Transmembrane sequences are determinants of immunoreceptor signaling.
To investigate structural features critical for signal initiation by Ag-stimulated immunoreceptors, we constructed a series of single-chain chimeric receptors that incorporate extracellular human Fc epsilonRIalpha for IgE binding, a variable transmembrane (TM) segment, and the ITAM-containing cytoplasmic tail of the TCR zeta-chain. We find that functional responses mediated by these receptors are strongly dependent on their TM sequences, and these responses are highly correlated to cross-link-dependent association with detergent-resistant lipid rafts. For one chimera designated alpha Fzeta, mutation of a TM cysteine abolishes robust signaling and lipid raft association. In addition, TM disulfide-mediated oligomerization of another chimeric receptor, alpha zetazeta, enhances signaling. These results demonstrate an important role for TM segments in immunoreceptor signaling and a strong correspondence between strength of signaling and cross-link-dependent partitioning into ordered membrane domains.
Gosse
Julie A
JA
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853, USA.
Wagenknecht-Wiesner
Alice
A
Holowka
David
D
Baird
Barbara
B
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002999:Clone Cells; D003432:Cross-Linking Reagents / Q000378:metabolism; D000939:Epitopes / Q000502:physiology*; D006801:Humans; D021962:Membrane Microdomains / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D017830:Octoxynol; D010766:Phosphorylation; D051381:Rats; D017455:Receptors, IgE / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D013395:Sucrose; D014443:Tyrosine / Q000378:metabolism
D003432:Cross-Linking Reagents; D000939:Epitopes; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D013395:Sucrose; D017830:Octoxynol; D002118:Calcium