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Abstract
The human high affinity IgE receptor (FcepsilonRI) is a central component of the allergic response and is expressed as either a trimeric alphagamma2 or tetrameric alphabetagamma2 complex. It has been previously described that the cytoplasmic domain (CD) of the alpha-chain carries a dilysine motif at positions -3/-7 from the C terminus that functions in intracellular retention prior to assembly with other FcepsilonRI subunits. In this report we have further explored the role of the -3/-7 dilysine signal in controlling steady-state alpha-chain transport by mutational analysis and found little surface expression of a -3/-7 dialanine alpha-chain mutant but significant Golgi localization. We compared the transport properties of a series of alpha-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 alpha-chain retention. By performing alanine-scanning mutagenesis we identified a dilysine sequence (Lys(212)-Lys(216)) proximal to the transmembrane domain (TMD) that is important for both alpha-chain cell-surface expression and intracellular stability. Furthermore, co-mutation of the Lys(212)-Lys(216) residues with the -3/-7 dilysine signal produced a dramatic increase in alpha-chain surface expression that was further increased by co-mutation of the lone charged residue (Asp(192)) in the TMD thereby defining three regions that function to regulate alpha-chain transport and in a highly synergistic manner.
Highlights
Naling by the presence of intracellular tyrosine activation motifs
It has recently been shown that assembly of the multimeric Fc⑀RI ␣␥2 complex is an important step in regulation of Fc⑀RI surface expression that in turn is closely linked to the magnitude of the allergic response [17]
In this work we re-evaluated the role of the Ϫ3/Ϫ7 dilysine motif in steady-state ␣-chain surface expression by first expressing variants of the C-terminal ␣-chain sequence PKPNPKNN fused to the C terminus of the Tac (CD25) membrane protein
Summary
Naling by the presence of intracellular tyrosine activation motifs. Assembly with the -chain 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. In addition it has been demonstrated that ␣␥2 receptor complexes show significantly higher intracellular accumulation than the corresponding tetrameric receptor isoform [17], suggesting the possibility that the factors responsible for intracellular retention of the Fc⑀RI ␣-chain may function in partial retention of the trimeric Fc⑀RI complex It has been previously shown using chimeric reporter molecules composed of the interleukin-2 receptor ␣-chain (CD25 and Tac) and C-terminal Fc⑀RI␣ cytoplasmic domain (CD) sequences (Tac-␣) that the two lysine residues near the C terminus (positions Ϫ3 and Ϫ7) function in ER retention during early biosynthesis [18]. N ␣D192A was produced using the PCR products using oligonucleotides 14 and 27 plus 15 and 28 in a second stage PCR using primers 14 and 15. Additional ER localization signal sequences have been identified, including diarginine-based (RXR) CD sequences [36, 37] as well as certain TMD sequences [38, 39]
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