8), Ig-anti-Ig complexes, or PBS for 2 min at 37°C prior to lysation and immunoprecipita-tion with anti-phosphotyrosine mAb 4G10 coupled to agarose beads. The precipitates were separated by 10% SDS-PAGE and analyzed in Western blotting with 4G10 (Fig. 5A). Treatment with cross-linked anti-CD16 mAb or Ig-anti-Ig complexes resulted in tyrosine phosphorylation of several proteins in both JGN-Fc␥WT and JGN-Fc␥t230 cells. Although no significant qualitative differences were observed in the tyrosine-phosphorylated proteins from the two cell lines, significant quantitative differences were seen. Most strikingly, the chain and two unidentified proteins with a molecular mass of approximately 40 and 55-60 kDa became only weakly phosphorylated in JGN-Fc␥t230 cells as compared with the corresponding proteins in JGN-Fc␥WT cells, suggesting that some of the tyrosine kinases activated by engagement of wild-type Fc␥RIIIA were not as efficiently (or not at all) activated by engagement of Fc␥RIIIA containing the tail-less Fc␥RIIIA␣ chain.
PLC-␥, and excluding that the lack of its detection with the anti-phosphotyrosine mAb in the precipitates from JGN-Fc␥t230 cells was due to its absence from these precipitates (Fig. 5B, lower panel).
These results strongly suggested that deletion of the cytoplasmic tail of Fc␥RIIIA␣ disturbed the coupling of the receptor to protein tyrosine phosphorylation events involving the phosphorylation of and PLC-␥1. Since the known components of Fc␥RIIIA lack intrinsic kinase activity, this indicated that the association and/or activation of non-receptor protein-tyrosine kinases involved in Fc␥RIIIA signaling is dependent on the cytoplasmic tail of Fc␥RIIIA␣. It has been demonstrated that stimulation of Fc␥RIIIA induces an increased tyrosine phosphorylation of the non-receptor protein-tyrosine kinases zap and syk (16,17). Accordingly, zap and syk were immunoprecipitated from JGN-Fc␥WT and JGN-Fc␥t230 cells treated with cross-linked anti-CD16 mAb (3G8), Ig-anti-Ig complexes, or PBS for 2 min at 37°C, and the immunoreactive material was analyzed in Western blotting with the anti-phosphotyrosine mAb 4G10 (Fig. 6). zap from JGN-Fc␥WT cells became strongly phosphorylated whereas zap from JGN-Fc␥t230 cells became only weakly phosphorylated following stimulation with cross-linked anti-CD16 mAb or Ig-anti-Ig complexes (Fig. 6A). Likewise, syk from JGN-Fc␥WT cells became strongly phosphorylated whereas syk from JGN-Fc␥t230 cells became only weakly phosphorylated following stimulation with cross-linked anti-CD16 mAb or Ig-anti-Ig complexes (Fig. 6B). In addition, phosphorylated was clearly coprecipitated with syk in JGN-Fc␥WT cells following stimulation with cross-linked anti-CD16 mAb or Ig-anti-Ig complexes but was only weakly coprecipitated with syk from JGN-Fc␥t230 cells following stimulation with cross-linked anti-CD16 mAb. DISCUSSION It has previously been shown that truncated Fc␥RIIIA␣ with only the four most membrane-proximal amino acids of the cytoplasmic tail left allows surface expression and signaling through Fc␥RIIIA (21)(22)(23). Our results are in agreement with these results. More importantly, we have extended these studies by demonstrating that further truncations of the cytoplasmic tail of Fc␥RIIIA␣ still allow F␥RIIIA␣-association and expression but severely affect the signaling capacity of Fc␥RIIIA. At first sight, our results might seem controversial as it has generally been believed that the short cytoplasmic tails of the ligand binding subunits of FcR, TCR, and BCR were not involved in signal transduction. However, our findings that the membrane-proximal part of the cytoplasmic tail of Fc␥RIIIA␣ is important for Fc␥RIIIA signaling are not unprecedented. Thus, a similar important role in signal transduction by the BCR has been described for the short cytoplasmic tail of membrane-bound IgM (mIgM). Point mutation of one of the three amino acids of the cytoplasmic tail of mIgM completely abrogated antigen-induced signal transduction by the BCR (26).
Several possibilities for the role of the membrane-proximal amino acids of the cytoplasmic tail of Fc␥RIIIA␣ may be suggested. The cytoplasmic tail of Fc␥RIIIA␣ may be directly involved in association with non-receptor protein-tyrosine kinases e.g. syk. syk is found in association with Fc␥RIIIA (17), Fc⑀RI (36), the BCR (37), and the TCR (38). In B cells, syk may associate with the BCR by two different mechanisms: syk may be bound to the resting BCR via the transmembrane and cytoplasmic domain of mIg, and syk may be bound to the tyrosinephosphorylated TAM of the Ig ␣ and ␤ chains of the activated BCR via its SH2 domains (39). Likewise, in T cells there is evidence that syk is constitutively bound to the resting TCR by a still unidentified mechanism (40) and that syk associates with the tyrosine-phosphorylated TAM of the chain of the activated TCR via its SH2 domains (38). Therefore, it is possible that syk binds to Fc␥RIIIA via the transmembrane and membrane-proximal part of the Fc␥RIIIA␣ chain. Alternatively, the transmembrane and membrane-proximal part of the Fc␥RIIIA␣ chain might be indirectly involved in the association with syk or other protein-tyrosine kinases via a third unidentified molecule.
An alternative but not mutually exclusive explanation might be that the membrane-proximal part of the cytoplasmic tail of Fc␥RIIIA␣ in response to ligand binding induces conformational changes in the chain promoting optimal binding and/or function of the TAM-associated kinases. Thus, this model implies that the membrane-proximal part of the cytoplasmic tail of Fc␥RIIIA␣ is involved in transfer of information from the ligand binding subunit to subunits involved in downstream signaling.
CD45 has been found to associate with Fc␥RIIIA at the cell surface (41). CD45 is a phosphotyrosine phosphatase that is crucial for the normal function of T and B cells and presumably other leukocytes as well (42)(43)(44)(45). In the absence of CD45, the rapid TCR-triggered tyrosine phosphorylation of cellular proteins normally seen is severely reduced resulting in impaired activation of PLC and rise in [Ca 2ϩ ] i (42)(43)(44). One mechanism by which CD45 seems to regulate these events is by regulating src family protein-tyrosine kinases including lck (reviewed in Ref. 46). A third role of the membrane-proximal part of the cytoplasmic tail of Fc␥RIIIA␣ could be to contribute to the association between Fc␥RIIIA and CD45 and thereby indirectly to regulate lck.
The roles of the membrane-proximal part of the cytoplasmic tail of Fc␥RIIIA␣ proposed above appear to be inconsistent with the finding that receptor chimeras containing the cytoplasmic tail of /␥ (22-25, 47, 48), CD3⑀ (49), and Ig-␣ (50) are effective signal transducers when cross-linked by mAb. It is, however, possible that cross-linking of such chimeric receptor molecules allows a closer "nonphysiological" contact of their cytoplasmic tails than cross-linking of the ligand binding subunits of intact receptors does. A closer contact of the TAM in chimeric receptors could facilitate activation of protein-tyrosine kinases constitutively bound to the TAM (51). Clearly, further studies are necessary to resolve the role of the cytoplasmic tail of Fc␥RIIIA␣ in signal transduction.