(Received for publication, January 24, 1995; and in revised form, June 30, 1995)
From the
Incubation of cells with growth hormone (GH) stimulates both
tyrosine phosphorylation of the Jak2 tyrosine kinase and, in some
cells, the transcription factor
Stat1(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 Stat1
. 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 Fc
R1 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)()(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, 7, 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, 10, 11, 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, 17, 18) . Tyrosine-phosphorylated
Stat proteins bind enhancers that are present in genes whose
transcription is rapidly activated by treatment of cells with
interferons and other
cytokines(16, 17, 18, 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, 17, 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, 17, 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.
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 (lane6) whose
mobility was different from that of GHSF (compare lanes2 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 (lanes3 and 7) and
a related sequence, which functions as an enhancer in the c-fos promoter termed SIE (20) (lanes4 and 8) but not with oligonucleotides to other enhancers such as
the interferon-stimulated response element (compare lanes3 and 5). Formation of GHSF was not seen in
cells that were not transfected with the receptor (data not shown).
Figure 1:
Growth
hormone and IFN treatment of FDC-P1 cells expressing the
full-length receptor activates the formation of a complex that binds to
the GRR. Cells were starved for 16 h in the absence of GH prior to the
addition of either GH (10 nM) or IFN
(10 ng/ml) for 10
min. Whole cell extracts were prepared, and EMSAs were performed using
a radiolabeled GRR oligonucleotide. Lane1, untreated
cells; lanes2-5, cells incubated 10 min with
GH; lanes6-9, cells incubated with IFN
for 10 min. In lanes3 and 7, a 200-fold
molar excess of unlabeled GRR probe was added to the binding reaction.
Similar oligonucleotides corresponding to the SIE element or the
IFN-stimulated response element were included at a 200-fold excess in lanes4 and 8 and lanes 5 and 9, respectively.
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, 10, 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 activation 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.
Figure 2:
Analysis of GH-stimulated GHSF formation
and tyrosine phosphorylation of Jak2 in cell lines containing deletions
of the intracellular domain of the GH receptor. A, diagram of
the receptor deletion constructs that were transfected into FDC-P1
cells. B, GH-stimulated formation of GHSF in the cell lines
containing the receptor truncations diagrammed in Fig. 2A. Cells were treated for 10 min with GH (lanes2, 4, 6, 8, and 10) or untreated (lanes1, 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 (lanes2, 4, 6, 8, and 10) or without GH (lanes1, 3, 5, 7, and 9). Cellular lysates were made,
and immunoprecipitations of Jak2 were performed. After
SDS-polyacrylamide 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).
Activation of GRR-binding Stat proteins has been described in
cell-free homogenates treated with a variety of
cytokines(22, 24, 25, 26, 27, 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, lanes1 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 (compare lanes3 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)
since the glutathione S-transferase receptor protein is not
tyrosine-phosphorylated (see below).
Figure 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 (lanes2, 3, and 4) for 10 min at 30 °C prior to stopping the reaction and assaying for the formation of GHSF by EMSA. In lanes3 and 4, homogenates were incubated for 1 h at 4 °C with 12 µM 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.
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.
Figure 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 (lanes1 and 3), or presence (lanes2 and 4) of GH. EMSAs were performed on extracts of these cells (lanes1 and 2), or tyrosine phosphorylation of immunoprecipitated Jak2 (lanes3 and 4) was analyzed as described in Fig. 2C.
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, 35, 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, 35, 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, 40, 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 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.