The Cytoplasmic Domain of Human FcγRIa Alters the Functional Properties of the FcγRI·γ-Chain Receptor Complex

Abstract

The ␥/-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding ␣-chain alters the biological properties of the receptor complex. Using macrophage Fc␥RIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human Fc␥RIa. Both receptors functionally associate with the endogenous murine ␥-chain. However, we have established that the CY of Fc␥RIa directly contributes to the functional properties of the receptor complex. Deletion of the Fc␥RIa CY leads to slower kinetics of receptorspecific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent Fc␥RIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates Fc␥RIa-specific secretion of interleukin-6 but does not affect production of interleukin-1␤. These results demonstrate a functional role for the CY of Fc␥RIa and provide a general model for understanding how multiple receptors that utilize the ␥-chain can generate diversity in function.Fc␥ receptors play a central role in the handling of immune complexes, regulation of inflammatory responses, antibody secretion, and T cell activity (1-4). Common to each of these functions is the initiation of tyrosine phosphorylation following receptor cross-linking (5) and the involvement of the ␥/ subunits leading to the view that Fc receptors subserve redundant signaling functions. However, recent evidence suggests that these receptors are not redundant. For example, Fc␥RIIIa appears necessary for initiating the Arthus inflammatory reaction (6, 7), while Fc␥RIa and Fc␣RI can down-regulate inflammatory responses by initiating the secretion of IL 1 -10 and IL-1ra, respectively (4, 8). The basis for these differences are unknown.Fc␥RI is expressed on the cell surface in association with the ␥-chain (9, 10). This association is not a prerequisite for transient receptor expression but is necessary for stable expression (11, 12). The ␥-chain cytoplasmic domain contains an immunoreceptor tyrosine activation motif (ITAM) and current data suggest that the ␥-chain cytoplasmic domain is both necessary and sufficient for Fc␥RIa induced functions (13-15). Biochemical studies have shown that cross-linking of the Fc␥RIa⅐␥-chain complex results in activation of a Src family kinase(s) and the tyrosine kinase p72Syk (2,5). Activation of these kinases results in tyrosine phosphorylation of the ␥-chain and the initiation of a signaling cascade that can culminate in the induction of degranulation, phagocytosis, an oxidative burst, ADCC activity and the induction of gene transcription. The association between Fc␥RIa and ␥-chain may also be important in the formation of a h...

A Comparison of Mediators Released or Generated by IFN-γ-Treated Human Mast Cells Following Aggregation of FcγRI or FcεRI

“…Furthermore, 10 g/ml of mouse F(abЈ) 2 of IgG did not affect the standard curves. The data were confirmed by an additional method described by Edberg et al (20). Wells of tissue culture plates were coated with absorbed protein (10 g/ml of goat F(abЈ) 2 of anti-mouse F(abЈ) 2 of IgG) in carbonate-bicarbonate buffer (Sigma) for 30 min at 37°C and overnight at 4°C.…”

“…With regard to this observation, recent evidence suggests that the cytoplasmic domain of Fc␥RI␣ may recruit distinct signaling elements to the receptor complex (20). It has been shown by comparison of responses in wild-type human Fc␥RI␣ with a cytoplasmic domain deletion mutant of Fc␥RI␣ expressed at comparable levels in stable transfectants of the murine macrophage cell line that the Fc␥RI␣-␥-chain complex-induced responses, such as IL-6 production and phagocytosis, are altered (20). The basis for these differences is still unknown, but this might explain significant enhancement of some cytokine mRNAs expression via Fc␥RI aggregation compared with Fc⑀RI aggregation.…”

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

“…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).…”

“…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.…”

Direct interaction between FcγRI (CD64) and periplakin controls receptor endocytosis and ligand binding capacity

“…The Fc␥RI cytosolic domain (Fc␥RI-CY) mediated MHC class II antigen presentation without active FcR ␥-chain signaling (10), whereas deletion of Fc␥RI-CY retarded kinetics of endocytosis and phagocytosis and abrogated Fc␥RI-triggered IL-6 secretion (11). Thus far, only actin-binding protein 280 (filamin A) has been described to bind Fc␥RI-CY under some conditions, but effects on receptor function have not been shown (12).…”