PHENOTYPIC KNOCKOUT OF THE HIGH-AFFINITY HUMAN INTERLEUKIN-2 RECEPTORBY INTRACELLULAR SINGLE-CHAIN ANTIBODIES AGAINST THE ALPHA-SUBUNIT OFTHE RECEPTOR

The experimental manipulation of peptide growth hormones and their cel lular receptors is central to understanding the pathways governing cel lular signaling and growth control. Previous work has shown that intra cellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing the ir transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high- affinity interleukin 2 receptor (IL-2R alpha). A single chain variable -region fragment of the anti-Tac monoclonal antibody was constructed w ith a signal peptide and a C-terminal ER retention signal. Intracellul ar expression of the single-chain antibody was found to completely abr ogate cell surface expression of IL-2R alpha in stimulated Jurkat T ce lls, IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain ant ibody lacking the ER retention signal was also able to inhibit cell su rface expression of IL-2R alpha although the mechanism appeared to inv olve rapid degradation of the receptor chain within the ER. These intr acellular antibodies will provide a valuable tool for examining the ro le of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha .