Proteolytic digestion of the beta and gamma subunits of the receptor for immunoglobulin E at the cytoplasmic face of the plasma membrane.
Cytoplasmically exposed portions of the high affinity receptor for immunoglobulin E were investigated with controlled proteolytic digestion of plasma membrane vesicles from rat basophilic leukemia cells. Hypotonic shock treatment results in vesicle inversion, thereby exposing the cytoplasmic portions of the approximately 32 kDa beta and approximately 8 kDa gamma subunits to surface labeling by lactoperoxidase-catalyzed 125I-iodination. These 125I-labeled protein segments disappeared after treating inverted vesicles with trypsin, and labeled components also disappeared when chloramine T mediated 125I-iodination was used to label receptors after inverted vesicles had been trypsin digested and solubilized. Biosynthetic labeling of receptors with 35S-methionine showed that a 17-19 kDa labeled fragment, designated beta', remains associated with alpha after trypsin digestion of inverted vesicles. This beta' fragment was confirmed to be the intramembranous portion of the beta subunit in experiments where receptors were labeled with the hydrophobic photoactivated probe 3-(trifluoro-methyl)-3-(m-[125I] iodophenyl)diazirine prior to digestion. Our experimental results are consistent with the amino acid sequence and topography of the beta subunit predicted from the recently cloned cDNA for this subunit (Kinet, J.-P., et al., Proc. Natl. Acad. Sci. 85, 6483, 1988). Since the cytoplasmically-exposed portions of the beta and gamma subunits can also be efficiently and selectively removed from solubilized receptors by trypsin, reconstitution experiments to examine the importance of these segments in mediating the delivery of the transmembrane signal are made possible.
Hammes
S
S
Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853.
Holowka
D
D
Baird
B
B
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte; D001345:Autoradiography; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D007073:Immunoglobulin E; D011233:Precipitin Tests; D011961:Receptors, Fc; D017455:Receptors, IgE; D014357:Trypsin
D000944:Antigens, Differentiation, B-Lymphocyte; D011961:Receptors, Fc; D017455:Receptors, IgE; D007073:Immunoglobulin E; D014357:Trypsin