The CY Domain of the FcγRIa α-Chain (CD64) Alters γ-Chain Tyrosine-based Signaling and Phagocytosis*

Although the cytoplasmic domain of the human FcγRIa α-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcγRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcγRIa, we developed a series of human FcγRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the α-chain of FcγRIa and suggest that the cytoplasmic domain of FcγRI regulates the different functional capacities of the FcγRIa-receptor complex.

The ␥-chain, initially described as a component of the Fc⑀RI signaling complex, is able to form multichain complexes with the ligand-binding ␣-chain of several Fc receptors (1)(2)(3). The Fc␥RIa (CD64), Fc␥RIIIa (CD16A), Fc␣RI (CD89), and Fc⑀RI ␣-chains associate with the ␥-chain as a common molecule in signal transduction. The stoichiometry of the assembly of the receptor complex is generally ␣␥ 2 , except in mast cells, which may have Fc⑀RI and Fc␥RIIIa complexes where there is, in addition, a ␤ chain (␣␤␥ 2 ) (4 -6). In all of these Fc receptor complexes, the ␥-chain with its tyrosine activation motif (ITAM) is necessary for receptor signaling (7)(8)(9). In the case of Fc⑀RI, the ␤-chain serves as an amplifier of receptor function (10,11).
Fc␥RIa, a receptor with high affinity for IgG (10 9 M Ϫ1 ) (12), has received attention over the past few years as a potential therapeutic target in malignancy. Targeting of tumors to Fc␥RI with bispecific mAbs 1 can facilitate tumor killing via Fc␥RIexpressing macrophages, and therapeutic humanized bispecific reagents targeting human Fc␥RIa are currently in clinical trials (13)(14)(15)(16)(17)(18)(19). Bispecific mAb-based antigen targeting to Fc␥RI can also enhance antigen presentation by dendritic cells with clear applications to enhanced immunization strategies (20).
Expression of the Fc␥RIa ␣-chain in the presence or absence of the ␥-chain has allowed an assessment of the functional capacity of each chain. For example, the ␥-chain is necessary for Fc␥RIa-mediated phagocytosis and Fc␥RIa-induced activation of tyrosine kinase activity (7)(8)(9). However, the ␣-chain is sufficient for endocytosis (7,9). Whereas expression of the Fc␥RIa ␣-chain without a CY domain can also induce pseudopod extension and endocytosis, recent data from our group and others have provided the first evidence that the ␣-chain of Fc␥RIa can alter receptor function downstream of IgG binding (21)(22)(23). Expression of an Fc␥RIa ␣-chain lacking the CY domain in a murine macrophage cell line results in quantitative differences in the phagocytic and endocytic capacity of the ␣␥ 2 receptor complexes compared with wild-type Fc␥RI. Lack of the CY domain also changes the Ca 2ϩ dependence of receptorspecific phagocytosis and abolishes the receptor-elicited IL-6 response. Expression of a similar receptor mutant in B cells results in alterations in the intracellular cycling of the internalized receptor (21). These data suggest a role for the ␣-chain CY domain in the regulation of Fc␥RI function.
We have begun to dissect the molecular basis for the ␣-chain involvement in Fc␥RI signaling and cell activation. We now show that the CY domain of Fc␥RIa is constitutively phosphorylated and that receptor engagement and cross-linking result in a time dependent dephosphorylation. Inhibition of the dephosphorylation with okadaic acid, an inhibitor of type 1 protein serine/threonine phosphatase and type 2A protein serine/ threonine phosphatase, blocks wild-type receptor-mediated phagocytosis and reduces tyrosine phosphorylation of the ␥-chain. In contrast, both truncation and mutation of CY serines to alanine abrogate the effect of OA on phagocytosis and ␥-chain tyrosine phosphorylation. These data suggest that serine phosphorylation of the Fc␥RIa ␣-chain inhibits early receptor-initiated tyrosine phosphorylation events. Furthermore, the selective reduction in IL6 production with truncation, but not with serine to alanine mutation, indicates that cytokine production is likely influenced by other elements engaged by the Fc␥RIa ␣-chain.

