Selection of internalization-deficient cells by interleukin-2-Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin-2 receptor beta chain does not contribute to internalization of interleukin-2.
To study the structural basis of ligand-induced receptor-mediated internalization of interleukin-2 (IL-2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine. IL-2 receptor (IL-2R) alpha- and beta-bearing EL4 cells, that express high-affinity IL-2R and internalize IL-2, were treated with low doses of IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40). This treatment resulted in isolation of a variant (CX1) that was unable to express high-affinity IL-2R or internalize IL-2. Transfection of CX1 with the IL-2R beta cDNA led to surface expression of IL-2R beta and high-affinity IL-2R as well as the ability to internalize IL-2. This finding indicates that the absence of the beta subunit was the sole defect in CX1 responsible for its failure to internalize IL-2. By transfecting CX1 with mutated beta cDNA, several CX1 transfectants were produced that expressed a beta-subunit that lacked all amino acids of the intracytoplasmic region. These transfectants expressed high-affinity IL-2R and internalized IL-2 at a rate comparable to cells expressing wild-type beta-chain. These results demonstrate that internalization of IL-2 is independent of any signals contained in the intracytoplasmic tail of the beta subunit and raise the possibility that such signals may be entirely contained within the gamma subunit.
Furse
R K
RK
Department of Microbiology and Immunology, University of Miami School of Medicine, FL 33101.
Malek
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry; D004705:Endocytosis; D005098:Exotoxins / Q000378:metabolism*; D000922:Immunotoxins / Q000378:metabolism*; D007376:Interleukin-2 / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D015375:Receptors, Interleukin-2 / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins
D005098:Exotoxins; C057961:IL-2-PE40 chimeric protein, recombinant; D000922:Immunotoxins; D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins