The hematopoietic system provides an attractive model for studying growth factor-controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony-stimulating factor receptor (G-CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G-CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G-CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G-CSFR mutants lacking K632 show a perturbed proliferation/differentiation balance in response to G-CSF. This is the first demonstration of SOCS-mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output.

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand–receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10 4 -fold increase of colonyforming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Truncated granulocyte colony-stimulating factor receptors (G-CSF-Rs) are implicated in severe congenital neutropenia (SCN) and the consecutive development of acute myeloid leukemia (AML). Mice expressing G-CSF-R truncation mutants (gcsfr-d715) show defective receptor internalization, an increased signal transducer and activator of transcription 5 (STAT5)/STAT3 activation ratio, and hyperproliferative responses to G-CSF treatment. We determined whether a lack of negative feedback by suppressor of cytokine signaling (SOCS) proteins contributes to the signaling abnormalities of G-CSF-R-d715. Expression of SOCS3 transcripts in bone marrow cells from G-CSF-treated gcsfr-d715 mice was approximately 60% lower than in wild-type (WT) littermates. SOCS3 efficiently suppressed STAT3 and STAT5 activation by WT G-CSF-R in luciferase reporter assays. In contrast, while SOCS3 still inhibited STAT3 activation by G-CSF-R-d715, STAT5 activation was no longer affected. This was due mainly to loss of the SOCS3 recruitment site Tyr729, with an additional contribution of the internalization defects of G-CSF-R-d715. Because Tyr729 is also a docking site for the Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2), which binds to and inactivates STAT5, we suggest a model in which reduced SOCS3 expression, combined with the loss of recruitment of both SOCS3 and SHP-2 to the activated receptor complex, determine the increased STAT5/STAT3 activation ratio and the re- IntroductionGranulocyte colony-stimulating factor (G-CSF) receptor is the major regulator of neutrophil production, both under steady-state conditions and during stages of bacterial infections. 1-3 G-CSF exerts its activity via a receptor (G-CSF-R) of the hematopoietin receptor superfamily. 4,5 Typical of this class of receptors, G-CSF-R has no intrinsic kinase activity but recruits cytoplasmic tyrosine kinases of both the Janus kinase (Jak) and Src kinase families and activates signal transducer and activator of transcription (STAT) proteins. 6-11 G-CSF activates STATs 1, 3, and 5. [12][13][14] Whereas the contribution of STAT1 to G-CSF responses remains unclear, STAT3 has been implicated in G-CSF-mediated growth arrest preceding differentiation, while activation of STAT5 has been linked to proliferation and survival signaling. [15][16][17] Four tyrosine residues (Tyr704, Tyr729, Tyr744, and Tyr764) in the G-CSF-R carboxy-terminus are involved in the recruitment of signaling molecules, such as the adapter molecules growth factor receptorbound protein 2 (Grb2) and Src homology and collagen protein (Shc) of the p21Ras-mitogen-activated protein (MAP) kinase pathway, and the Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2). [18][19][20] In addition, activation of STAT3 depends on its recruitment to the G-CSF-R via tyrosines 704 or 744. [20][21][22] At higher G-CSF concentrations, STAT3 can also be activated in a tyrosine-independent way via the G-CSF-R Cterminus. 22,23 In contrast, activation of STAT1 and STAT5 is achieved via the membrane-proximal...