Ubiquitin-independent binding of Hrs mediates endosomal sorting of the interleukin-2 receptor beta-chain.
Several lines of evidence have revealed that ubiquitylation of membrane proteins serves as a signal for endosomal sorting into lysosomes or lytic vacuoles. The hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) interacts with ubiquitylated cargoes through its ubiquitin-interacting-motif domain (UIM domain), and plays an essential early role in endosomal sorting. Here, we show that the C-terminal region of Hrs, which does not contain the UIM domain, can bind to interleukin-2 receptor beta (IL-2Rbeta). We found a direct interaction between bacterially expressed IL-2Rbeta and Hrs in GST pull-down assays, indicating that their binding is independent of ubiquitin. Trafficking and degradation assays revealed that, similarly to wild-type IL-2Rbeta, an IL-2Rbeta mutant lacking all the cytoplasmic lysine residues is sorted from Hrs-positive early endosomes to LAMP1-positive late endosomes, resulting in degradation of the receptor. By contrast, an IL-2Rbeta mutant lacking the Hrs-binding region passes through early endosomes and is mis-sorted to compartments positive for the transferrin receptor. The latter mutant exhibits attenuated degradation. Taken together, these results indicate that precise sorting of IL-2Rbeta from early to late endosomes is mediated by Hrs, a known sorting component of the ubiquitin-dependent machinery, in a manner that is independent of UIM-ubiquitin binding.
Yamashita
Yuki
Y
Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan.
Kojima
Katsuhiko
K
Tsukahara
Tomonori
T
Agawa
Hideyuki
H
Yamada
Koichiro
K
Amano
Yuji
Y
Kurotori
Naoki
N
Tanaka
Nobuyuki
N
Sugamura
Kazuo
K
Takeshita
Toshikazu
T
D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D053646:Interleukin-2 Receptor beta Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051907:Lysosomal Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1
D056827:Endosomal Sorting Complexes Required for Transport; D053646:Interleukin-2 Receptor beta Subunit; D051907:Lysosomal Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate