Stimulation of Stat5 by Granulocyte Colony-Stimulating Factor (G-CSF) Is Modulated by Two Distinct Cytoplasmic Regions of the G-CSF Receptor Fan Dong, 1 * ² Xiuwen Liu, ‡ John P. de Koning, § Ivo P. Touw, § Lothar Henninghausen, ‡ Andrew Larner, 1,2² and Philip M. Grimley* In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cyto- plasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell prolifer- ation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival. The Journal of Immunology, 1998, 161: 6503– 6509. G ranulocyte CSF (G-CSF) 3 plays a critical role in regu- lating the proliferation and differentiation of myeloid progenitor cells and maintaining neutrophil levels in pe- ripheral blood (1, 2). G-CSF exerts its biological activities via binding to a cell surface receptor that is a member of the cytokine receptor superfamily (1, 2). Incubation of hemopoietic cells with G-CSF leads to the activation of the Jak/STAT pathway and the Ras/Raf/MAP kinase pathway (2). Activation of Jak1 and Jak2 requires the membrane-proximal cytoplasmic region of the G-CSF receptor (3–7). Jaks permit tyrosine phosphorylation of the STAT transcription factors, which then translocate to the nucleus, bind enhancer elements, and stimulate the transcription of cellular genes (8). In contrast, tyrosine phosphorylation of Shc and subsequent initiation of Ras/Raf/MAP kinase signaling appear to require the carboxyl-terminal region of the G-CSF receptor (9 –12). Activated MAP kinases translocate to the nucleus and phosphorylate several transcription factors that induce the expression of immediate early genes that are distinct from those activated by STATs (13). Several recent studies have identified distinct cytoplasmic re- gions of the G-CSF receptor that are involved in transducing sig- nals for cell proliferation and differentiation (14, 15). The mem- brane-proximal region is required and sufficient for mitogenic signaling, whereas the distal carboxyl tail of the receptor mediates growth-suppressing signals and is involved in induction of termi- nal granulocytic maturation. Truncations of the carboxyl-terminal region of the G-CSF receptor as a result of point mutations have been reported in patients with severe congenital neutropenia and acute myeloid leukemia (16 –19). However, little is known about the mechanisms whereby the carboxyl-terminal region of the G- CSF receptor mediates granulocytic maturation and growth-sup- pressing signals. In other related receptors such as the erythropoi- etin receptor, proliferation is also down-regulated by the receptor carboxyl terminus, which appears to provide the binding site for the PTPase SHP-1 that inactivates Jak2 (20). Although Stat5 was originally identified as a mammary gland factor that is regulated by prolactin (21), recent studies have shown that Stat5 is activated by other cytokines as well, including IL-2, IL-3, IL-5, granulocyte-macrophage CSF, growth hormone, thrombopoietin, and G-CSF (22–26). Activation of Stat5 by IL-3 appears to be associated with cell proliferation (27). In this study, we show that Stat5 activation by G-CSF is regulated by distinct cytoplasmic regions of the G-CSF receptor. We further demon- strate that Stat5 signaling pathway is critical for G-CSF-mediated cell proliferation and survival. Materials and Methods Cells Murine BAF3 and 32D cells, stably transfected with cDNAs encoding either the wild-type or the truncated forms of the human G-CSF receptor, have been described (15, 28). Cells were grown in RPMI 1640 medium *Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; ² Division of Cytokine Biology, Center for Biologics, Evalua- tion and Research, Food and Drug Administration, Bethesda, MD 20892; ‡ Laboratory of Biochemistry and Metabolism, National Institute of Diabetes, Digestive, and Kid- ney Diseases, National Institutes of Health, Bethesda, MD 20892; and § Institute of Hematology, Erasmus University, Rotterdam, The Netherlands Received for publication May 18, 1998. Accepted for publication August 11, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Current address: Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. 2 Address correspondence and reprint requests to Dr. Andrew Larner, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, FFb-37, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: LarnerA@cesmtp.ccf.org 3 Abbreviations used in this paper: G-CSF, granulocyte colony-stimulating factor; CHX, cycloheximide; EMSA, electrophoretic mobility shift assay; GAS, gamma ac- tivated site; GFP, green fluorescent protein; MAP, mitogen-activated protein; PTPase, protein tyrosine phosphatase; SHP, Src homology 2 (SH2)-containing phosphatase. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00