Identification of Distinct Roles for a Dileucine and a Tyrosine Internalization Motif in the Interleukin (IL)-13 Binding Component IL-13 Receptor α2 Chain

doi:10.1074/jbc.M100936200

Identification of Distinct Roles for a Dileucine and a Tyrosine Internalization Motif in the Interleukin (IL)-13 Binding Component IL-13 Receptor α2 Chain*

From the ^‡Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, and ^§Retroviral Immunology Section, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

Abstract

Interleukin (IL)-13 receptor α2 (IL-13Rα2) chain is an essential binding component for IL-13-mediated ligand binding. Recently, we have demonstrated that this receptor chain also plays an important role in the internalization of IL-13. To study the mechanism of IL-13 internalization, we generated mutated IL-13Rα2 chains that targeted trileucine residues (Leu³³⁵, Leu³³⁶, and Leu³³⁷) in the transmembrane domain and a tyrosine motif (Tyr³⁴³) in the intracellular domain and transfected these cDNAs in COS-7 cells. Cells that expressed a C-terminally truncated IL-13Rα2 chain (Δ335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13Rα2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Rα2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Rα2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Rα2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonasexotoxin (IL13-PE38QQR) to cells that expressed IL-13Rα2 chain mutants. We further demonstrate that the IL-13Rα2 chain is not ubiquitinated and that internalization of IL-13Rα2 did not depend on ubiquitination. Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Rα2 internalization in distinct manners.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Bldg. 29B, Rm. 2NN10, 29 Lincoln Dr. MSC 4555, Bethesda, MD 20892. Tel.: 301-827-0471; Fax: 301-827-0449; E-mail: puri@cber.fda.gov.
Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M100936200
↵2 B. H. Joshi and R. K. Puri, unpublished results.
Abbreviations:

IL

interleukin

IL-6R and IL-13R

IL-6 and IL-13 receptor, respectively

IL-13Rα2

interleukin-13 receptor α2 chain

DMEM

Dulbecco's modified Eagle's medium

IL13-PE38QQR

a recombinant fusion protein composed of IL-13 and a truncated form of Pseudomonas exotoxin A

STAT

signal transducers and activators of transcription

PCR

polymerase chain reaction

RT-PCR

reverse transcriptase-PCR
- Received January 31, 2001.
- Revision received May 11, 2001.
The American Society for Biochemistry and Molecular Biology, Inc.