Mapping of the cytoplasmic domain of the human growth hormone receptor required for the activation of Jak2 and Stat proteins.

Incubation of cells with growth hormone (GH) stimulates both tyrosine phosphorylation of the Jak2 tyrosine kinase and, in some cells, the transcription factor Stat1α(1, 2, 3, 4). When the promyeloid cell line FDC-P1 is transfected with the human growth hormone receptor, these cells can grow in the presence of GH and in the absence of interleukin-3. Growth hormone treatment of cells expressing the human growth hormone receptor did not activate Stat1α. However, a complex is present in extracts prepared from growth hormone-treated cells that binds to the response region, an enhancer present in the promoter of the high affinity FcR1 receptor to which cytokine-activated Stat complexes bind. When truncations of the cytoplasmic domain of the receptor are expressed in FDC-P1 cells only the membrane-proximal 80 amino acids (containing box 1 and box 2) are required for activation of both a GH-stimulated binding activity (GHSF) and tyrosine phosphorylation of Jak2. Activation of GHSF can be inhibited in a cell-free system by the addition of a glutathione S-transferase fusion protein containing these 80 amino acids. Replacement of the one tyrosine in this region of the receptor with a phenylalanine does not alter the activation of either GHSF or Jak2, suggesting that tyrosine phosphorylation of the receptor is not required for GH activation of GHSF. Moreover, a cell line expressing a receptor with only the 54 membrane-proximal amino acids of the intracellular domain (including box 1) shows constitutively tyrosine-phosphorylated Jak2 as well as GHSF binding. With this truncated receptor, there is little if any additional GH-induced tyrosine phosphorylation of Jak2 or induced binding to the response region. These results define the importance of the membrane-proximal 80 amino acids of the GH receptor (with the conserved box 1 and box 2 domains) with regard to GH activation of both Jak2 and Stat(s). They also suggest that within these domains there may be positive and negative elements that regulate Jak2 function.

Fat metabolism, insulin action, and long bone and soft tissue growth are among many biological responses that are regulated by growth hormone (GH) 1 (5). The GH receptor (GHR) is a single transmembrane-spanning protein that is a member of the cytokine receptor superfamily that includes the prolactin, interleukin (IL)-2, IL-7, erythropoietin, leukemia inhibitory factor, ciliary neurotropic factor, and the interferon receptors (6 -8). The receptors for growth hormone and other members of this family have several conserved features including cysteine residues within their extracellular domains and two subdomains (termed box 1 and box 2) adjacent to the transmembrane region (9 -12). Although these receptors have no intrinsic tyrosine kinase activity, binding of ligand has been shown to induce rapid tyrosine phosphorylation of several cellular proteins (13)(14)(15). Most of the cytokine receptor family interacts with members of the Jak family of tyrosine kinases. Tyrosine-phosphorylated Jak2 has been shown to associate with the GHR after the addition of ligand (1). Incubation of cells with GH in addition to many other cytokines also stimulates tyrosine phosphorylation of the Stat (signal transducers and activators of transcription) family of transcription factors (16 -18). Tyrosinephosphorylated Stat proteins bind enhancers that are present in genes whose transcription is rapidly activated by treatment of cells with interferons and other cytokines (16 -19). One of these enhancers is the ␥ response region (GRR) present in the promoter of the Fc␥R1 receptor gene. This enhancer, which is required for IFN␥ activated transcription of the Fc␥R1 receptor gene, has a sequence similar to those enhancers required for activation of cellular genes by a variety of cytokines (16 -18, 20). GRR-binding activity can be measured in many cells in response to growth hormone treatment and serves as an assay for tyrosine phosphorylation of Stat proteins (16 -18, 21). In many cases, the Stat proteins that bind to the GRR have been defined. However, in other examples the proteins that are induced to bind to the GRR or related sequences by cytokine treatment of cells are presumed to be members of the Stat family, and in the cases thus far examined this has proved to be correct (2,19). In order to elucidate the domains in the GH receptor required for activation of Stat(s) and Jak2, cell lines containing deletions in the cytoplasmic domain of the human receptor were analyzed for GH-stimulated tyrosine phosphorylation of Jak2 and GRR-binding activity. From these studies we have been able to demonstrate the importance of the 80-amino acid region containing box 1 and box 2 in GH activation of Jak2 kinase and the Stat transcription factors.

