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J. Biol. Chem., Vol. 277, Issue 43, 41287-41293, October 25, 2002

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The CY Domain of the FcRIa -Chain (CD64) Alters -Chain Tyrosine-based Signaling and Phagocytosis^*

Jeffrey C. Edberg^§¶, Hongwei Qin^§, Andrew W. Gibson, Arthur M. F. Yee, Patricia B. Redecha, Zena K. Indik^**, Alan D. Schreiber^**, and Robert P. Kimberly

From the  Departments of Medicine and Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama 35294,  Department of Medicine, Hospital for Special Surgery and Weill Medical College of Cornell University, New York, New York 10021, and ^** Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, August 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although the cytoplasmic domain of the human FcRIa -chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcRIa 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 FcRIa, we developed a series of human FcRIa 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 -chain of FcRIa and suggest that the cytoplasmic domain of FcRI regulates the different functional capacities of the FcRIa-receptor complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The -chain, initially described as a component of the FcRI signaling complex, is able to form multichain complexes with the ligand-binding -chain of several Fc receptors (1-3). The FcRIa (CD64), FcRIIIa (CD16A), FcRI (CD89), and FcRI -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 FcRI and FcRIIIa 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 (7-9). In the case of FcRI, the -chain serves as an amplifier of receptor function (10, 11).

FcRIa, a receptor with high affinity for IgG (10⁹ M¹) (12), has received attention over the past few years as a potential therapeutic target in malignancy. Targeting of tumors to FcRI with bispecific mAbs¹ can facilitate tumor killing via FcRI-expressing macrophages, and therapeutic humanized bispecific reagents targeting human FcRIa are currently in clinical trials (13-19). Bispecific mAb-based antigen targeting to FcRI can also enhance antigen presentation by dendritic cells with clear applications to enhanced immunization strategies (20).

Expression of the FcRIa -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 FcRIa-mediated phagocytosis and FcRIa-induced activation of tyrosine kinase activity (7-9). However, the -chain is sufficient for endocytosis (7, 9). Whereas expression of the FcRIa -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 FcRIa can alter receptor function downstream of IgG binding (21-23). Expression of an FcRIa -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 FcRI. Lack of the CY domain also changes the Ca²⁺ dependence of receptor-specific 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 FcRI function.

We have begun to dissect the molecular basis for the -chain involvement in FcRI signaling and cell activation. We now show that the CY domain of FcRIa 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 FcRIa -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 FcRIa -chain.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 FcRIa (WT) or a mutant form of FcRIa containing a stop codon after the first amino acid of the cytoplasmic domain (Lys³¹⁵Stop 315) (CY) as we have described previously (8, 22). FcRIa constructs encoding serine to alanine mutations (S328A/S331A, S339A/S340A, and S328A/S331A/S339A/S340A (S₄A₄)) were prepared by overlap PCR and stably transfected as described previously (22, 24). In all cases, two independently prepared cell lines stably expressing each FcRI construct were analyzed. P388D1 cells transfected with human FcRIIa 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 ³²P studies, cells were cultured for 24 h in phosphate-free RPMI 1640 medium in the presence of 5 mCi of ³²P_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(ab')₂ fragments of the anti-FcRIa mAb 22.2 and Fab fragments of the anti-FcRIIa mAb IV.3 were obtained from Medarex (Annandale, NJ). Mouse F(ab')₂ fragments and F(ab')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 huFcRI expression was matched for cells expressing the WT and the cytoplasmic domain deletion mutant by fluorescence-activated cell sorting using anti-FcRI 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 FcRIa 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.

Immunoprecipitation and Phosphotyrosine Analysis-- FcRI was immunoprecipitated from the transfected cell lines or U937 cells using either mAb 22.2 or mAb 197 (kindly provided by Dr. Paul Guyre, Dartmouth University Medical School) (26) prebound to protein G-agarose (Pharmacia Corp.). -Chain from transfected cells was immunoprecipitated by polyclonal rabbit anti--chain Abs bound to protein G-agarose. Cells (10-20 × 10⁶ cells/ml) were lysed in phosphate-buffered saline containing 1% Nonidet P-40 (Sigma) and protease inhibitors (EDTA/pepstatin/aprotinin/sodium orthovanadate/pefabloc). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting.

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 anti-rabbit 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(ab')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.

P388D1 cells, adhered to round glass coverslips at 5 × 10⁵ cells/ml, were incubated with anti-FcRIa mAb 22.2 F(ab')₂-coated bovine erythrocytes (E-22.2) or anti-FcRIIa mAb IV.3 Fab-coated bovine erythrocytes (E-IV.3) in RPMI 1640 medium/20% fetal calf serum (50 µl at 5 × 10⁷ bovine erythrocytes/ml) for 1 h at 37 °C. Noninternalized bovine erythrocytes were lysed by brief immersion of the coverslip in distilled H₂O followed by immersion in buffer. Phagocytosis was quantitated by light microscopy and expressed as the phagocytic index (number of bovine erythrocytes internalized per 100 P388D1 cells).

