(Received for publication, November 27, 1995; and in revised form, February 13, 1996)
From the
The binding of growth hormone (GH) to its receptor results in
its dimerization followed by activation of Jak2 kinase and tyrosine
phosphorylation of the GH receptor itself, as well as Jak2 and the
transcription factors Stat1, -3, and -5. In order to study the role of
GH receptor tyrosine phosphorylation in intracellular signaling, we
constructed GH receptors in which combinations of tyrosines were
mutated to phenylalanines. We identified three tyrosine residues at
positions 534, 566, and 627 that were required for activation of
GH-stimulated transcription of the serine protease inhibitor (Spi) 2.1
promoter. Any of these three tyrosines is able to independently mediate
GH-induced transcription, indicating redundancy in this part of the GH
receptor. Tyrosine phosphorylation was not required for GH stimulation
of mitogen-activated protein (MAP) kinase activity or for GH-stimulated
Ca channel activation since these pathways were
normal in cells expressing a GH receptor in which all eight
intracellular tyrosines were mutated to phenylalanines. Activation of
Stat5 by GH was, however, abolished in cells expressing the GH receptor
lacking intracellular tyrosines. This study demonstrates that specific
tyrosines in the GH receptor are required for transcriptional signaling
possibly by their role in the activation of transcription factor Stat5.
Pituitary growth hormone (GH) ()is the major
regulator of postnatal growth(1) . The actions of GH at the
cellular level include direct mitogenic
effects(2, 3) , insulin-like and insulin-antagonizing
metabolic effects(4) , as well as gene regulatory
actions(5, 6, 7) . All of these effects are
initiated by the binding of GH to its receptor, which belongs to the
cytokine receptor superfamily. Members of this family of receptors
activate cytoplasmic tyrosine kinases of the Jak family, and these
activated kinases are required for most receptor-initiated signaling
pathways (8) . The activated Jak2 kinase has been shown to
phosphorylate several intracellular substrates including the GH
receptor itself, as well as transcription factors of the Stat
family(9, 10, 11) .
Not only does GH
activate the Jak/Stat pathway in which specific tyrosine
phosphorylation of Stat1, -3, and -5 occurs in response to GH,
resulting in dimerization, nuclear translocation, and binding to
-interferon activated sequence-like
elements(11, 12, 13) , but we and others have
previously identified several alternative signaling pathways induced by
the activated GH receptor. Stimulation and tyrosine phosphorylation of
MAP kinase by GH have been studied both in cultured cells (14, 15) and in vivo(16) . In
addition to activation of MAP kinase, its translocation to the nucleus
has also been demonstrated. The activation of MAP kinase by GH is
dependent upon the proline-rich box 1 domain of the GH receptor that
presumably is directly involved in the binding of Jak2. When the box 1
domain is deleted or the prolines are mutated to alanines, the GH
receptor is no longer able to mediate GH-induced MAP kinase activity.
The functional role of MAP kinase activation in GH signaling is not
known; however, it has been speculated that this activity is important
for protein synthesis and possibly cell proliferation(14) . The
mechanism by which GH activates MAP kinase is also largely unknown, but
the fact that GH can activate SHC and Grb2 indicates a mechanism
similar to that utilized by receptor tyrosine kinases. We have also
reported that GH is able to activate voltage-dependent Ca
channels. This activity is independent of Jak2 (17) and
cannot be inhibited by tyrosine kinase inhibitors(18) . The
GH-induced Ca
response consists of oscillations in
intracellular free Ca
concentrations of varying
frequency and amplitude and has been demonstrated to be required for
GH-induced transcription of the insulin gene(17) . We have also
shown that a region in the rat GH receptor located between residues 454
and 506 is required for the Ca
-signaling ability of
the GH receptor. Finally, GH-induced tyrosine phosphorylation of
cytosolic proteins has also been described, and this response has been
found to be dependent on a 40-amino acid domain located between
residues 476 and 516 of the porcine GH receptor(19) .
