The granulocyte colony-stimulating factor receptor (G-CSF-R) induces proliferation, survival and differentiation of myeloid progenitor cells in a tightly controlled temporal fashion. These responses depend on multiple signaling mechanisms that are activated via distinct regions in the cytoplasmic domain of wild type (WT) G-CSF-R. About 20% of severe congenital neutropenia patients acquire mutations that truncate the C-terminus of G-CSF-R, which is often associated with disease progression to acute myeloid leukemia. Myeloid cells expressing these truncated G-CSF-R hyperproliferate and are hampered in differentiation in response to G-CSF. Multiple mechanisms have been linked to perturbed signaling of truncated G-CSF-R. Specifically, defective internalization has been associated with a prolonged activation status of the truncated G-CSF-R, which is e.g. reflected by strongly increased and sustained activation of STAT5. We found that internalized WT G-CSF-R are rapidly targeted to lysosomes, suggesting that lysosomal degradation is a major mechanism for signal attenuation. Lysine (K) residues in the cytoplasmic tail of transmembrane receptors are often determinants for ubiquitin-mediated sorting into multi-vesicular bodies and lysosomes. We observed that G-CSF-R is ubiquitinated and subsequently assessed the contribution of K residues in G-CSF-R routing and signaling. To this end, we generated mutant G-CSF-R-K5R, in which all five conserved cytoplasmic K residues were replaced by arginine (R). To study differential localization of internalized K5R and WT G-CSF-R, we introduced constitutively active Rab5 (Rab5-Q79L) to enlarge early endosomes. Under these conditions, internalized WT G-CSF-R colocalized extensively with endosomal microdomains containing the endosome-to-lysosome sorting protein Hrs. In contrast, internalized G-CSF-R-K5R was mostly found outside these domains. Consequently, lysosomal routing of G-CSF-R-K5R was severely impaired, which resulted in strongly increased receptor protein levels. Upon removal of G-CSF, downregulation of STAT5 activity in 32D/K5R cells was significantly delayed compared to 32D/WT cells, establishing that the lysine residues are not only crucial for receptor stability, but also for duration of signaling. Moreover, in the continuous presence of G-CSF, 32D/K5R cells showed strongly increased STAT5 activity and proliferation and reduced granulocytic differentiation compared to 32D/WT. Similar results were obtained in colony assays with g-csfr deficient primary bone marrow progenitors transduced with K5R or WT G-CSF-R. To substantiate a relationship between signal duration and receptor degradation, we inhibited lysosomal degradation by pharmacological inhibitors and by knockdown of Hrs and the related protein Tsg101 by RNA interference. As expected, receptor stability was significantly increased by these treatments. Surprisingly however, this did not influence STAT5 activity, suggesting that signal attenuation predominantly occurred in a pre-lysosomal compartment. We conclude from these data that ubiquitinated lysine residues of G-CSF-R are required to target internalized receptors for lysosomal degradation. However, they also appear to be involved in contracting an as yet unidentified inhibitor, perhaps a phosphatase, to a specific Hrs-positive subendosomal compartment, thereby switching off G-CSF-R already prior to its degradation in lysosomes.