Transport of the IgE Receptor α-Chain Is Controlled by a Multicomponent Intracellular Retention Signal

Abstract

The human high affinity IgE receptor (Fc⑀RI) is a central component of the allergic response and is expressed as either a trimeric ␣␥2 or tetrameric ␣␤␥2 complex. It has been previously described that the cytoplasmic domain (CD) of the ␣-chain carries a dilysine motif at positions ؊3/؊7 from the C terminus that functions in intracellular retention prior to assembly with other Fc⑀RI subunits. In this report we have further explored the role of the ؊3/؊7 dilysine signal in controlling steady-state ␣-chain transport by mutational analysis and found little surface expression of a ؊3/؊7 dialanine ␣-chain mutant but significant Golgi localization. We compared the transport properties of a series of ␣-chain cytoplasmic domain truncation mutants and observed that truncation mutants lacking 23 or more C-terminal residues showed a dramatic increase in steady-state transport suggesting a role for the membrane-proximal CD sequence in ␣-chain retention. By performing alanine-scanning mutagenesis we identified a dilysine sequence (Lys The high affinity IgE receptor (Fc⑀RI) 2 is a multisubunit complex comprised of either a tetrameric ␣␤␥2 complex or an alternative trimeric ␣␥2 isoform (1, 2). Aggregation of the tetrameric receptor on mast cells and basophils occurs upon cross-linking of receptor-bound IgE with a multivalent antigen that initiates Fc⑀RI-dependent signaling, culminating in cellular degranulation and the ensuing clinical symptoms of hypersensitivity. Expression of the trimeric Fc⑀RI isoform occurs on a number of cells, including monocytes, epidermal Langerhans cells, and dendritic cells, and a role for the ␣␥2 receptor in antigen presentation has now been established (3). The Fc⑀RI ␣-chain is exclusively involved in IgE binding, whereas the associated ␥-and ␤-chains function in signaling by the presence of intracellular tyrosine activation motifs. Assembly with the ␤-chain also functions to amplify Fc⑀RI cell-surface expression relative to the ␣␥2 isoform (4, 5) and represents a fundamental mechanism for the regulation of Fc⑀RI cell-surface expression. It has long been known that binding of IgE to cell-surface Fc⑀RI leads to enhanced receptor expression (6), an effect that has now been amply demonstrated for both receptor isoforms (6 -9) and is thought to involve receptor-ligand surface stabilization and possibly increased export of ␣-chain from an intracellular pool (10, 11). Additional mechanisms that regulate Fc⑀RI expression have also been identified, including the effects of interleukin-4 in mast cells (7, 12) and transforming growth factor-␤1 in both dendritic cells (13) and mast cells (14). Because the sensitivity of the allergic response is critically linked to Fc⑀RI surface expression (15) that in turn is dependent on Fc⑀RI subunit transport characteristics (16) and assembly (17), it remains an important goal to fully elucidate the Fc⑀RI structural features that regulate these processes to thereby provide a better understanding of the molecular origins of the allergic response.The underlying factors th...

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

“…Thus, the single-chain isoform of the receptor could act as an IgE-receptor, which reacts to serum IgE levels and shuttles antigen into antigen presentation compartments. Removal of the cytosolic ER retention signals as well as mutating the transmembrane ER retention signal suggest that the signal peptide regulates ER retention in a concerted fashion with formerly described ER retention motifs (15,18). The fact that the regulatory influence of the signal peptide could still be observed when other ER retention signals were removed argues against a simple overexpression artifact.…”

“…Signals-ER retention/retrieval by dilysine motifs in the cytosolic tail of Fc⑀RI-␣ has been proposed as a key regulatory mechanism to prevent ER exit in the absence of the Fc⑀RI-␥ chain (15,16). Thus, we generated cytosolic tail truncations of endo-␣ and K b -␣ to dissect the contributions of the signal peptide from the ER retention signals of the cytosolic tail of the ␣-chain.…”

“…We also investigated whether the modification of the transmembrane ER retention signal Asp 192 (15) (Fig. 3D) at equal mRNA expression levels (Fig.…”

“…This is different from assembly mechanisms defined for other activating immune receptors, such as the T or B cell receptors, that do not depend on coordinated translation of their subunits for receptor complex formation (4,20). After the removal of all known transmembrane and cytosolic retention signals in Fc⑀RI-␣, a substantial amount of the protein still remains intracellular (15,18,21). These findings suggest that an as yet undefined sequence element prevents surface expression of the Fc⑀RI ␣-chain in the absence of Fc⑀RI-␥.…”

“…, in the cytosolic tail and the charged transmembrane amino acid Asp 192 (15,16). In human cells, these ER retrieval signals are overcome by the assembly of Fc⑀RI-␣ with Fc⑀RI-␥, the common Fc receptor ␥-chain (3).…”