Cells-
The FDC-P1 cell line was transfected with the cytoplasmic truncations of the human growth hormone receptor cDNA (11). The receptor-expressing cell lines were characterized previously for GH binding and receptor size and number (11). Cell lines were grown in RPMI 1640 supplemented with 10% fetal calf serum, 50 M 2-mercaptoethanol, 50 g/ml gentamicin, 800 g/ml G418, and 5 nM human growth hormone (11). Cells were starved overnight in the absence of GH prior to being incubated with 10 nM GH for the times indicated.
Electrophoretic Mobility Shift Assay (EMSA)-The EMSA was performed as described previously using whole cell extracts (see above) (2,19,21). The GRR (␥ response region) (5Ј-AGCATGTTTCAAGGATTT-GAGATGTATTTCCCAGAAAAG-3Ј) of the promoter of the Fc␥R1 gene was end-labeled using polynucleotide kinase and [␥-32 P]ATP and used in all EMSAs. Competitive inhibition experiments were performed using a 200-fold molar excess of unlabeled oligonucleotides: the GRR; the IFN-stimulated response element of the ISG15 gene (5Ј-GATCCATGC-CTCGGGAAAGGGAAACCGAAACTGAAGCC-3Ј); and the SIF-E element in the promoter of the c-fos gene, which has been optimized for Stat binding (5Ј-AGCTTAGGGATTTACGGGAAATGA-3Ј) (20).
Glutathione S-Transferase Fusion Proteins-The DNA encoding the C-terminal six amino acids of the transmembrane domain and the following 80 amino acids of the cytoplasmic domain of the human GHR (Phe 265 -Ser 350 ) was prepared by polymerase chain reaction. The DNA was ligated in pGEX-5X-1, and the entire insert DNA was sequenced. The identity of the glutathione S-transferase fusion protein was confirmed by amino acid sequencing the 17 N-terminal amino acids of the protein.
Immunoprecipitations-Starved cells (3 ϫ 10 6 ) were treated with or without GH for 10 min at 37°C. Cells were washed one time with ice-cold phosphate-buffered saline containing 1 mM vanadate, and lysed at 4°C in 0.5 ml of 50 mM Tris (pH 7.5), 137 mM NaCl, 2 mM EDTA, 1 mM vanadate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, and 5 g/ml leupeptin (lysis buffer). After centrifugation, the supernatant was incubated with 5 l of anti-Jak2 antisera (Upstate Biotechnologies) for 2 h at 4°C. Protein G-Sepharose (50 l) was added and incubated 2 h at 4°C. The beads were washed 2 times in lysis buffer and boiled in SDS sample buffer, and the eluted proteins were analyzed by 6% SDS-polyacrylamide gel electrophoresis followed by blotting to nitrocellulose. The membranes were then probed with anti-phosphotyrosine antibody (4G10) and developed using alkaline phosphatase (Promega).

RESULTS
FDC-P1 cells transfected with cDNA to the full-length human growth hormone receptor were starved overnight in the absence of GH and then exposed to GH for 10 min prior to the preparation of whole cell extracts. Extracts were analyzed for both activation of DNA binding proteins that interact with the GRR and for tyrosine phosphorylation of Jak2. Incubation of cells with GH resulted in both tyrosine phosphorylation of the Jak2 kinase (see below) and induction of a complex that interacted with the GRR enhancer as assayed by EMSA (Fig. 1). The GH-inducible complexes that bound to the GRR probe are labeled GHSF. Extracts from IFN␥-treated cells contained a GRR-binding complex (lane 6) whose mobility was different from that of GHSF (compare lanes 2 and 6). The IFN␥-induced complex contains Stat1␣ (data not shown). As with other complexes that bind to ␥ activation sequence-like sequences as a result of GH treatment of cells, the GHSF is specific in that it is displaced by unlabeled oligonucleotide corresponding to the GRR (lanes 3 and 7) and a related sequence, which functions as an enhancer in the c-fos promoter termed SIE (20) (lanes 4 and 8) but not with oligonucleotides to other enhancers such as the interferon-stimulated response element (compare lanes 3 and 5). Formation of GHSF was not seen in cells that were not transfected with the receptor (data not shown).
