Tyrosine-Dependent and -Independent Mechanisms of STAT3 Activation by the Human Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Differentially Utilized Depending on G-CSF Concentration

Abstract

The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-▵715). These findings suggest that G-CSF–induced STAT3 activation during basal granulopoiesis (low G-CSF) and “emergency” granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.

Suppressor of cytokine signaling 3 controls lysosomal routing of G-CSF receptor

“…Subsequently, we assessed the consequences of removal of the internalization domain and substitution of the lysine residues for G-CSF-induced proliferation and differentiation. As demonstrated previously, 32D cells expressing wt G-CSFR proliferated 6-8 days and differentiated into neutrophilic granulocytes from day 6 onwards ( Figure 4D and E) (Ward et al, 1999a). In contrast, 32D cells expressing d749-769 or K5R continued with proliferation in response to G-CSF at the expense of terminal granulocytic differentiation ( Figure 4D and E).…”

Novel Point Mutation in the Extracellular Domain of the Granulocyte Colony-Stimulating Factor (G-Csf) Receptor in a Case of Severe Congenital Neutropenia Hyporesponsive to G-Csf Treatment

“…Regardless of the exact composition, this ligand concentration-dependent transition to a higher order complex involving the Ig domain contrasts with other hematopoietin receptors, such as growth hormone receptor, which only requires its CRH region to form a simple, symmetrical 2:1 receptor-ligand complex (13,49). In addition, we have recently shown that the ligand sensitivity for STAT3 activation is ‫1ف‬ log higher than for activation of STATs 1 and 5 from the wild-type G-CSF-R (22). This suggests that different intracellular signaling complexes are also formed depending on ligand concentration: at low concentrations, one that can activate STAT3, and at high concentrations, one that can activate all three STATs.…”

“…When expressed in myeloid cells, these truncated receptors transduce a strong growth signal but are defective in maturation signaling (26,28). This is due to sustained receptor activation caused by defective internalization and an altered balance of STAT activation (22,29,30). However, in SCN patients with this mutation, G-CSF treatment is able to reverse the neutropenia (25)(26)(27).…”

Granulocyte colony-stimulating factor and its receptor in normal myeloid cell development, leukemia and related blood cell disorders

“…On the other hand, investigations in Ba/F3 cells and primary bone marrow cultures have established that at saturating G-CSF concentrations STAT3 can also be activated via a tyrosine-independent route. The latter mechanism requires the presence of the membrane-distal region of the G-CSF-R (77,78). Although the exact nature of this tyrosine independent route is still unclear, it was suggested that different mechanisms for STAT3 activation might be involved in the control of steady-state granulopoiesis at a low G-CSF level (mainly tyrosinedependent) versus "emergency" granulopoiesis initiated by an increased level of G-CSF (tyrosine-independent) (77).…”