Although it has been known for some time that binding of GH to its receptor leads to rapid phosphorylation of tyrosine residues in the GH receptor(9) , the functional importance of GH receptor tyrosine phosphorylation remains relatively unknown. In the promyeloid cell line FDC-P1, it has recently been shown that tyrosine phosphorylation of the GH receptor is not required for GH-stimulated proliferation(20) . However, we have recently demonstrated that phosphorylation of tyrosines located close to the transmembrane region of the GH receptor is required for certain metabolic effects of GH(10, 21) . For other members of the cytokine receptor superfamily, tyrosine phosphorylation has been implicated in signaling. In the IL-4 (22) and leukemia inhibitory factor (LIF) receptors, as well as in the signal transducer gp130(23) , phosphorylation of specific tyrosine residues is required for binding and activation of Stat factors. Furthermore, a single tyrosine located in the C-terminal domain of the prolactin receptor has been found to be crucial for transcriptional signaling(24) .
While it has been possible to identify
specific regions of the GH receptor as being important for various
signaling pathways stimulated by GH by using deletion and substitution
mutations, we now address the question of the role of GH receptor
tyrosine phosphorylation in GH-mediated signal transduction. In this
study, we have constructed a series of GH receptor mutants in which we
have systematically substituted all intracellular tyrosines with
phenylalanines and have analyzed their ability to mediate GH-stimulated
transcription, activation of MAP kinase, Ca signaling, as well as activation of Stat5.
CHO cells were transiently transfected using the calcium
phosphate procedure with 3 µg of the -galactosidase-encoding
plasmid, 1.5 µg of the construct containing the bacterial CAT
coding sequence linked to three copies of the sequence -147 to
-103 of the Spi 2.1 promoter(1, 2) , and 1.5
µg of the different mutated GH receptor plasmids. After 4 h, the
cells were subjected to a glycerol shock (14% glycerol in Opti-MEM I)
for 1 min and then washed twice before fresh GC-3 medium, with or
without 20 nM hGH (Novo Nordisk, Gentofte, Denmark), was
added. Following 48 h of culture at 37 °C, the cells were scraped
off the plate, and extracts were prepared by three consecutive
freeze-thaw cycles followed by centrifugation at 15,000
g for 10 min. GH receptor expression was analyzed in cells cultured
for 48 h, and all GH receptor constructs were found to be expressed at
the cell surface.
The CHO cells were stably transfected with 5
µg of pGH-R cDNA using LipofectAMINE (Life Technologies, Inc.)
according to the manufacturer's suggestions (75 µg of
LipofectAMINE). After 8 h of exposure to the LipofectAMINE-DNA complex,
the medium was changed to Ham's F-12 medium supplemented with 10%
fetal calf serum, 100 units of penicillin/ml, and 100 µg of
streptomycin/ml. Following 24 h of additional culture, the cells were
split (1:4) by trypsin treatment and cultured in the presence of 1 mg
of G418/ml. After 6 days, colonies were picked and analyzed for hGH
binding. Three different clones expressing the pGH-R and three clones expressing the pGH-RmC8 were isolated and
analyzed.
In order to determine the role of GH receptor tyrosine
phosphorylation in transcriptional signaling, we constructed a number
of mutated porcine GH receptors as shown schematically in Fig. 1. The GH receptor cDNAs were co-transfected into CHO cells
together with a CAT reporter construct containing three copies of the
GH-responsive element from the Spi 2.1 promoter. After 48 h in culture
with or without 20 nM GH, the functional activity of the
transfected GH receptor was analyzed by its ability to direct
GH-induced expression of CAT activity. In the series of truncated GH
receptors shown in Fig. 1A, it was found that the 80
most C-terminal amino acids could be deleted without loss of GH
induction, but when 122 amino acids were deleted (GHR 1-516), no
GH stimulation of CAT activity was observed. The mutation of individual
tyrosines to phenylalanines in the intracellular domain of the GH
receptor did not affect the signaling ability of the GH receptor since
all eight receptors were able to mediate GH-stimulated CAT activity (Fig. 1B). Similar results were obtained using the rat
GH receptor in which the intracellular tyrosines were individually
mutated to phenylalanines. ()In contrast, when tyrosine
residues were mutated to phenylalanines in combination and sequentially
from the C terminus, signaling by the GH receptor was lost when the
four most C-terminal tyrosines were mutated to phenylalanines (Fig. 1C). These results indicate a role for two or
more of these tyrosines in signaling. Finally, we constructed mutated
GH receptors in which seven out of the eight cytoplasmic tyrosines were
mutated to phenylalanines. The results from this series of GH receptor
mutants revealed that any one of three tyrosines, 534, 566, or 627,
when present as the only cytoplasmic tyrosine in the GH receptor, can
generate a functional GH receptor (Fig. 1D). Comparison
of the amino acid sequences surrounding the three tyrosines shows no
obvious sequence homology (Fig. 2).
Figure 1: Growth hormone-induced CAT activity in CHO cells expressing wild-type and mutated GH receptors. The various mutated GH receptors are shown schematically. N denotes the amino terminus, and the shaded box represents the transmembrane domain of the GH receptor. The position of the eight intracellular tyrosines (Y) is indicated and identified by the position number. The -fold induction of CAT activity by GH (20 nM) in cells cotransfected with the indicated GH receptor and the Spi 2.1/CAT reporter is shown. Results represent the average of three to eight experiments.
Figure 2: Amino acid sequence of the porcine GH receptor domains containing the three tyrosine residues found to be involved in transcriptional signaling. The number above the tyrosine refers to the position of the tyrosine residue in the porcine GH receptor precursor.
The role of GH receptor tyrosine phosphorylation in GH-induced MAP kinase activation was evaluated in CHO cells stably transfected with either the wild-type or the GH receptor mC8 mutant lacking all intracellular tyrosines. The number of GH receptors in the two clones shown in Fig. 3was comparable, expressing approximately 40,000 receptors/cell. GH-induced MAP kinase activity was the same in cells expressing either the wild-type or the mC8 mutant GH receptor, but no MAP kinase activation was seen in untransfected CHO cells (Fig. 3). These results demonstrate that tyrosine phosphorylation of the GH receptor is not required for GH-induced activation of MAP kinase activity.
Figure 3: MAP kinase activity in GH-stimulated CHO cells expressing wild-type or mC8 mutant GH receptors. MAP kinase activity was measured as described under ``Experimental Procedures,'' and cells were stimulated for 0, 5, or 10 min with 20 nM GH. Results shown are from one representative experiment.
The
domain of the GH receptor required for GH-induced oscillations in
intracellular free Ca concentrations has been found
to overlap with the domain required for transcriptional signaling,
suggesting a role for Ca
in GH-induced
transcriptional signaling. We have, therefore, examined the ability of
the wild-type and the mC8 mutant GH receptors to mediate GH-induced
Ca
oscillations by fluorescence image microscopy of
Fura-2-loaded CHO cells. Untransfected CHO cells (Fig. 4A), cloned cell lines expressing the wild-type (Fig. 4B), or the mC8 mutant (Fig. 4C)
GH receptor exhibited GH-induced Ca
oscillations
similar to those described previously. Similarly, pools of transfected
CHO cells expressing wild-type (Fig. 4D) or mutant (Fig. 4E) GH receptors showed the typical GH-induced
Ca
oscillations of varying frequency and amplitude.
The Ca
response was observed in the majority of GH
receptor-expressing cells analyzed but was never seen in untransfected
CHO cells.
Figure 4:
Growth hormone-induced
Ca oscillations in CHO cells expressing wild-type or
mC8 mutant GH receptors. Intracellular free Ca
concentrations were measured by fluorescence image analysis of
Fura-2 loaded cells. GH (20 nM) was added as indicated by the arrow. Recordings are from untransfected CHO cells (A) and clones of CHO cells expressing the wild-type (B) or mC8 mutant (C) GH receptor. Recordings are
from pools of CHO cells transfected with wild-type (D) or mC8
mutant GH receptor cDNA (E). Panels B-E show
three representative single cell recordings from three different
experiments.
Since cytokine receptor phosphorylation has been
implicated in Stat factor activation for the IL-4 and LIF receptors, we
investigated the role of GH receptor phosphorylation in the activation
of Stat5 by gel retardation assay. Nuclear extracts from control and
GH-stimulated cells expressing wild-type or mC8 mutant GH receptors
were isolated. Using the GH response element from the Spi 2.1 promoter
as a probe, a GH-induced band was observed using nuclear extracts from
cells expressing the wild-type GH receptor (Fig. 5A, lane 4). In contrast, no specific bands were observed using
nuclear extracts from cells expressing the mC8 mutant GH receptor (Fig. 5A, lanes 5 and 6). The
GH-induced band was specific since it could be inhibited by unlabeled
oligonucleotide but not by an unrelated oligonucleotide (Fig. 5C, lanes 15 and 16). The
presence of Stat5 in the observed complexes was tested by using Stat5
antibodies in gel retardation assays. A supershift of the GH-induced
bands was observed when the Stat5 antibody was present in the binding
reaction, whereas a preimmune serum did not affect the formation of the
GH-induced complexes (Fig. 5C, lanes 17 and 18). Antibodies against Stat1 or Stat3 did not cause a
supershift of the GH-induced bands. The quality of the nuclear extracts
was tested by DNA binding activity to the cyclic AMP response element
from the -chorionic gonadotropin promoter. All extracts were found
to contain proteins capable of binding to this promoter element (Fig. 5B).
Figure 5:
Electrophoretic mobility shift assay of
GH-stimulated nuclear extracts from CHO cells expressing wild-type or
mC8 mutant GH receptors. Panel A shows the effect of GH
stimulation on Spi 2.1 GLE binding activity. Cells were cultured with
or without 20 nM GH for 10 min, and nuclear extracts were
prepared. Arrows indicate the position of the GH-induced band. B, the same nuclear extracts as used in lanes 1-6 were incubated with an -chorionic gonadotropin promoter probe
containing a cyclic AMP response element. Panel C shows
characterization of the GH-induced Spi 2.1 GLE binding activities. One
hundred-fold excess unlabeled competitor (lane 15) or
unrelated competitor (lane 16) was included. Control or
anti-Stat5 antiserum was included in lanes 17 and 18,
respectively.
We have demonstrated in this study that three tyrosine
residues in the intracellular domain of the GH receptor are involved in
transcriptional signaling. Specific tyrosine phosphorylation of GH
receptors in response to GH has been previously observed in several
different cell types(9, 29, 30) , and even
highly purified GH receptor preparations were found to exhibit tyrosine
kinase activity phosphorylating the GH receptor(9) . However,
it was recently shown that the GH receptor-associated tyrosine kinase
was Jak2 (31) and that binding and activation of Jak2 by GH was
dependent upon a proline-rich domain in the GH receptor(15) .
The proline-rich domain is required for receptor signaling since
deletion of this domain results in a receptor that is unable to
activate signal transduction(17, 32, 33) . We
and others have previously identified the C-terminal 184 amino acids of
the rat GH receptor as being required for GH-stimulated transcription
of the Spi 2.1 (32, 33) and insulin (34) genes. However, the signaling properties of this domain
are not known, although it has been implicated in GH-stimulated
Ca channel activation(17) . By way of
co-transfection of truncated porcine GH receptor cDNA with an Spi 2.1
promoter/CAT construct, we found that GH stimulation of CAT activity
can be abolished when 122 amino acids were deleted from the C terminus
of the porcine GH receptor. In order to examine the possible effect of
GH receptor tyrosine phosphorylation on transcriptional signaling, we
substituted individual tyrosine residues with phenylalanines. All GH
receptors containing single mutations were able to mediate GH-induced
CAT activity. The variation in GH induction observed (Fig. 1B) does not reflect a true variation in
signaling ability, but it most likely reflects differences in
expression levels of the transfected GH receptors caused by differences
in plasmid purity. When different preparations of the wild-type GH
receptor encoding plasmid were used in the transcription assay, GH
induction varied from 4- to 12-fold.
Mutation of multiple tyrosines
to phenylalanines as shown in Fig. 1, C and D,
revealed that three tyrosine residues at positions 534, 566, and 627
are involved in signaling and that each of these tyrosines can function
independently. A similar redundancy has been observed in receptors for
IL-4(22) , LIF, and the signal transducer gp130(23) .
Three tyrosines in the intracellular domain of the LIF receptor and
four tyrosines in gp130 were found to be involved in the activation of
Stat3. Furthermore, in the IL-4 receptor, two tyrosines have been
implicated in the activation of Stat6. In all cases, it has been
implicated that a direct interaction between the SH2 domain of the Stat
factor and the phosphorylated tyrosine on the receptor is a
prerequisite for Stat activation. The specificity of SH2 domain
interaction with phosphorylated tyrosines appears to reside within the
three amino acids C-terminal to the phosphorylated tyrosine (35) . In the LIF receptor and gp130, all five tyrosines found
to be involved in Stat3 activation have conserved amino acids in the
+1 to +3 positions. Similarly, in the IL-4 receptor, the
identical amino acids are present in the +1 and +3 positions
relative to the tyrosines involved in Stat6 activation. In the
prolactin receptor that is 35% identical to the GH receptor, a single
tyrosine residue has been identified as being responsible for
activation of -casein gene transcription. Interestingly, this
tyrosine is conserved in prolactin and GH receptors of all species
examined so far. Considering that both prolactin and GH stimulate Stat5
factors and that Stat5 has been shown to bind to hormone-responsive
elements in the
-casein (36, 37) and Spi 2.1
promoters, it seems likely that this C-terminal tyrosine is directly
involved in Stat5 activation. In contrast to the LIF and IL-4 receptors
and gp130 in which the tyrosines involved in Stat activation have very
similar +1 to +3 positions, the three tyrosines identified in
this study have varying C-terminal flanking amino acids. Except for the
threonine residue at the +3 position relative to tyrosines 627 and
566, no other identities or similarities exist among these positions.
However, the C-terminal flanking amino acids are conserved in all
species of cloned GH receptors until now. This raises the possibility
that the three tyrosines use different signaling pathways for mediating
GH-stimulated Spi 2.1 transcription by binding separate signaling
molecules having different SH2 domains. Another explanation could be
that all three tyrosines are involved in Stat5 activation, but each
tyrosine activates only one of the Stat5 isoforms, A or
B(38, 39) .
In CHO cells expressing the GH receptor
mutant lacking all intracellular tyrosines (mC8), no activation of
Stat5 could be observed, whereas in cells expressing the wild-type GH
receptor GH was able to induce binding of Stat5 to the Spi 2.1
promoter. In contrast it was previously shown that in FDC-P1 promyeloid
cells expressing wild-type or tyrosine-deficient GH receptors
intracellular tyrosines are not required for GH activation of Stat1 or
-3 binding to the sis-inducible element from the c-fos promoter (20) . In the same study, GH-induced Jak2
activation in FDC-P1 cells was not affected by the lack of
intracellular tyrosines in the GH receptor. We also observed that GH
can induce Jak2 activation in CHO cells expressing the
tyrosine-deficient GH receptor mutant as well as
GH-stimulated MAP kinase activity. GH-induced oscillations in
[Ca
]
were similarly unaffected
by the mutation of all intracellular tyrosines to phenylalanines. This
is in agreement with our previous observation that Jak2 kinase activity
is not required for GH-stimulated Ca
signaling and
that tyrosine kinase inhibitors are not able to block the GH-induced
rise in
[Ca
]
(17, 18) .
In conclusion we have shown that three tyrosines in the C-terminal
domain of the GH receptor function as mediators of GH-induced
transcription of the Spi 2.1 gene. These tyrosines are, however, not
required for GH-induced MAP kinase activity nor for GH-induced
Ca signaling. The transcriptional signaling pathway
presumably involves Stat5 activation by specific binding of Stat5 to
the phosphorylated tyrosines followed by phosphorylation of Stat5 that
leads to dimerization and DNA binding. Whether Stat5 binds directly to
the phosphorylated tyrosines in the GH receptor or via adapter proteins
is not known at present. The observation that tyrosine-deficient GH
receptors can still mediate GH effects on proliferation and Stat1 and
-3 activation in FDC-P1 cells (20) suggests that GH activates
different Stat proteins by distinct signaling pathways. The activation
of Stat1 and -3 may be mediated by phosphorylated tyrosines on Jak2
itself, whereas Stat5 activation requires phosphotyrosines in the
C-terminal domain of the GH receptor. This would imply that the
proliferative effects of GH are mediated at least in part by Stat1 and
-3 activation, whereas the effects of GH on cell-specific gene
transcription require Stat5.