Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.
The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their 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 with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. 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 antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.
Richardson
J H
JH
Department of Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Sodroski
J G
JG
Waldmann
T A
TA
Marasco
W A
WA
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D002451:Cell Compartmentation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015316:Genetic Therapy; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000494:pharmacology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000494:pharmacology*; D015458:Leukemia, T-Cell / Q000628:therapy; D008969:Molecular Sequence Data; D010641:Phenotype; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology
D000906:Antibodies; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; C055966:immunoglobulin Fv