(Received for publication, October 28, 1994; and in revised form, January 4, 1995)
From the
Ligand binding and dimerization of the growth hormone (GH)
receptor leads to the rapid tyrosine phosphorylation of the
intracellular kinase, Jak2, to the tyrosine phosphorylation and
activation of STAT protein(s) and to the tyrosine phosphorylation of
the receptor itself. Expression of the human GH receptor in the mouse
promyeloid, interleukin-3-dependent cell line, FDC-P1, shows that this
receptor can signal ligand-dependent proliferation in these cells as
well as induce the tyrosine phosphorylation of Jak2 and the activation
of transcription factors. We now examine the requirement for tyrosine
phosphorylation of the GH receptor for these three events by expression
of a receptor without tyrosine residues in the intracellular domain.
Six of the seven intracellular tyrosine residues were removed by a
carboxyl-terminal truncation, and the remaining tyrosine was changed to
phenylalanine to yield the GH receptor D351Stop/Y314F. When expressed
in FDC-P1 cells, this receptor retained its ability to induce the
tyrosine phosphorylation of Jak2, to induce the activation of
transcription factors, and to signal ligand-dependent cell
proliferation. Thus, tyrosine phosphorylation of the GH receptor is not
essential for the signaling of these three events at least in this
system. This finding contrasts with that for the interferon-
receptor system where data indicate that the specific tyrosine
phosphorylation of the interferon-
receptor leads to an
association with the STAT protein, p91, that is the mechanism by which
ligand couples the receptor to the signal transduction system.
The GH ()receptor is a 130-kDa glycoprotein that
transduces the signal for a variety of the biological actions of GH,
including the regulation of long bone and soft tissue growth, fat
metabolism, and insulin action(1) . The human GH receptor
contains an extracellular hormone binding domain of 246 amino acids, a
single transmembrane domain of 24 amino acids, and a 350-amino acid
cytoplasmic domain(2) . It is a member of a family of proteins
that includes the receptors for GH, prolactin, interleukins 2-7
and 11, G-CSF, GM-CSF, erythropoietin, thrombopoietin, ciliary
neurotrophic factor, leukemia inhibitory factor, and oncostatin
M(3, 4, 5) . Structural, mutational, and
functional studies provide strong evidence that dimerization of
receptors of this family by a single ligand molecule is the first step
in receptor activation that ultimately leads to signal
transduction(6, 7, 8) .
Studies of
intracellular signaling events induced via the GH receptor (and by
several other receptors of the GH/cytokine receptor family) show that
tyrosine phosphorylation is an early consequence of ligand binding and
receptor dimerization(9) . In various cell lines and in
vivo, GH induces the rapid tyrosine phosphorylation of several
proteins, including the GH receptor itself, the tyrosine kinase Jak2,
the STAT protein p91 (or a related molecule), as well as other
proteins(10, 11, 12, 13, 14, 15, 16, 17, 18, 19) .
Recently, a direct signaling pathway utilizing tyrosine kinases of the
Jak family and STAT proteins has been delineated for the IFN- and
IFN-
receptors(20, 21, 22) . These
experiments and data from the GH system support the hypothesis that
Jak2 associates with the GH receptor following ligand binding and
receptor dimerization(14) . This leads to activation of the
Jak2 kinase which results in tyrosine phosphorylation of Jak2 itself,
of the GH receptor, and of STAT proteins. The activated STAT proteins,
possibly in a complex with other transcription factors, then migrate to
the nucleus and bind DNA elements responsible for the modulation of
gene transcription(10) . For the IFN-
receptor,
mutagenesis and phosphorylated peptide data show that Tyr
of the intracellular domain of the
chain is essential for
signaling(23, 24) . These data also show that the STAT
protein, p91, specifically interacts with the phosphorylated receptor,
indicating that this inducible association is the mechanism by which
the ligand couples the receptor to the signal transduction
system(24) .
Expression of the human GH receptor in the promyeloid cell line, FDC-P1, or in the pro-B cell line, Ba/F3, which both normally require IL-3 for maintenance and growth, leads to lines that proliferate in response to GH(25, 26) . We have used this system to demonstrate that GH receptor truncations containing as few as 54 amino acids of the cytoplasmic domain will signal a proliferative response upon ligand binding(27) . This proliferative response is preceded in these systems by the tyrosine phosphorylation of Jak2 and the activation of transcription factors(25) . Mutation of amino acids in the remaining 54-residue signaling region shows that there is a correlation of Jak2 phosphorylation, transcription factor activation, and cell proliferation, a finding that is consistent with the hypothesis that the Jak-STAT signaling pathway is required for the signaling of proliferation in this system(25) .
In the current work we examine the requirement for tyrosine phosphorylation of the GH receptor for the signaling of proliferation and for the activation of the Jak-STAT pathway. We find that the human GH receptor without tyrosine residues in the intracellular domain is capable of signaling the proliferation of FDC-P1 cells and that Jak2 tyrosine phosphorylation and transcription factor activation appear unaffected.
Figure 1: Outline of the human GH receptor mutants. The location of the exons is shown. Numbers indicate the extent of the extracellular, transmembrane (TM), and cytoplasmic domain; residue 1 is the first amino acid of the mature protein(2) . The location of the cysteine residues (C) in the extracellular domain and of regions conserved in the GH/cytokine receptor family (WSXWS, Box 1, and Box 2) are indicated. The WSXWS motif is less conserved in the human GH receptor where the sequence is YGEFS. The amino acid sequence of the cytoplasmic domain of the D351Stop truncated GH receptor is shown as well as the location of the Y314F mutation.
In order to determine whether tyrosine phosphorylation of the
cytoplasmic domain of the GH receptor is essential for the signaling of
a ligand-dependent growth response, we utilized plasmids encoding the
three forms of the receptor shown in Fig. 1. Previous work has
shown that the D351Stop truncated receptor (which lacks 77% of the
cytoplasmic domain and six of the seven tyrosine residues) gives a
growth response similar to that found for the full-length receptor when
expressed in FDC-P1 or Ba/F3 cells(25, 27) . The one
remaining tyrosine in the cytoplasmic domain, Tyr, was
changed to phenylalanine in the D351Stop truncated GH receptor to
eliminate all the tyrosines in the cytoplasmic domain. Characterization
of the GH binding properties of the receptors when stably expressed in
FDC-P1 cells gives affinities of K
= 0.61,
0.21, and 1.8 nM and receptor numbers of 5600, 500, and 3500
sites/cells for the full-length, D351Stop, and D351Stop/Y314F
expressing cell lines, respectively (data not shown)(27) .
Tyrosine phosphorylation of the GH receptor in the expressing cell
lines was assessed by immunoprecipitation of the receptor followed by
phosphotyrosine immunoblot (Fig. 2). For the full-length
receptor, a broad, GH-dependent band of the expected size (130
kDa) (2, 31) was found (FFR); this band was absent
with untransfected cells (FDC-P1). These findings indicate that the GH
receptor is tyrosine-phosphorylated in this system. No phosphorylated
receptor band was found for the D351Stop/Y314F receptor, as expected
(F51.5). No phosphorylated receptor band was found as well for the
D351Stop receptor (F351), suggesting the possibility that much of the
tyrosine phosphorylation found for the full-length receptor is in the
distal six tyrosine residues.
Figure 2: Tyrosine phosphorylation of GH receptor mutants expressed in FDC-P1 cells. Whole cell extracts prepared from cells treated without(-) or with (+) 10 nM human GH were immunoprecipitated with an anti-GH receptor monoclonal antibody (263) and analyzed by phosphotyrosine immunoblot (8% polyacrylamide gel). FDC-P1, parental cells; FFR, cells expressing the full-length GH receptor; F351, cells expressing the D351Stop GH receptor; F51.5, cell expressing the D351Stop/Y314F GH receptor.
FDC-P1 cells expressing the three forms of the GH receptor were assayed for GH-dependent proliferation in the presence of saturating concentrations of ligand (Fig. 3). No response was found without GH or IL-3. All three receptor expressing lines, including the line expressing the GH receptor without tyrosine residues in the cytoplasmic domain (F51.5), show a proliferation response that equals that for IL-3. A full proliferative response was also found for FDC-P1 cells expressing the Y314F mutation in the full-length receptor (data not shown). Thus, tyrosine phosphorylation of the cytoplasmic domain of the human GH receptor is not required to signal a growth response in this system.
Figure 3: Proliferation of FDC-P1 cells expressing mutants of the GH receptor. Parental FDC-P1 cells and the GH receptor-expressing cell lines were assayed for GH-dependent growth as described under ``Materials and Methods.'' The standard deviation of triplicates is shown.