MATERIALS AND METHODS
Cell Culture and Reagents-The murine macrophage cell line P388D1 (obtained from American Type Culture Collection, Manassas, VA) was stably transfected with a cDNA encoding human Fc␥RIa (WT) or a mutant form of Fc␥RIa containing a stop codon after the first amino acid of the cytoplasmic domain (Lys 315 3 Stop 315) (CYϪ) as we have described previously (8,22). Fc␥RIa constructs encoding serine to alanine mutations (S328A/S331A, S339A/S340A, and S328A/S331A/ S339A/S340A (S 4 3 A 4 )) were prepared by overlap PCR and stably transfected as described previously (22,24). In all cases, two independently prepared cell lines stably expressing each Fc␥RI construct were analyzed. P388D1 cells transfected with human Fc␥RIIa have been described previously (22,24). Cell lines were maintained as adherent cultures (Corning tissue culture dishes) in RPMI 1640 medium as described previously (24). The human myelomonocytic cell line U937 (American Type Culture Collection) was maintained as a suspension culture in RPMI 1640 medium. All tissue culture reagents were from Invitrogen. For 32 P studies, cells were cultured for 24 h in phosphatefree RPMI 1640 medium in the presence of 5 mCi of 32 P i .
The protein tyrosine kinase inhibitor genistein was obtained from Invitrogen. The type 1/2A protein serine/threonine phosphatase inhibitor okadaic acid was obtained from Calbiochem. An okadaic acid analog, 1-Nor-okadaone, that does not possess protein phosphatase inhibitory activity (25) was obtained from Alexis Biochemicals (San Diego, CA). F(abЈ) 2 fragments of the anti-Fc␥RIa mAb 22.2 and Fab fragments of the anti-Fc␥RIIa mAb IV.3 were obtained from Medarex (Annandale, NJ). Mouse F(abЈ) 2 fragments and F(abЈ) 2 goat anti-mouse IgG (GAM) were obtained from Jackson ImmunoResearch (West Grove, PA). Mouse IgG was obtained from Sigma. All other reagents were from Sigma. Quantitative huFc␥RI expression was matched for cells expressing the WT and the cytoplasmic domain deletion mutant by fluorescence-activated cell sorting using anti-Fc␥RI mAb 22.2-fluorescein isothiocyanate (Medarex). Polyclonal anti-␥-chain Abs prepared in rabbits immunized with a C-terminal peptide sequence that is shared by both human and murine ␥-chain were used for immunoprecipitations and blotting as we have described previously (22). A polyclonal rabbit antiserum raised against the C-terminal 11 amino acids of the cytoplasmic domain of Fc␥RIa was prepared. Anti-phosphotyrosine mAb 4G10 was obtained from Upstate Biotechnology (Lake Placid, NY). A rabbit polyclonal anti-phosphoserine Ab, soluble phosphoserine, and epidermal growth factor-stimulated A431 cell lysate (used as a positive control) were obtained from Zymed Laboratories Inc.
For immunoblotting analysis, protein immunoprecipitates were separated by SDS-PAGE and blotted onto nitrocellulose membranes (22,27). Membranes were blocked with 10% nonfat milk or 3% bovine serum albumin followed by incubation with the blotting Ab/mAb. Blots were washed three times with phosphate-buffered saline-0.1% Tween 20 and probed with horseradish peroxidase-conjugated anti-mouse IgG or antirabbit IgG (Amersham Biosciences or Jackson ImmunoResearch). After three more washes, bound horseradish peroxidase-conjugated Ab was detected using ECL (Amersham Biosciences) according to the manufacturer's directions. Membranes were stripped by incubation with Tris-HCl, pH 2.3, for 30 min at room temperature and then reprobed as described above.
Phagocytosis-Phagocytosis by transfected P388D1 cells was determined in an adherent assay system (22,24). Biotinylated mAb 22.2 F(abЈ) 2 , mAb IV.3 Fab, and biotinylated bovine erythrocytes were prepared as described previously (22,24). Biotinylated bovine erythrocytes were saturated with streptavidin and washed. The resulting bovine erythrocytes were coated with biotinylated mAb, and the level of mAb binding was verified by flow cytometry.
