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-13R alpha2) chain is an essential b inding component for IL-13-mediated ligand binding. Recently, we have demon strated that this receptor chain also plays an important role in the intern alization of IL-13. To study the mechanism of IL-13 internalization, we gen erated mutated IL-13R alpha2 chains that targeted trileucine residues (Leu( 335), Leu(336), and Leu(337)) in the transmembrane domain and a tyrosine mo tif (Tyr(343)) in the intracellular domain and transfected these cDNAs in C OS-7 cells. Cells that expressed a C-terminally truncated IL-13R alpha2 cha in (Delta 335) did not bind IL-13, suggesting that the trileucine region mo dulates IL-13 binding. Truncation of IL-13R alpha2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13R alpha2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13R alpha2 chain transfected cells; howev er, dissociation of cell surface IL-13 was faster compared with wild type I L-13R alpha2 transfectants. These results were further confirmed by determi ning the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR) to cells that expressed IL-13R alpha2 chain mutants. We further demonstrate that the IL-13R alpha2 chain is not ubiquitinated and that internalization of IL-13R alpha2 did not depe nd on ubiquitination, Together, our findings suggest that the dileucine mot if in the trileucine region and tyrosine motif participate in IL-13R alpha2 internalization in distinct manners.