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Molecular characterization of the dimer formation of Fcα/μ receptor (CD351)

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Abstract

Fcα/μR (CD351) is an Fc receptor for both IgA and IgM and forms an atypical dimer that is resistant to reduction by 2-mercaptoethanol or boiling. We previously demonstrated that the cytoplasmic portion of Fcα/μR is required for dimer formation and for its efficient cell-surface expression. However, the biochemical nature of these phenomena has not been determined. By using a BW5147 mouse cell line expressing deletion mutants of the cytoplasmic region of Fcα/μR, we found that the region spanning amino acids 504–523 was required for efficient cell-surface expression, whereas the region spanning amino acids 481–490 was required for dimmer formation. Immunoblotting analyses of transfectants simultaneously expressing Flag-tagged Fcα/μR and hemagglutinin-tagged Fcα/μR suggested that Fcα/μR does not form homodimers. Instead, our data suggest that Fcα/μR forms heterodimers with an as-yet-unknown molecule with a molecular weight of 60–70 kDa.

Introduction

Fc receptors recognize the Fc portion of immunoglobulins and mediate a variety of immune responses upon binding to immune complexes of immunoglobulins and antigens (Daeron, 1997). Several Fc receptors for IgG and IgE have been shown to play pivotal roles in IgG- or IgE-mediated immune responses, such as antibody-dependent cellular cytotoxicity, mast cell degranulation, phagocytosis, cell proliferation, antibody secretion, and antigen presentation (Daeron, 1997, Lin et al., 1994, Ravetch, 1997). In contrast, the functional characteristics of the Fc receptors for IgM are not as well understood. We previously identified Fcα/μR (CD351), an Fc receptor for IgA and IgM (Sakamoto et al., 2001, Shibuya et al., 2000). Because the Fcα/μR gene is located near the polymeric IgR on chromosome 1 (1F in mice and 1q32.3 in humans), these receptors seem to be closely related (Kaetzel, 2005, Shibuya et al., 2000, Shimizu et al., 2001). Fcα/μR is expressed not only on hematopoietic cells such as B cells and macrophages but also on follicular dendritic cells (FDCs) within follicles of lymphoid organs (Honda et al., 2009). Fcα/μR mediates endocytosis of the ligands IgA and IgM, for which the cytoplasmic portion of Fcα/μR is responsible (Shibuya et al., 2000, Yang et al., 2009). By using Fcα/μR-deficient mice, we reported that Fcα/μR negatively regulates T-independent antigens retention by FDCs, leading to suppression of humoral immune responses against T-independent antigens (Honda et al., 2009).

We have also reported that the cytoplasmic portion of Fcα/μR is important for the formation of dimers with a molecular weight of ∼130 kDa. These dimers are resistant to boiling in SDS and to reduction by 2-mercaptoethanol (2-ME). The cytoplasmic portion is also important for the efficient cell-surface expression of Fcα/μR (Cho et al., 2010). In this study, we further examined this cytoplasmic region by using BW5147 transfectants expressing deletion mutants of the cytoplasmic portion of Fcα/μR. We also examined the formation of Fcα/μR dimers and show that Fcα/μR does not form homodimers, as we expected, but instead forms heterodimers with a molecule that has a molecular weight of 60–70 kDa.

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Cells and transfectants

BW5147 transfectants stably expressing wild-type Fcα/μR or mutant Fcα/μR lacking the cytoplasmic portion from amino acids 481–535 (ΔCyt.Fcα/μR), 491–535 (Δ490.Fcα/μR), 504–535 (Δ503.Fcα/μR) or 524–535 (Δ523.Fcα/μR) and tagged with Flag or hemagglutinin (HA) at the N-terminus were established as described previously (Cho et al., 2010, Shibuya et al., 2000). To generate site-directed mutations of the Fcα/μR di-Leucine motif at residues Leu519 and Leu520 and of the Gln482 and Gln486 residues

The region spanning amino acids 504–523 of the cytoplasmic portion is necessary for the efficient cell-surface expression of Fcα/μR

In previous reports, we showed that the cytoplasmic portion of Fcα/μR is required for both dimer formation and efficient cell-surface expression (Cho et al., 2010). To determine the region responsible for these features, we established a BW5147 transfectant expressing mutated Fcα/μR, in which the cytoplasmic portion of Fcα/μR was deleted after the 503rd amino acid (Δ503.Fcα/μR) or the 523rd amino acid (Δ523.Fcα/μR) (Fig. 1). Consistent with our previous report (Cho et al., 2010), flow cytometry

Acknowledgments

We thank S. Mitsuishi for secretarial assistance. This research was supported in part by grants provided by the Ministry of Education, Science and Culture of Japan and the Program for the Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO).

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    Citation Excerpt :

    Fcα/μR (CD351) is an Fc receptor for IgM as well as IgA (Sakamoto et al., 2001; Shibuya et al., 2000). Fcα/μR forms an atypical dimer, for which the cytoplasmic region of Fcα/μR is required (Cho et al., 2010; Takagaki et al., 2013). Fcα/μR is moderately expressed on B cells and macrophages (Sakamoto et al., 2001; Shibuya et al., 2000), and most strongly on follicular dendritic cells (FDCs) (Cho et al., 2006; Honda et al., 2009; Usui et al., 2012), which is involved in germinal center (GC) formation (Chaplin and Zindl, 2006; Park and Choi, 2005).

1

These authors contributed equally to this work.

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