Treatment of cells with genistein (100 nM) to block protein tyrosine kinase or with okadaic acid (1 M) to block protein type 1 and 2A protein serine/threonine phosphatase activity was performed by preincubating the coverslip-adherent cells for 30 min or 10 min, respectively, at 37°C followed by addition of E-22.2 or E-IV.3 in RPMI 1640 medium/20% fetal calf serum as described above. Controls included loading cells with 0.1-1% Me 2 SO (depending on the concentration of inhibitor) for the same period of time.
Flow Cytometry-Aliquots of cells at 5 ϫ 10 6 cell/ml were incubated with saturating concentrations of primary mAb for 30 min at 4°C followed by two washes. For indirect immunofluorescence, the cells were then incubated with saturating concentrations of fluorescein isothiocyanate-conjugated goat anti-mouse IgG F(abЈ) 2 at 4°C for another 30 min. After washing, the cells were analyzed immediately for immunofluorescence using a FACScan (BD Biosciences).
Statistical Analysis-Analysis of flow cytometry listmode data was performed using CellQuest (BD Biosciences). Statistical comparisons were performed with the paired t test. A probability of 0.05 was used to reject the null hypothesis that there is no difference between the samples.

Regulation of Phagocytosis by the Cytoplasmic Domain of
Human Fc␥RI-The cytoplasmic domain of the ligand binding ␣-chain of the human Fc␥RIa receptor complex (␣␥ 2 ) exerts a quantitative influence on receptor-specific phagocytosis (22). The quantitative phagocytic capacity of a cytoplasmic domainlacking mutant of huFc␥RIa is lower than that of wild-type huFc␥RIa expressed in the murine macrophage cell line P388D1. Likewise, the kinetics of phagocytosis are slower in the tail minus mutant form of huFc␥RIa compared with WT huFc␥RIa in these cells. To explore possible mechanisms for this effect, we quantitated huFc␥RIa-specific phagocytosis in these cell lines in the presence of several kinase and phosphatase inhibitors. Previous work has demonstrated that Fc␥Rmediated phagocytosis is dependent on tyrosine kinase activation. For Fc␥RIa, phagocytosis is dependent on tyrosine phosphorylation of the ␥-chain. In P388D1 cell lines stably expressing either WT huFc␥RIa or the cytoplasmic tail minus mutant form of huFc␥RIa (CYϪ), phagocytosis was completely inhibited by pretreatment of the cells with the tyrosine kinase inhibitor genistein (Fig. 1A). Cell viability was unaffected by genistein during the time course of these studies, establishing a clear role for tyrosine phosphorylation in Fc␥RI phagocytosis. Receptor-specific phagocytosis was reestablished to normal levels in the WT cells during the 60-min time course of the phagocytosis assay if genistein was removed after the initial incubation period (Fig. 1A). Interestingly, only 70% of the phagocytic capacity of the tail minus mutant was restored during the same time period. This observation was highly reproducible (p Ͻ 0.017, n ϭ 9 pairs).
Although the CY domain of huFc␥RIa lacks tyrosine residues, it does contain four serine residues. We hypothesized that alteration in the phosphorylation status of the CY domain of huFc␥RI might be important in regulation of receptor complex (␣␥ 2 ) function. The type 1 and 2A protein serine/threonine phosphatase inhibitor OA significantly blocked WT huFc␥RIaspecific phagocytosis (treated versus untreated cells, p Ͻ 0.001, n ϭ 15 pairs). In contrast, OA had no effect on phagocytosis mediated by mutant huFc␥RIa lacking the CY domain (treated versus untreated cells, p Ͼ 0.05, n ϭ 15 pairs) (Fig. 1A). The addition of OA to the cells did not alter cell viability of either cell line during the 1-h phagocytic assay. Furthermore, pretreatment of the WT Fc␥RIa-expressing cell line with the OA analog 1-Nor-okadaone, which is structurally similar to OA but does not inhibit type 1 and 2A protein serine/threonine phosphatase (25), did not alter the phagocytic response. As an additional control for the OA treatment, we incubated P388D1 cells expressing human Fc␥RIIa with OA, and no inhibition of Fc␥RIIa-specific phagocytosis was observed (Fig. 1A). These results strongly suggest that OA does not have nonspecific effects on the cells over the time course of these experiments and that serine dephosphorylation is important in regulation of huFc␥RIa-specific phagocytosis in the presence of an intact Fc␥RIa cytoplasmic domain.
