(Received for publication, March 8, 1995; and in revised form, May 30, 1995)
From the
We have examined the involvement of tyrosine residues 333 and
338 of the growth hormone (GH) receptor in the cellular response to GH.
Stable Chinese hamster ovary (CHO) cell clones expressing a receptor
with tyrosine residues at position 333 and 338 of the receptor
substituted for phenylalanine (CHO-GHRY333F,
Y338F) were generated by cDNA transfection. Compared with the wild type
receptor the Y333F,Y338F mutant possessed normal high affinity ligand
binding, hormone internalization, and ligand-induced receptor
down-regulation. GH activation of mitogen-associated protein kinase was
also similar in CHO clones expressing similar wild type and Y333F,Y338F
receptor number. However, two GH-regulated cellular events
(lipogenesis, and protein synthesis) were deficient in the tyrosine
substituted receptor. In contrast, transcriptional regulation by GH (as
evidenced by chloramphenicol acetyltransferase cDNA expression driven
by the GH-responsive region of the SPI 2.1 gene) was not affected by
Y333F,Y338F substitution. Thus we provide the first experimental
evidence that specific tyrosine residues of the GH receptor are
required for selected cellular responses to GH.
The GH ()receptor is a member of the cytokine
receptor superfamily (Bazan, 1990; Cosman et al., 1990;
Kitamura et al., 1994). One characteristic of this family of
receptors is the lack of a canonical sequence for intrinsic tyrosine
kinase activity (Leung et al., 1987; Mathews et al.,
1989; Smith et al., 1989; Kitamura et al., 1994).
However, ligand binding to a number of these receptors, including the
GH receptor, induces tyrosyl phosphorylation of cellular proteins
(Möller et al., 1992; Wang et
al., 1992, 1993). This tyrosyl phosphorylation of cellular
proteins is attributed to the association with Janus kinase 2 (JAK2)
for the GH receptor (Argetsinger et al., 1993) and also the
receptors for erythropoietin, prolactin, interleukin-3, interleukin-6,
granulocyte-colony-stimulating factor, granulocyte-macrophage
colony-stimulating factor, leukocyte inhibitory factor, oncostatin M,
and ciliary neurotrophic factor (Silvennoinen et al., 1993;
Stahl et al., 1994; Witthuhn et al., 1993; Narazaki et al., 1994; Dusanter-Fourt et al., 1994; DaSilva et al., 1994). Pharmacological inhibition of cellular tyrosine
phosphorylation does inhibit certain GH-stimulated cellular events
(Campbell et al., 1993; Möller et
al., 1994; Sliva et al., 1994). Although the GH receptor
is itself tyrosine-phosphorylated (Foster et al., 1988), it
has not been demonstrated that such receptor phosphorylation is
required for the biological response to GH.
Studies involving truncation of the GH receptor intracellular domain has suggested functional specificity for different regions of the intracellular domain. Thus the membrane proximal portion alone (truncated at amino acid 454) is capable of mediation of GH-stimulated mitogenesis (Möller et al., 1992; Colosi et al., 1993), lipid synthesis (Möller et al., 1994), protein synthesis (Billestrup et al., 1994), MAP kinase activation (Möller et al., 1992), c-fos expression (VanderKuur et al., 1994) and hormone internalization (Möldrup et al., 1991). In contrast the carboxyl-terminal portion of the GH receptor is required for full transcriptional activation (Goujon et al., 1994) and phosphorylation of p97 (a putative GH-dependent transcription factor) (VanderKuur et al., 1994). The membrane proximal portion of the intracellular domain contains 4 tyrosine residues but a single set of paired tyrosine residues at position 333 and 338 of the receptor. Phosphorylation of specific tyrosyl residues in receptor molecules serve as docking sites for for one or more proteins containing SH2 domains (Koch et al., 1991; Cantley et al., 1991; Mayer and Baltimore, 1993; Nishimura et al., 1993). Concordant with functional specificity of other receptor molecules being determined by phosphorylation of specific tyrosine residues (Mohammadi et al., 1992; Pawson and Gish, 1992), we have investigated the importance of tyrosine 333 and 338 of the GH receptor for the biologic response to GH. We show here that phosphorylation of tyrosine 333 and 338 are required for selected biological functions of GH.