Treatment of cells with genistein (100 nM) to block protein tyrosine kinase or with okadaic acid (1 µM) 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% Me₂SO (depending on the concentration of inhibitor) for the same period of time.

Cytokine Analysis-- Cells were stimulated in 96-well tissue culture plates (Corning) with either phorbol 12-myristate 13-acetate, surface-absorbed rabbit IgG, or surface-adsorbed F(ab')₂ GAM + mAb 22.2 F(ab')₂. Wells were coated with adsorbed protein (20 µg/ml rabbit IgG or F(ab')₂ GAM) for 2 h at 37 °C. For anti-FcRI stimulation, mAb 22.2 F(ab')₂ at 20 µg/ml was added to a suspension of cells for 30 min at 4 °C followed by two washes to remove unbound mAb. Cells (1-2.5 × 10⁵ cells/ml) were added to the wells and cultured for varying periods of time. The level of murine IL-1 or IL-6 in diluted culture supernatants was quantitated by enzyme-linked immunosorbent assay. For IL-1 determination, recombinant standard, capture Ab (polyclonal rabbit Ab) and biotinylated detection and neutralization mAb (clone 1400.24.17) were obtained from Endogen (Woburn, MA). For IL-6 determination, recombinant standard, capture mAb (clone MP5-2-F3) and biotinylated detection mAb (clone MP5-32C11) were obtained from Pharmingen. Horseradish peroxidase-conjugated streptavidin (Jackson ImmunoResearch) and then 3,3',5,5'-tetramethylbenzidine substrate were added, and the A₄₅₀ was determined.

Flow Cytometry-- Aliquots of cells at 5 × 10⁶ 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(ab')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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Regulation of Phagocytosis by the Cytoplasmic Domain of Human FcRI-- The cytoplasmic domain of the ligand binding -chain of the human FcRIa receptor complex (₂) exerts a quantitative influence on receptor-specific phagocytosis (22). The quantitative phagocytic capacity of a cytoplasmic domain-lacking mutant of huFcRIa is lower than that of wild-type huFcRIa expressed in the murine macrophage cell line P388D1. Likewise, the kinetics of phagocytosis are slower in the tail minus mutant form of huFcRIa compared with WT huFcRIa in these cells. To explore possible mechanisms for this effect, we quantitated huFcRIa-specific phagocytosis in these cell lines in the presence of several kinase and phosphatase inhibitors. Previous work has demonstrated that FcR-mediated phagocytosis is dependent on tyrosine kinase activation. For FcRIa, phagocytosis is dependent on tyrosine phosphorylation of the -chain. In P388D1 cell lines stably expressing either WT huFcRIa or the cytoplasmic tail minus mutant form of huFcRIa (CY), 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 FcRI 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 < 0.017, n = 9 pairs).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Quantitation of receptor-specific phagocytosis by P388D1 cells stably expressing transfected WT huFcRIa, cytoplasmic domain lacking (CY) huFcRIa, or human FcRIIa. Data are presented as a percentage of control (vehicle-treated cells). A, cells were pretreated with 100 nM genistein for 30 min followed by addition of E-22.2 in the continued presence of genistein for 60 min (+Genistein), pretreated with genistein for 30 min followed by addition of E-22.2 in the absence of genistein for 60 min (+Genistein Recovery), pretreated with 1 µM OA for 10 min followed by addition of E-22.2 in the continued presence of OA for 60 min (+Okadaic Acid), or pretreated with 1 µM 1-Nor-okadaone for 10 min followed by addition of E-22.2 in the continued presence of 1-Nor-okadaone for 60 min (+1-Nor-Okadaone). B, stably transfected P388D1 cells were saturated with murine IgG2a followed by determination of FcRIa-specific phagocytosis (+OA) as described in A.

Although the CY domain of huFcRIa lacks tyrosine residues, it does contain four serine residues. We hypothesized that alteration in the phosphorylation status of the CY domain of huFcRI might be important in regulation of receptor complex (₂) function. The type 1 and 2A protein serine/threonine phosphatase inhibitor OA significantly blocked WT huFcRIa-specific phagocytosis (treated versus untreated cells, p < 0.001, n = 15 pairs). In contrast, OA had no effect on phagocytosis mediated by mutant huFcRIa lacking the CY domain (treated versus untreated cells, p > 0.05, n = 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 FcRIa-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 FcRIIa with OA, and no inhibition of FcRIIa-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 huFcRIa-specific phagocytosis in the presence of an intact FcRIa cytoplasmic domain.

We considered the possibility that engagement of the ligand-binding site of FcRIa, 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 huFcRIa to OA, nor did it change the lack of inhibition of CY huFcRIa phagocytosis (Fig. 1B).

The CY Domain of FcRIa Is Dephosphorylated upon Receptor Activation-- To directly assess serine phosphorylation of the CY domain of the -chain of FcRIa, we examined anti-phosphoserine mAb binding to huFcRIa immunoprecipitates from transfected P388D1 cells. Constitutive serine phosphorylation of huFcRIa 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 FcRIa (data not shown). As additional controls for the specificity of the anti-phosphoserine Ab, human FcRIa in which all four cytoplasmic serine residues were mutated to alanine (stably transfected into P388D1 cells, see below) and the mutant FcRIa lacking a cytoplasmic domain were immunoprecipitated and were nonreactive with this blotting Ab. Reprobing of the membrane with a blotting anti-human FcRIa Ab confirmed loading of the serine-mutated form of the receptor (Fig. 2A).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Activation-dependent serine dephosphorylation of huFcRIa. A, FcRIa from stably transfected P388D1 cells was immunoprecipitated with the anti-FcRIa mAb 197 bound to protein G-agarose. Serine phosphorylation was detected with an anti-phosphoserine mAb in resting CY FcRI, S4A4 FcRI, and WT FcRI transfected cells (top panel), followed by stripping of the blot and reprobing with a polyclonal anti-FcRI Ab (bottom panel) as described under "Materials and Methods." B, a time course of serine dephosphorylation upon FcRIa cross-linking in WT huFcRIa stably transfected P388D1 cells. C, FcRIa from 32P-loaded U937 cells was immunoprecipitated with mAb 197 bound to protein G-agarose from unstimulated control cells (lane 1), from cells treated with irrelevant murine IgG F(ab')2 fragments + F(ab')2 GAM for 5 min (lane 2), or from cells stimulated with mAb 22.2 F(ab')2 + F(ab')2 GAM for 5 min in the presence (lane 3) or absence (lane 4) of 1 mM OA.

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-huFcRI Ab confirmed equivalent levels of immunoprecipitated FcRIa at each time point (Fig. 2B). To verify the results of the anti-phosphoserine mAb, we also preloaded U937 cells with ³²P_i and examined the level of phosphorylation of immunoprecipitated huFcRI. Again, constitutive levels of ³²P-labeled FcRIa 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 ³²P-labeled FcRI immunoprecipitate (Fig. 2C). Treatment of the cells with OA before receptor cross-linking resulted in the preservation of FcRI phosphorylation, demonstrating that the OA is acting, at least in part, at the level of the FcRI -chain.

Functional Role of Serine Phosphorylation of the CY Domain of FcRIa-- To directly demonstrate that dephosphorylation of the CY domain of FcRIa is important in receptor function, we prepared three different constructs of huFcRIa 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 (S₄A₄). These three variant forms of huFcRI 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 CY mutant FcRIa 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 FcRIa-mediated phagocytosis.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A, expression of the S328A/S331A, S339A/S340A, and S4A4 mutant FcRIa constructs on the surface of stably transfected P388D1 cells (thin solid line, S4A4; thick solid line, S328A/S331A; dashed line, S339A/S340A; dotted line, isotype control). B, expression of the WT and CY mutant FcRIa constructs on the surface of stably transfected P388D1 cells (thin solid line, WT; thick solid line, CY; dotted line, isotype control). Cells were incubated with a saturating concentration of the anti-human FcRIa mAb 22.2-fluorescein isothiocyanate and analyzed by flow cytometry.

View this table: [in this window] [in a new window]   Table I FcRIa-specific phagocytic index in P388D1 cells

We demonstrated previously that the CY domain of FcRIa is required for early FcRIa-induced IL-6 secretion (22). In contrast to the CY domain truncation form of FcRIa, which did not elicit IL-6 secretion, receptor-specific cross-linking of the S328A/S331A, S339A/S340A, and S₄A₄ mutants resulted in both IL-1 and IL-6 secretion 8 h after stimulation (Table II). We attempted to determine the sensitivity of FcRIa-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 FcRIa function that requires dephosphorylation of the FcRIa CY domain for receptor-mediated cell activation.