Growth hormone-stimulated tyrosine phosphorylation of Stat1␣ (p91) occurs in both cultured cell lines and livers isolated from rats injected with GH. However, in both 3T3 cells and rat liver, evidence suggests that another Stat in addition to Stat1 may be activated by GH (2)(3)(4)23). In addition, in human IM9 lymphocytic cells, antisera specific for p91 do not recognize the GH-stimulated tyrosine-phosphorylated protein that interacts with the GRR (2, 23). Since it had not been determined whether FDC-P1 cells containing the full-length growth hormone receptor activated Stat1␣ in response to GH, initial assays were performed to characterize whether the GHSF that interacted with the GRR contained Stat1. Interestingly, in a manner similar to IM9 cells, GHSF from this cell line did not "supershift" with Stat1␣-specific antisera or with antisera to Stat2 or Stat4 (data not shown). In some experiments, Stat3 and Stat5 appeared to be components of GHSF, and this result is under investigation. Immunoprecipitations of Stat1␣ orStat3 from GH-stimulated cells also did not show any tyrosine phosphorylation of these Stats, while Stat5 was tyrosine-phosphorylated in these cells with GH treatment (data not shown). Additionally, Stat1␣ was tyrosine-phosphorylated after treatment of these cells with IFN␥ (data not shown).
In order to define the regions of the GHR required for GH activation of GHSF and Jak2, FDC-P1 cell lines expressing receptors with deletions in the cytoplasmic domain have been established (11). These cell lines all require GH to proliferate (11). A schematic of the receptor deletions is diagrammed in  Fig. 2A, with the location of the conserved box 1 and box 2 motifs indicated. These motifs are present in many receptors of the cytokine family (9 -11). Cells were incubated with GH for 10 min prior to lysis and subsequent analysis for GHSF and tyrosine phosphorylation of Jak2 (Fig. 2, B and C). Truncation of the cytoplasmic tail such that only the membrane-proximal 80 amino acids remained (containing box 1 and box 2) permitted full activation of both GHSF and Jak2 as measured by tyrosine phosphorylation. However, when the box 2 domain of the receptor was deleted, there was constitutive GHSF activa-tion and Jak2 tyrosine phosphorylation. Enhanced activation of GHSF and tyrosine phosphorylation of Jak2 in the presence of GH was variable in the 325 cell line but in many experiments did not occur. The cell line expressing the 325 truncation, however, remained dependent upon GH for survival in spite of constitutive GHSF and tyrosine-phosphorylated Jak2. For all of the receptor truncations studied, there was a direct correlation between GH-activated GHSF and tyrosine-phosphorylated Jak2.
Activation of GRR-binding Stat proteins has been described in cell-free homogenates treated with a variety of cytokines (22, 24 -28). Such a system was used to demonstrate that activation of Stat1 by IFN␥ can be inhibited with a peptide that contains a tyrosine-phosphorylated residue of the ␣ chain of the IFN␥ receptor (27). To confirm that the membrane-proximal 80 amino acids of the growth hormone receptor are critical for activation of GHSF, cell homogenates were prepared from FDC-P1 cells expressing the full-length receptor. Homogenates from these cells were incubated with or without GH for 10 min prior to terminating the reaction and measuring GHSF activity (Fig. 3). GHSF formed with homogenates of cells incubated with GH was similar to that seen previously with intact cells (Fig. 3, lanes 1 and 2). The specificity of binding was also identical (data not shown). We next incubated homogenates with a glutathione S-transferase fusion protein corresponding to the 80-amino acid membrane-proximal region of the receptor or with glutathione S-transferase alone. The homogenates were then treated with or without GH, and GHSF activity was assayed. Addition of glutathione S-transferase protein alone had little or no effect on activation of GHSF, while addition of the receptor domain inhibited formation of the complex (com- pare lanes 3 and 4). These results not only confirm the importance of this region but indicate that in vitro activation of the complex may provide a rapid method to screen for residues within this region that may be critical for signaling in vivo. These findings also indicate that tyrosine phosphorylation of the receptor itself may not be required for activation of Stat(s)  2, 4, 6, 8, and 10) or untreated (lanes 1, 3, 5, 7 and 9) prior to preparation of whole cell extracts. GHSF formation was assayed by EMSA as in Fig. 1. IFN␥ activated formation of a complex that bound to the GRR in all of these cell lines as in Fig. 1 (data not  shown). C, GH-stimulated tyrosine phosphorylation of Jak2 in the same cell lines used in Fig. 2A. Cells were treated for 10 min with (lanes 2, 4, 6, 8, and 10) or without GH (lanes 1, 3, 5, 7, and 9). Cellular lysates were made, and immunoprecipitations of Jak2 were performed. After SDSpolyacrylamide gel electrophoresis and transfer to polyvinylidene difluoride, the blot was probed with antiphosphotyrosine antibody and developed with ECL. The concentrations of Jak 2 were equal in all cell lines (data not shown).