For estimation of
ligand-induced receptor down-regulation, cell monolayers in six-well
plates were incubated in the presence of different concentrations of
hGH in serum-free medium for 12 h (Roupas and Herington, 1988). The
cells were placed on ice and associated hGH removed by acid wash (0.15 M NaCl, 0.05 M glycine, pH 2.5) and further washed
three times in a wash buffer containing 10 mM HEPES, pH 7.4,
124 mM NaCl, 4 mM CaCl, and 1.5 mM MgCl
. The cells were then incubated for 90 min at room
temperature in binding medium (10 mM HEPES, pH 7.4, 1% human
serum albumin) containing
I-hGH (approximately 100,000
cpm). The cells were washed four times in wash buffer and solubilized
with 1 ml of 1 M NaOH. Radioactivity was counted in a
counter. Nonspecific binding was estimated in the presence of 1
µg/ml unlabeled hGH and was <5%.
Figure 1:
Receptor-mediated
hormone internalization in CHO cells stably transfected with cDNA for
the wild type GH receptor (GHR) and for the
Y333F,Y338F receptor (GHR
Y333F,Y338F).
Internalization assays were performed as described under
``Experimental Procedures'' using
I-hGH
(approximately 100,000 cpm). Results are presented as the percentage of
cell surface-bound hormone internalized. Each point is the mean
± S.E. of triplicate determinations. Data shown are derived from
CHOA cells (GHR
) and
GHR
Y333F,Y338F clone 23. Similar results were
obtained with the use of CHO4 cells and clone 3 of
GHR
Y333F,Y338F.
Figure 2:
Ligand-induced receptor down-regulation in
CHO cells stably transfected with cDNA for the wild type GH receptor
(GHR ) and for the Y333F,Y338F receptor
(GHR
Y333F,Y338F). Estimation of receptor
down-regulation was performed as described under ``Experimental
Procedures'' using
I-hGH (approximately 100,000
cpm). Results are presented as the mean ± S.E. of quadruplicate
determinations. Data shown are derived from CHOA cells
(GHR
) and GHR
Y333F,Y338F
clone 23. Similar results were obtained with the use of CHO4 cells and
clone 3 of GHR
Y333F,Y338F.
Figure 3:
GH activation of MAP kinase activity in
CHO cells and in CHO cells stably transfected with cDNA for the wild
type GH receptor (GHR) and for the Y333F,Y338F
receptor (GHR
Y333F,Y338F (clones 3 and 23)). MAP
kinase activity in immunoprecipitates of MAP kinase was analyzed 15 min
after stimulation with GH using myelin basic protein as substrate.
Results are presented as mean ± S.D. from triplicate
determinations. Results for GHR
presented are
derived from CHOA cells. Similar results were obtained with CHO4 cells
and also have been reported previously (Möller et al., 1992).
Figure 4:
GH stimulation of protein synthesis in CHO
cells stably transfected with cDNA for the wild type GH receptor (GHR ), a receptor expressing only 5 amino acids in
the intracellular domain (GHR
), and receptors in
which tyrosines 333 and 338 have been substituted with phenylalanine
(GHR
Y333F,Y338F (clones 3 and 23)). Confluent
cells were incubated in serum-free medium for 24 h before addition of
100 nM hGH for 12 h. Protein synthesis was estimated by the
incorporation of [
H]leucine during a 2-h
pulse-chase. [
H]Leucine incorporated into
proteins was precipitated by trichloroacetic acid and collected on
glass fiber filters. Radioactivity was estimated by scintillation
counting. Results are presented as mean ± S.D. from triplicate
determinations of the -fold stimulation above untreated cells. Results
for GHR
presented here were derived from CHO4
cells with similar results being obtained with CHOA
cells.
