Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor beta (IL-2R beta) and IL-4 receptor alpha (IL-4R alpha), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2R beta and IL-4R alpha in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2R beta and IL-4R alpha as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2R alpha was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2R beta together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2R beta. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation. (C) 2013 Elsevier Inc. All rights reserved.