FIG. 3. GH activates GHSF binding to the GRR in homogenates prepared from cells expressing the full-length receptor.
Homogenates were prepared from cells as described under "Materials and Methods." Homogenates were incubated in the absence (lane 1) or presence of 50 nM GH (lanes 2, 3, and 4) for 10 min at 30°C prior to stopping the reaction and assaying for the formation of GHSF by EMSA. In lanes 3 and 4, homogenates were incubated for 1 h at 4°C with 12 M of either glutathione S-transferase protein alone or glutathione S-transferase containing amino acids 265 to 350 of the human GH receptor. Subsequently, the homogenates were incubated 10 min more at 30°C with 50 nM GH prior to EMSA. since the glutathione S-transferase receptor protein is not tyrosine-phosphorylated (see below).
Previous studies have reported that activation of Stat1 or IL-4 Stat require specific tyrosine-phosphorylated residues within the cytoplasmic domains of the IFN␥ or IL-4 receptor (27,29). Growth hormone-stimulated tyrosine phosphorylation of its receptor has also been described (30), and we have confirmed this observation in cells expressing the full-length receptor (data not shown). However, the glutathione S-transferase fusion protein containing box 1 and 2 inhibited GHSF in cell homogenates, suggesting that a tyrosine-phosphorylated receptor may not be required for Stat activation in this system. To examine directly the potential role of tyrosine phosphorylation of the GHR for GHSF activation, we used the 351 deletion mutant, which displays normal activation of GHSF and which contains only one tyrosine (at amino acid 314) in its intracellular region. A cell line was established using the 351 truncation, which contained a substitution of its single intracellular tyrosine residue with a phenylalanine. Incubation of these cells with growth hormone induced both tyrosine phosphorylation of Jak2 and activation of Stat as assayed by binding to the GRR (Fig. 4). Thus it appears that for GH activation of Stat proteins a tyrosine-phosphorylated residue in the intracellular domain of the receptor is not required. Rather other mechanisms must be invoked to permit a presumed association of the Stat with the GHR/Jak2 complex.

DISCUSSION
Growth hormone, in a manner similar to many other cytokines, is known to activate members of the Jak family of tyrosine kinases and stimulate tyrosine phosphorylation of Stat proteins (1)(2)(3)(4). Association of the GH receptor with Jak2 requires that cells be exposed to ligand (1). Although several groups have described regions in the GHR that are involved in Jak2 activation, the domain(s) within the receptor required for Stat activation have yet to be fully delineated (31)(32)(33). Using the full-length human growth hormone receptor and a series of deletions of the carboxyl terminus of the cytoplasmic region, it has been possible to demonstrate that 80 amino acids adjacent to the membrane are sufficient for activation of this Jak/Stat pathway. Cell homogenates prepared from cells expressing the full-length receptor can be treated with GH to stimulate the formation of GHSF in a manner similar to that seen in vivo. In addition, a glutathione S-transferase fusion protein consisting of these 80 amino acids, when added to homogenates, prevented GH-stimulated GRR binding. This region contains box 1 and box 2 motifs, which are present in many cytokine receptors of this class including receptors for erythropoietin, prolactin, and granulocyte-macrophage colony-stimulating factor. All of these receptors have been shown to use the same domains to bind Jak2 (34 -36). These domains have also been shown to be essential for growth hormone-induced mitogenesis as well as activation of cellular genes by GH (11,37). Although box 1 and box 2 motifs are present in receptors that couple to Jak2, there appear to be differences between these cytokine receptors with regard to the requirement of box 1 and box 2 for activation of Jak2. For example, granulocyte-macrophage colony-stimulating factor activation of Jak2 requires only the box 1 domain of its receptor, while activation of Jak2 by the prolactin requires both box 1 and box 2 (34,38). GH-stimulated tyrosine phosphorylation of Jak2 in the receptor truncations described here indicates that while both box 1 and box 2 are needed for activation of Jak2 by GH, constitutively activated Jak2 is present in cells expressing only box 1 (Fig. 2). These results imply that a negative