To test whether some of the intracellular signaling events coupled to receptor activation are affected by the lack of tyrosine phosphorylation of the GH receptor, we examined Jak2 tyrosine phosphorylation (Fig. 4) and transcription factor activation (Fig. 5) in response to GH binding. The GH-stimulated tyrosine phosphorylation of Jak2, and the band shifts induced with the DNA elements SIE or GRR, are unaffected in the D351Stop/Y314F mutant (F51.5). Thus, these two intracellular signals also do not require tyrosine phosphorylation of the GH receptor.
Figure 4: Jak2 tyrosine phosphorylation in GH receptor expressing FDC-P1 cell lines. Whole cell extracts prepared from cells treated without(-) or with (+) 10 nM human GH were immunoprecipitated with anti-Jak2 antiserum and analyzed by phosphotyrosine immunoblot as described under ``Materials and Methods.''
Figure 5: Transcription factor complex formation in GH receptor expressing FDC-P1 cell lines. The binding of whole cell extracts to labeled DNA elements was performed as described under ``Materials and Methods.'' Binding was performed with extracts from lines treated without(-) or with (+) 10 nM human GH (GH) or unlabeled oligonucleotide (Cold Oligo). A, binding to the element SIE. B, binding to the element GRR.
Following dimerization of the GH receptor by a single
molecule of GH, there is a rapid tyrosine phosphorylation of a number
of proteins in the cytoplasm, including the GH receptor
itself(15, 17) . Recently, Jak2 has been identified as
the tyrosine kinase likely to be responsible for these phosphorylation
events(14) . Jak2 associates with the receptor following ligand
binding and is activated by tyrosine phosphorylation(14) . The
activation of Jak2 and the related kinases, Jak1 and Tyk2, have been
implicated in the intracellular signaling pathways for many if not all
of the receptors of the GH/cytokine receptor family(9) . Direct
evidence that the Jak family kinases are an essential component of the
signaling pathway has been provided for the IFN- and IFN-
receptors where cell lines lacking these kinases or STAT proteins fail
to respond to ligand
stimulation(20, 21, 22) .
We find that the full-length GH receptor when expressed in FDC-P1 cells is rapidly tyrosine phosphorylated in response to GH stimulation, as has been found for other systems(16, 17, 18, 19) . In order to determine whether this tyrosine phosphorylation is an essential step in the signaling of proliferation and in the activation of the Jak-STAT signaling pathway, we have expressed a truncated form of the human GH receptor that lacks tyrosine residues in the cytoplasmic domain. This receptor is able to signal a hormone-dependent biological response (e.g. proliferation), as well as stimulate the phosphorylation of Jak2 and the activation of STAT protein(s). Thus, at least in FDC-P1 cells, tyrosine phosphorylation of the GH receptor is not an essential step in these signaling events.
Work is in progress to identify which STAT protein or proteins are activated by growth hormone in FDC-P1 cells. Published data from other systems indicate that p91 and STAT3 may be the specific STAT proteins activated by the GH receptor(11, 32) .
Recently described experiments
show that Tyr of the
chain of the IFN-
receptor is required for signal transduction in a mouse fibroblast cell
line, SCC16-5(23) . Furthermore, activation of the STAT
protein, p91 (as assayed by electrophoretic mobility shift assay with
homogenates of the human cell line, Colo-205), can be specifically
inhibited by a 9-amino acid phosphorylated peptide having the sequence
surrounding Tyr
; the unphosphorylated peptide fails to
block p91 activation(24) . Although no direct binding of p91 to
the phosphorylated IFN-
receptor has been reported, the docking of
p91 to Tyr
of the receptor has been proposed as a key
step in its activation that is followed by its tyrosine
phosphorylation, dissociation, and translocation to the
nucleus(24) . Recent, phosphorylated peptide inhibition
experiments with the IL-4 receptor and the newly identified IL-4 STAT
protein support a similar model for IL-4 STAT binding to two
phosphotyrosine-containing sequences of the IL-4 receptor cytoplasmic
domain(33) . On the other hand, mutants of the IL-4 receptor
lacking tyrosine residues in the cytoplasmic domain are able to signal
proliferation in Ba/F3 cells, although STAT activation was not
determined(34) . Although additional studies will be required
to resolve these apparently divergent actions, perhaps there are
phosphotyrosine-containing STAT binding sites on proteins other than
the receptors (for example on ancillary molecules, on the Jak proteins
themselves, or on receptors for other ligands) that can be used as
alternative sites for STAT docking and activation. Such a hypothesis
would predict that deletion of any one site would have little or no
effect, whereas competition for all the sites would inhibit STAT
activation and signaling.