We considered the possibility that engagement of the ligandbinding site of Fc␥RIa, which can augment receptor function (28), might alter the sensitivity of phagocytic to OA. However, saturation of the transfected P388D1 with murine IgG2a (22) did not change the sensitivity of WT huFc␥RIa to OA, nor did it change the lack of inhibition of CYϪ huFc␥RIa phagocytosis (Fig. 1B).
The CY Domain of Fc␥RIa Is Dephosphorylated upon Receptor Activation-To directly assess serine phosphorylation of the CY domain of the ␣-chain of Fc␥RIa, we examined anti-phosphoserine mAb binding to huFc␥RIa immunoprecipitates from transfected P388D1 cells. Constitutive serine phosphorylation of huFc␥RIa was clearly apparent in the murine P388D1 cells (Fig. 2A). The specificity of the anti-phosphoserine Ab was confirmed by the ability of soluble phosphoserine to completely block the reactivity of the Ab with Fc␥RIa (data not shown). As additional controls for the specificity of the anti-phosphoserine Ab, human Fc␥RIa in which all four cytoplasmic serine residues were mutated to alanine (stably transfected into P388D1 cells, see below) and the mutant Fc␥RIa lacking a cytoplasmic domain were immunoprecipitated and were nonreactive with this blotting Ab. Reprobing of the membrane with a blotting anti-human Fc␥RIa Ab confirmed loading of the serine-mutated form of the receptor (Fig. 2A).
Upon receptor-specific cross-linking, the level of phosphoserine decreased over a 5-min time period (Fig. 2B). Reprobing of the membranes with a polyclonal rabbit anti-huFc␥RI Ab confirmed equivalent levels of immunoprecipitated Fc␥RIa at each time point (Fig. 2B). To verify the results of the antiphosphoserine mAb, we also preloaded U937 cells with 32 P i and examined the level of phosphorylation of immunoprecipitated huFc␥RI. Again, constitutive levels of 32 P-labeled Fc␥RIa were detectable in resting cells, and, in agreement with the immunoblotting studies, cross-linking of the receptor resulted in a decrease in the level of 32 P-labeled Fc␥RI immunoprecipitate ( Fig. 2C). Treatment of the cells with OA before receptor crosslinking resulted in the preservation of Fc␥RI phosphorylation, demonstrating that the OA is acting, at least in part, at the level of the Fc␥RI ␣-chain.
Functional Role of Serine Phosphorylation of the CY Domain of Fc␥RIa-To directly demonstrate that dephosphorylation of the CY domain of Fc␥RIa is important in receptor function, we prepared three different constructs of huFc␥RIa in which we mutated 1) the two membrane proximal serines to alanine (S328A/S331A), 2) the two membrane distal serines to alanine (S339A/S340A), and 3) all four cytoplasmic domain serines to alanine (S 4 3 A 4 ). These three variant forms of huFc␥RI were stably expressed in P388D1 cells (Fig. 3A), and all three receptors were functional, as demonstrated by their ability to mediate receptor-specific phagocytosis (Table I). For comparison, expression of the WT and CYϪ mutant Fc␥RIa is shown in Fig.  3B. To determine whether dephosphorylation of any of the specific serine residues (or of some combination of serine residues) is required for receptor function, we examined the OA sensitivity of phagocytosis by each of these variant receptors. Like the CY domain truncation construct, receptor-specific phagocytosis mediated by each of the three serine mutant receptor forms was resistant to OA treatment (Table I). As expected, pretreatment of all lines with genistein resulted in complete inhibition of receptor-specific phagocytosis. These results, taken together, demonstrate a role for dephosphorylation of serine residues in the CY domain in Fc␥RIa-mediated phagocytosis.
We demonstrated previously that the CY domain of Fc␥RIa is required for early Fc␥RIa-induced IL-6 secretion (22). In contrast to the CY domain truncation form of Fc␥RIa, which did not elicit IL-6 secretion, receptor-specific cross-linking of the S328A/S331A, S339A/S340A, and S 4 3 A 4 mutants resulted in both IL-1␤ and IL-6 secretion 8 h after stimulation (Table II). We attempted to determine the sensitivity of Fc␥RIa-induced IL-1␤ and IL-6 secretion to OA, but the incubation of cells with OA for the time periods necessary for cytokine production was toxic to the cells and thereby precluded measurement of cytokine release. These results are consistent with a model of Fc␥RIa function that requires dephosphorylation of the Fc␥RIa CY domain for receptor-mediated cell activation.
