Site-Specific Ubiquitination Determines Lysosomal Sorting and Signal Attenuation of the Granulocyte Colony-Stimulating Factor Receptor

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-beta-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

Functional interaction between mutations in the granulocyte colony-stimulating factor receptor in severe congenital neutropenia

Most severe congenital neutropenia (SCN) cases possess constitutive neutrophil elastase mutations; a smaller cohort has acquired mutations truncating the granulocyte colony-stimulating factor receptor (G-CSF-R). We have described a case with constitutive extracellular G-CSF-R mutation hyporesponsive to ligand. Here we report two independent acquired G-CSF-R truncation mutations and a novel constitutive neutrophil elastase mutation in this patient. Co-expression of a truncated receptor chain restored STAT5 signalling responses of the extracellular G-CSF-R mutant, while constitutively-active STAT5 enhanced its proliferative capacity. These data add to our knowledge of SCN and further highlight the importance of STAT5 in mediating proliferative responses to G-CSF