regulatory element may reside between amino acids 54 and 80 that prevents Jak2 and Stat activation in the absence of hormone. Since growth hormone is required to maintain the 325 cell line, these results also suggest that activation of this Jak/Stat pathway is not sufficient by itself to promote the mitogenic response. Activation of mitogenesis by erythropoietin also appears to require other signal(s) besides activation of Jak2 (38). It is interesting to note that deletional analysis of the rabbit GHR indicates that box 1 alone is sufficient for some GH-induced tyrosine phosphorylation of Jak2 (31,32). In COS-7 cells, the receptor truncation expressing only box 1 appeared to have increased basal tyrosine phosphorylation of Jak2 (31), while in Chinese hamster ovary cells expressing a similar construct, stimulation of Jak2 by GH was clearly diminished when compared with the wild-type receptor (32). It thus appears that the box 1 domain and amino acids adjacent to this motif may contain binding sites for cell-specific and possibly species-specific factors (since our lines contained the human GHR) that can regulate basal and GH-stimulated Jak2 activation.
Although activation of Jak2 has been mapped in the cytokine receptor systems described above, the domains needed for their activation of Stat proteins (which occurs with treatment of cells with these other cytokines) have yet to be defined (34 -36). In the studies presented here, there is a strict correlation between activation of the GHSF and tyrosine phosphorylation of Jak2. This is not surprising in light of the fact that cell lines defective in IFN␣-or IFN␥-activated gene expression (and Stat activation) have been characterized that are lacking in Jak kinase(s) (39 -41). It still remains to be determined whether these kinases use the Stat proteins as substrates for their activity or whether they are intermediates in the signaling cascade.
It is rather surprising that Stat activation by growth hormone does not require a tyrosine to be phosphorylated in the cytoplasmic domain of the receptor (Fig. 4). This result is different from that reported for IFN␥ activation of Stat1, where a critical tyrosine in the ␣ chain of the receptor needs to be phosphorylated for Stat 1 to be tyrosine-phosphorylated. Based on this observation and related findings with regard to IL-4 Stat activation, it has been proposed that tyrosine phosphorylation of cytokine receptors permits the SH2 domains in the Stat proteins to bind to the receptor and subsequently leads to their FIG. 4. Growth hormone stimulates GHSF formation and tyrosine phosphorylation of Jak2 in cells that express the 351 receptor where tyrosine 314 has been substituted with a phenylalanine. Cells were incubated for 10 min in the absence (lanes 1 and 3), or presence (lanes 2 and 4) of GH. EMSAs were performed on extracts of these cells (lanes 1 and 2), or tyrosine phosphorylation of immunoprecipitated Jak2 (lanes 3 and 4) was analyzed as described in Fig. 2C. tyrosine phosphorylation (27,29). Such a model is not consistent with GH activation of the Stat protein(s) that bind to the GRR in these cells. Several mechanisms could be invoked to explain activation of Stat proteins that do not require tyrosine phosphorylation of the receptor. These would include other tyrosine-phosphorylated proteins in the complex that act as docking sites for the Stats or mechanisms where the SH2 domain of the Stat proteins is not required for its recruitment into the signaling complex. Such a domain, termed PTB, has been reported in the N-terminal region of SHC, which selectively interacts with some tyrosine-phosphorylated proteins stimulated by treatment of cells with platelet-derived growth factor (42). Further experiments will be needed to define this interaction. It is also unclear why Stat1␣ is activated by GH treatment of some types of cells and not by others. While GH discriminates in a cell-specific fashion between the Stat proteins that it tyrosine phosphorylates, Jak2 is tyrosine-phosphorylated upon GH treatment of all cells thus far examined (1,4,23). It is also notable that Stat1 is tyrosine-phosphorylated by IFN␥ in all cells thus far examined including those cell lines where GH activates GRR-binding complexes that do not contain Stat1 (1,4,23). Characterization of those Stat proteins that are activated by GH in FDC-P1 cells will allow the mechanisms to be clarified by which GH activates Stat proteins in a cell-specific manner.