The Signal Peptide of the IgE Receptor α-Chain Prevents Surface Expression of an Immunoreceptor Tyrosine-based Activation Motif-free Receptor Pool

Abstract

The high affinity receptor for IgE, Fc epsilon receptor I (Fc⑀RI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded Fc⑀RI ␣-chains induces cell activation via immunoreceptor tyrosinebased activation motifs in associated signaling subunits, such as Fc⑀RI ␥-chains. Here we show that the human Fc⑀RI ␣-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K b . This singlechain isoform of Fc⑀RI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of Fc⑀RI-␣. Once the Fc⑀RI ␣-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the Fc⑀RI ␥-chain, this single-chain Fc⑀RI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric Fc⑀RI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the Fc⑀RI ␣-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.

A Soluble Form of the High Affinity IgE Receptor, Fc-Epsilon-RI, Circulates in Human Serum

“…Immunoprecipitation was next performed with NIP-beads (Sigma) as previously described [36], [42]. Proteins were eluted from beads in non-reducing Laemmli sample buffer and samples were separated on 12% non-reducing SDS-PAGE gels, transferred to PVDF membrane (Pierce, Thermo Fisher Scientific, Rockford, IL) and probed with anti-FcεRI-alpha (mAb 19-1 or CRA1 for reducing conditions, both 0.5 mg/ml) followed by peroxidase (HRP)-conjugated goat-anti-mouse IgG for detection of precipitated α-chain.…”

Development and validation of a standardized ELISA for the detection of soluble Fc-epsilon-RI in human serum

“…Immunoprecipitation was performed as described using cIgE and anti-NIP sepharose (Cat#N-1199-5) from Biosearch Technologies (Novato, CA) (Platzer and Fiebiger, 2010). Precipitated rsFcεRI was eluted in reducing Laemmli buffer, samples were run on 12% SDS-PAGE gels, transferred to PVDF Transfer Membrane (Cat#88518) from Thermo Scientific and probed with 0.5 mg/ml CRA1, followed by detection with goat-anti-mouse IgG HRP conjugated (1:2000, Cat#31430) from Pierce.…”

Cloning and Characterization of CD300d, a Novel Member of the Human CD300 Family of Immune Receptors

“…Nevertheless, this regulation does not occur constitutively because in eosinophils and megakaryocytes, Fc⑀R␣ is accumulated in intracellular compartments despite the presence of Fc⑀R␥ in those cells (26,27). It is of note that additional mechanisms controlling the surface expression of Fc⑀R␣ related to its signal peptide (28) and to the N-glycosylation of asparagine residues within its immunoglobulin domain have been described (29). Signal peptide is not responsible for CD300d retention.…”