Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor alpha 2 chain.
Interleukin (IL)-13 receptor alpha2 (IL-13Ralpha2) 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-13Ralpha2 chains that targeted trileucine residues (Leu(335), Leu(336), and Leu(337)) in the transmembrane domain and a tyrosine motif (Tyr(343)) in the intracellular domain and transfected these cDNAs in COS-7 cells. Cells that expressed a C-terminally truncated IL-13Ralpha2 chain (Delta335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13Ralpha2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Ralpha2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Ralpha2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Ralpha2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR) to cells that expressed IL-13Ralpha2 chain mutants. We further demonstrate that the IL-13Ralpha2 chain is not ubiquitinated and that internalization of IL-13Ralpha2 did not depend on ubiquitination. Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Ralpha2 internalization in distinct manners.
Kawakami
K
K
Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Takeshita
F
F
Puri
R K
RK
D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D001665:Binding Sites; D019556:COS Cells; D005098:Exotoxins / Q000235:genetics; D006801:Humans; D018793:Interleukin-13 / Q000378:metabolism*; D053661:Interleukin-13 Receptor alpha1 Subunit; D007930:Leucine / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism*; D053660:Receptors, Interleukin-13; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D018793:Interleukin-13; D053661:Interleukin-13 Receptor alpha1 Subunit; D007930:Leucine; D009842:Oligopeptides; D018123:Receptors, Interleukin; D053660:Receptors, Interleukin-13; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D014452:Ubiquitins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C508181:IL13RA1 protein, human; C036671:leucyl-leucyl-leucine