View this table: [in this window] [in a new window]   Table II FcRIa-specific IL-1 and IL-6 production by P388D1 cells

Okadaic Acid Treatment Prevents FcRIa-induced Tyrosine Phosphorylation-- Among the earliest signaling events that occur after cross-linking of FcRIa are the activation of Src family tyrosine kinases such as Hck and tyrosine phosphorylation of the -chain (29-32). Given the importance of serine dephosphorylation for FcRIa 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 FcRI-expressing 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 FcRI-expressing cells, incubation of the tail minus mutant FcRIa or the S₄A₄ mutant FcRIa 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 FcRIa which in turn reduces WT FcRIa mediated tyrosine phosphorylation of the -chain.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   OA blocks tyrosine phosphorylation of the -chain after cross-linking of WT huFcRIa (A) but not the cytoplasmic domain lacking (CY) mutant or the S4A4 mutated FcRIa (B). The -chain was immunoprecipitated with a polyclonal anti--chain antiserum bound to protein G-agarose at the indicated time points after cross-linking of the transfected receptor in the presence or absence of OA. Blots were probed with the anti-phosphotyrosine mAb 4G10 followed by stripping and reprobing with the anti--chain Ab.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The cytoplasmic domain of the FcRIa -chain can modulate the kinetics of both receptor-mediated endocytosis and phagocytosis (22) and make receptor-specific phagocytosis insensitive to changes in [Ca²⁺]_i (22, 24). These observations suggest that the FcRIa -chain interacts with intracellular molecules that can modulate receptor signaling elements and function. Previous work has shown that murine FcRIa is constitutively phosphorylated on serine (33), and we now show that the serine residues in the CY domain of human FcRIa are actively phosphorylated and dephosphorylated in relation to receptor cross-linking and that this phosphorylation is important in regulation of FcRIa function. The constitutive phosphorylation of the huFcRIa -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 FcRIa -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 FcRIa or the FcRIa serine to alanine mutants. Furthermore, OA does not alter FcRIIa-specific phagocytosis. Taken together with the observation that OA alters the phosphorylation state of the FcRIa -chain, these data indicate a direct effect of OA on the phosphorylation state of the FcRIa -chain and suggest that this effect is responsible for OA altering FcRIa-specific phagocytosis.

The kinase(s) responsible for phosphorylation of the CY domain is currently unknown. Although protein kinase C activity is required for FcR phagocytosis (36, 37), it is unlikely that protein kinase C isoforms are directly involved in modulating the phosphorylation of the CY domain of FcRIa 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.isb-sib.ch/software/PFSCAN_form.html) does not reveal any potential 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 FcR-induced respiratory burst and that the novel isoforms protein kinase C  and/or protein kinase C  are involved in FcR 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 FcRIa CY domain and casein kinase II constructs using the yeast two-hybrid system has not shown any evidence of interaction between FcRIa 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 FcRIa.

The identity of the phosphatase(s) responsible for activation-induced 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 FcRIa 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 FcRIa and non-muscle filamin-280 (actin-binding protein ABP-280) regulates the localization of FcRIa in the membrane (41). The dissociation of this protein upon receptor engagement may change the relationship of FcRIa 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 FcR (42-48).

Although the specific kinase(s) and phosphatase(s) targeting the FcRIa -chain remain unclear, our data indicate that the previous model suggesting that the -chain is both necessary and sufficient for FcRIa function requires revision. We propose a model in which constitutive serine phosphorylation of the CY domain of FcRIa 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 receptor-induced cell activation proceeds normally. In the FcRIa 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 FcRIa 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 FcRIa 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 FcRIIA, FcRIIIA, and FcRIIIB alter ligand binding and impact upon autoimmune disease susceptibility and severity (49-53). In contrast, the CY domain of FcRIIA, 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 FcRIa (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 FcRIa 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 FcRIa cytoplasmic domain and its serine residues on receptor function.

	ACKNOWLEDGEMENTS

We thank Ka Chen, Jessica T. Leonard, Dana Lau, Paul Palavin, and James J. Moon for technical assistance and Andrew J. Beavis for flow cytometric analysis and cell sorting.

	FOOTNOTES

^* This work was supported by Grants RO1-AR33062, RO1-AR42476, and AI-22193 from the National Institutes of Health (NIH). Flow cytometry was supported in part by NIH Core Grant P60-AR20614 (to the University of Alabama at Birmingham).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

^¶ To whom correspondence should be addressed: University of Alabama at Birmingham, 1530 3rd Ave. S., THT433A, Birmingham, AL 35294-0006. Tel.: 205-934-0894; Fax: 205-934-1564; E-mail: JEdberg@uab.edu.

Published, JBC Papers in Press, August 27, 2002, DOI 10.1074/jbc.M207835200

	ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; Ab, antibody; OA, okadaic acid; huFcRIa, human FcRIa; IL, interleukin; WT, wild-type; GAM, goat anti-mouse IgG; S₄A₄, S328A/S331A/ S339A/S340A.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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