Title

Identification and characterization of subunits of the murine IL-2 receptor complex

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Thomas R. Malek, Committee Chair

Abstract

Variant T cells deficient in the internalization of IL-2 were generated and used to determine the identity of the molecular species associated in the IL-2R complex and study the mechanism of ligand-induced internalization of IL-2. This strategy employed treatment of high affinity IL-2R bearing EL4 cells with IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40) to selectively eliminate wild type cells. One resulting cell line, CX1, which was determined to be IL-2R$\beta$ deficient, was transfected with truncated $\beta$ cDNA resulting in cells which expressed a $\beta$ subunit lacking the intracytoplasmic region (CX$\beta$t.1). CX$\beta$t.1 expressed high affinity, rapidly internalizing IL-2R demonstrating that internalization of IL-2 is independent of the IL-2R$\beta$ intracytoplasmic tail. Additionally, biochemical analysis of cross-linked complexes from CX$\beta$t.1 revealed that the $\beta$ subunit of the murine IL-2 receptor is a 90-100 kDa protein.Subsequent to the identification of the $\beta$ subunit, further characterization of the receptor complex was accomplished through cloning of the murine IL-2R$\gamma$ subunit and using that clone in the characterization of mutant cells. Murine IL-2R$\gamma$ was determined to be encoded by an 1107 base ORF, as is the human IL-2R$\gamma$. The cloned IL-2R$\gamma$ was used in the characterization of cells which were chemically mutagenized then IL-2-PE40 selected. These cells (1F1) did not bear high affinity IL-2R or rapidly internalize bound IL-2. 1F1 were determined to bear a mutant IL-2R$\gamma$ gene which does not encode the transmembrane domain of the molecule. Biochemical analyses of the 1F1 cell line revealed that the $\gamma$ subunit of the IL-2 receptor is a 70-75 kDa protein.This study has defined the identity of the $\beta$ and the $\gamma$ subunits of the receptor and has clarified their roles in the function of the complex. A complete study of the $\gamma$ subunit and its relationship with this and other receptor complexes is of obvious import in molecular immunology. This mutant cell line is appropriate for further studies of this molecule in a T cell environment.

Keywords

Biology, Molecular; Biology, Cell; Biology, Microbiology; Health Sciences, Immunology

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9540971