title: Transport of the IgE receptor alpha-chain is controlled by a multicomponent intracellular retention signal.
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.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Cauvi
authors.forename: Xufang
authors.initials: X
authors.lastname: Tian
authors.forename: Katharina
authors.initials: K
authors.lastname: von Loehneysen
authors.forename: Michael W
authors.initials: MW
authors.lastname: Robertson
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D005434:Flow Cytometry; D017930:Genes, Reporter; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008239:Lysine / Q000737:chemistry*; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D009841:Oligonucleotides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017455:Receptors, IgE / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013499:Surface Properties; D013997:Time Factors; D014162:Transfection
chemical_list: C475664:FCER1A protein, human; D009841:Oligonucleotides; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; D009355:Neomycin; D008239:Lysine; D000409:Alanine