Ubiquitination of cytokine receptors controls intracellular receptor routing and signal duration, but the underlying molecular determinants are unclear. The suppressor of cytokine signaling protein SOCS3 drives lysosomal degradation of the granulocyte colony-stimulating factor receptor (G-CSFR), depending on SOCS3-mediated ubiquitination of a specific lysine located in a conserved juxtamembrane motif. Here, we show that, despite ubiquitination of other lysines, positioning of a lysine within the membrane-proximal region is indispensable for this process. Neither reallocation of the motif nor fusion of ubiquitin to the C-terminus of the G-CSFR could drive lysosomal routing. However, within this region, the lysine could be shifted 12 amino acids toward the C-terminus without losing its function, arguing against the existence of a linear sorting motif and demonstrating that positioning of the lysine relative to the SOCS3 docking site is flexible. G-CSFR ubiquitination peaked after endocytosis, was inhibited by methyl-β-cyclodextrin as well as hyperosmotic sucrose and severely reduced in internalization-defective G-CSFR mutants, indicating that ubiquitination mainly occurs at endosomes. Apart from elucidating structural and spatio-temporal aspects of SOCS3-mediated ubiquitination, these findings have implications for the abnormal signaling function of G-CSFR mutants found in severe congenital neutropenia, a hematopoietic disorder with a high leukemia risk.

Additional Metadata
Keywords Congenital neutropenia, G-CSF receptor, Leukemia, Lysosomal routing, Suppressor of cytokine signaling, Ubiquitination
Persistent URL dx.doi.org/10.1111/j.1600-0854.2009.00928.x, hdl.handle.net/1765/24858
Journal Traffic (Malden): the international journal of intracellular transport
Wölfler, A, Irandoust, M, Meenhuis, J.C.M, Gits, J, Roovers, O, & Touw, I.P. (2009). Site-specific ubiquitination determines lysosomal sorting and signal attenuation of the granulocyte colony-stimulating factor receptor. Traffic (Malden): the international journal of intracellular transport, 10(8), 1168–1179. doi:10.1111/j.1600-0854.2009.00928.x