Okadaic Acid Treatment Prevents Fc␥RIa-induced Tyrosine Phosphorylation-Among the earliest signaling events that occur after cross-linking of Fc␥RIa are the activation of Src family tyrosine kinases such as Hck and tyrosine phosphorylation of the ␥-chain (29 -32). Given the importance of serine dephos- phorylation for Fc␥RIa phagocytosis and the dependence of phagocytosis on the tyrosine phosphorylation of the ␥-chain, we reasoned that OA pretreatment might alter the tyrosine phosphorylation of the ␥-chain. Indeed, pretreatment of WT Fc␥RIexpressing cells with OA resulted in a dramatic decrease in the level of tyrosine phosphorylation of the ␥-chain (Fig. 4A). Comparable loading of ␥-chain in all lanes was confirmed by sequential analysis of the same blots with the anti-␥-chain Ab. In contrast to the WT Fc␥RI-expressing cells, incubation of the tail minus mutant Fc␥RIa or the S 4 3 A 4 mutant Fc␥RIa expressing P388D1 cells with OA showed no demonstrable effect on receptor-specific activation-dependent tyrosine phosphorylation of the ␥-chain (Fig. 4B). These results support the model that OA maintains serine phosphorylation of the CY domain of Fc␥RIa which in turn reduces WT Fc␥RIa mediated tyrosine phosphorylation of the ␥-chain.

DISCUSSION
The cytoplasmic domain of the Fc␥RIa ␣-chain can modulate the kinetics of both receptor-mediated endocytosis and phago-cytosis (22) and make receptor-specific phagocytosis insensitive to changes in [Ca 2ϩ ] i (22,24). These observations suggest that the Fc␥RIa ␣-chain interacts with intracellular molecules that can modulate receptor signaling elements and function. Previous work has shown that murine Fc␥RIa is constitutively phosphorylated on serine (33), and we now show that the serine residues in the CY domain of human Fc␥RIa are actively phosphorylated and dephosphorylated in relation to receptor crosslinking and that this phosphorylation is important in regulation of Fc␥RIa function. The constitutive phosphorylation of the huFc␥RIa ␣-chain was observed both in transfected murine macrophages and in the human myelomonocytic cell line U937. Upon activation, the receptor is transiently dephosphorylated. Inhibition of serine dephosphorylation with the type 1 and 2A protein serine/threonine phosphatase inhibitor, okadaic acid, results in a marked decrease in receptor function, including both phagocytosis and tyrosine phosphorylation of the ␥-chain. Mutagenesis of the four cytoplasmic domain serine residues suggests that at least several of these serines are involved in this modulation of Fc␥RIa ␣-chain function.
Okadaic acid likely alters the phosphorylation of multiple  intracellular targets, and we considered the possibility that OA might mediate its effects on phagocytosis by altering serine and threonine phosphorylation of the ␥-chain. Indeed, the ␥-chain is constitutively phosphorylated on threonine and upon receptor activation becomes phosphorylated on serine and tyrosine (34,35). It is unlikely, however, that the inhibitory effect of OA on receptor-specific phagocytosis is due to alterations in ␥-chain phosphorylation. OA does not alter phagocytosis in either the tail minus mutant form of Fc␥RIa or the Fc␥RIa serine to alanine mutants. Furthermore, OA does not alter Fc␥RIIaspecific phagocytosis. Taken together with the observation that OA alters the phosphorylation state of the Fc␥RIa ␣-chain, these data indicate a direct effect of OA on the phosphorylation state of the Fc␥RIa ␣-chain and suggest that this effect is responsible for OA altering Fc␥RIa-specific phagocytosis.
The kinase(s) responsible for phosphorylation of the CY domain is currently unknown. Although protein kinase C activity is required for Fc␥R phagocytosis (36,37), it is unlikely that protein kinase C isoforms are directly involved in modulating the phosphorylation of the CY domain of Fc␥RIa during receptor activation. Notably, the receptor is dephosphorylated upon cross-linking, and analysis of the sequence of the CY domain using ProfileScan of the Prosite data base (www.isrec.isbsib.ch/software/PFSCAN_form.html) does not reveal any po-tential protein kinase C consensus sites. Of course, it is possible that there are protein kinase C sites not identified by such analysis, but the observations that the classical protein kinase C ␣ isoform is important in the Fc␥R-induced respiratory burst and that the novel isoforms protein kinase C ␦ and/or protein kinase C ⑀ are involved in Fc␥R phagocytosis (37) suggest that protein kinase C family members play a role downstream of the receptor per se. Interestingly, motif analysis does indicate two consensus sites for casein kinase II, a kinase implicated in CD5 signaling (38). However, analysis of the Fc␥RIa CY domain and casein kinase II constructs using the yeast two-hybrid system has not shown any evidence of interaction between Fc␥RIa and either the ␣or ␤-subunits of casein kinase II. Future studies will be required to determine the nature of the kinase responsible for constitutive phosphorylation of Fc␥RIa.
The identity of the phosphatase(s) responsible for activationinduced dephosphorylation is also unknown. Our data with the protein phosphatase 1/2A inhibitor, okadaic acid, strongly implicate these phosphatases. Screens of two human leukocyte cDNA libraries for binding partners to the Fc␥RIa CY domain have not identified candidate phosphatases, but these serine/ threonine phosphatases may be targeted to a signaling complex rather than the phosphorylated target itself (39,40). Perhaps the known interaction between Fc␥RIa and non-muscle fil- amin-280 (actin-binding protein ABP-280) regulates the localization of Fc␥RIa in the membrane (41). The dissociation of this protein upon receptor engagement may change the relationship of Fc␥RIa with the actin cytoskeleton. As with other receptor systems, associations with cytoskeletal elements may be important in allowing localization of the receptor with a phosphatase(s) and other signaling elements activated by Fc␥R (42)(43)(44)(45)(46)(47)(48).
Although the specific kinase(s) and phosphatase(s) targeting the Fc␥RIa ␣-chain remain unclear, our data indicate that the previous model suggesting that the ␥-chain is both necessary and sufficient for Fc␥RIa function requires revision. We propose a model in which constitutive serine phosphorylation of the CY domain of Fc␥RIa regulates the ability of the receptor to initiate the tyrosine kinase-based signaling cascade necessary for receptor function. Upon serine dephosphorylation, the tyrosine-based signaling cascade is fully engaged, and receptorinduced cell activation proceeds normally. In the Fc␥RIa CY truncation mutant, the inhibitory influence of the phosphorylated tail is removed, allowing receptor function to proceed. However, the functional differences between this mutant form of the receptor and the wild-type receptor also suggest that the CY domain of Fc␥RIa facilitates signaling and is required for full receptor function, including the induction of IL-6 secretion (22) and sorting of internalized receptor to endosomes (21). Thus, in addition to facilitating association with cytoskeletal components, the Fc␥RIa CY domain may serve as a scaffold for the binding of signaling elements critical for full receptor function.
Our data also emphasize the potential for genetic variants of the CY domain to influence receptor function. Polymorphic variants of the extracellular domains of Fc␥RIIA, Fc␥RIIIA, and Fc␥RIIIB alter ligand binding and impact upon autoimmune disease susceptibility and severity (49 -53). In contrast, the CY domain of Fc␥RIIA, which contains a tyrosine activation motif, is invariate (54), and little attention has been focused on the CY domain of the ␥-chain-associated receptors. We have recently reported two single-nucleotide polymorphisms in the CY domain of Fc␥RIa (55). By their proximity to the serines at 339 and 340, we can now speculate that these single-nucleotide polymorphisms may alter quantitative phosphorylation and resultant receptor function. An understanding of the biology of these single-nucleotide polymorphisms will no doubt provide insights into the molecular mechanisms of Fc␥RIa signaling and also into genetic susceptibility factors for altered immune function. Nonetheless, they provide evidence for the potential clinical significance of our current observations on the role of the Fc␥RIa cytoplasmic domain and its serine residues on receptor function.