Proteolytic Digestion of the β and γ Subunits of the Receptor for Immunoglobulin E at the Cytoplasmic Face of the Plasma Membrane

Abstract

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 ∼32 kDa β and ∼8 kDa γ 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 ³⁵-methionine showed that a 17-19 kDa labeled fragment, designated β', remains associated with α after trypsin digestion of inverted vesicles. This β' fragment was confirmed to be the intramembranous portion of the β subunit in experiments where receptors were labeled with the hydrophobic photoactivated probe 3-(trifluoro-methyl)-3-(m-[¹²⁵I] iodopheny1)diazirine prior to digestion. Our experimental results are consistent with the amino acid sequence and topography of the β 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 β and γ 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.