Selection of internalization‐deficient cells by interleukin‐2‐Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin‐2 receptor β chain does not contribute to internalization of interleukin‐2

Abstract

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) α‐ and β‐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‐2Rβ cDNA led to surface expression of IL‐2Rβ and high‐affinity IL‐2R as well as the ability to internalize IL‐2. This finding indicates that the absence of the p subunit was the sole defect in CX1 responsible for its failure to internalize IL‐2. By transfecting CX1 with mutated β cDNA, several CX1 transfectants were produced that expressed a β‐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 β‐chain. These results demonstrate that internalization of IL‐2 is independent of any signals contained in the intracytoplasmic tail of the β subunit and raise the possibility that such signals may be entirely contained within the γ subunit.