Surface expression of mutated subunits of the high affinity mast cell receptor for IgE.
The mast cell receptor with high affinity for IgE consists of four transmembrane polypeptides which are held together by detergent-sensitive interactions: an IgE-binding alpha chain, a single beta chain, and a disulfide-linked dimer of gamma chains. Now that the cDNAs that code for each of the subunits have been isolated, it should be possible to probe by site-specific mutations, which portions of the receptor are critical for transmembrane signaling. One prerequisite for such studies is that the mutant receptors be expressible on the cell surface. We have explored this issue by transiently transfecting COS 7 cells with mutant subunits and assessing surface expression by IgE binding. Removal of any single cytoplasmic domain of the receptor's subunits had little influence on surface expression, and even receptors missing all five cytoplasmic domains were expressed, albeit less efficiently. Minor changes within the transmembrane domains (TMs) sometimes produced major effects and more drastic changes in the TMs ablated surface expression entirely. These data suggest that the TMs are critical loci for receptor display. Cys7 (residue 2 in the gamma TM) was shown to form the inter-gamma disulfide bond and to be nonessential for surface expression. By localizing this bond, residues in the TM of gamma that are buried in the interface between the gamma subunits could be predicted. Consistent with observations on other membrane proteins (Rees, D. C., DeAntonio, L., and Eisenberg, D. (1989) Science 245, 510-513), maximal interspecies conservation was observed for those residues in the gamma TM predicted to be buried. This was also true for those residues in the alpha and beta TMs predicted to be buried by analysis of the TM hydrophobic moments.
Varin-Blank
N
N
Section on Chemical Immunology, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Metzger
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D002678:Chimera; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D046911:Macromolecular Substances; D008407:Mast Cells / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D051381:Rats; D011961:Receptors, Fc / Q000235:genetics* / Q000378:metabolism; D017455:Receptors, IgE; D015183:Restriction Mapping; D012397:Rosette Formation; D014162:Transfection
D000944:Antigens, Differentiation, B-Lymphocyte; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011961:Receptors, Fc; D017455:Receptors, IgE; D007073:Immunoglobulin E