Figure 5:
GH
stimulation of lipid synthesis in CHO cells stably transfected with
cDNA for the wild type GH receptor (GHR ), a
receptor expressing only 5 amino acids in the intracellular domain
(GHR
) and receptors in which tyrosines 333 and
338 have been substituted with phenylalanine (GHR
Y333F,Y338F (clones 3 and 23)). Confluent cells were incubated in
serum-free medium containing 1% BSA for 24 h before commencement of
assay. Lipogenesis was estimated by the incorporation of
[
H]glucose into the lipid-soluble fraction of the
cell. Radioactivity was estimated by scintillation counting. Results
are presented as mean ± S.D. from triplicate determinations of
the -fold stimulation (in the presence of 100 nM hGH) above
untreated cells. Results presented here for GHR
are derived from CHO4 cells.
Figure 6:
GH stimulation of CAT activity driven by
the SPI-GH-responsive element in untransfected CHO cells, in CHO cells
transiently transfected with cDNA for the wild type GH receptor
(GHR), and in CHO cells transiently transfected
with cDNA for the Y333F,Y338F receptor
(GHR
Y333F,Y338F). CHO cells were transiently
transfected by the calcium phosphate procedure and also with a
construct containing bacterial CAT coding sequence linked to
-149/-103 of the serine protease inhibitor (SPI 2.1) gene
promotor. The cells were processed, and CAT assays were performed as
described under ``Experimental Procedures.'' Results are mean
± S.D. from triplicate
determinations.
We demonstrate here that specific tyrosine residues within
the GH receptor are required for GH to elicit selective cellular
function. We refer specifically to Tyr and Tyr
of the GH receptor and their involvement in GH-mediated protein
synthesis and lipogenesis. The selective retention of certain
GH-stimulated functions within the cell (also see VanderKuur et
al., 1995b) further substantiates the specific involvement of
these residues in GH signal transduction.
Several studies have
implicated the existence of distinct receptor domains regulating
GH-stimulated events (Moldrup et al., 1991;
Möller et al., 1992, 1994; Colosi et
al., 1993; Enberg et al., 1994; Goujon et al.,
1994; VanderKuur et al., 1994). Truncation of the
intracellular domain of the receptor has allowed definition of two
distinct macrodomains within the receptor with differential involvement
in transcription and metabolic events. A complete transcriptional
response to GH requires the presence of the carboxyl-terminal half of
the intracellular domain. In contrast certain presumed
``metabolic'' events such as mitogenesis lipogenesis and
protein synthesis are mediated by the membrane proximal portion of the
intracellular domain. That specific tyrosine residues are necessary for
selected functions further reinforces the concept of distinct receptor
domains. However, since no absolute delineation exists (e.g. both MAP kinase activation and protein synthesis require only the
membrane-proximal portion of the intracellular domain, yet only protein
synthesis is deficient in the Y333F,Y338F receptor), then regional
functional specificity of the receptor is likely to be dictated by the
association of specific signaling molecules to discrete phosphorylated
tyrosine residues. Such is the case for the better characterized
epidermal growth factor and platelet-derived growth factor receptors,
where specific phosphorylated tyrosine residues serve as docking sites
for proteins containing SH2 domains (Nishimura et al., 1993).
That transcriptional up-regulation is not affected by Y333F,Y338F
substitution in the receptor indicates that some more distal tyrosine
residue(s) may be involved in this event. GH has recently been
demonstrated to activate transcription by phosphorylation of a factor
binding to a GAS-like DNA element (Meyer et al., 1994;
Finbloom et al., 1994; Sliva et al., 1994). It has
been reported that this factor is p91 (STAT1)- and STAT3-like for the
SIE element (Meyer et al., 1994; Finbloom et al.,
1994; Campbell et al., 1995) and STAT5 like for the SPI 2.1
GAS-like element (Wood et al., 1995). The -interferon
receptor requires a distal tyrosine residue (Tyr
) for
activation of STAT 1 and presumably functions through direct
receptor-STAT1 interaction (Greenlund et al., 1994). The GH
receptor could also hypothetically participate in transcriptional
activation by providing a distal tyrosine residue for docking and
subsequent phosphorylation of the STAT protein. Also receptor
dimerization (Cunningham et al., 1991; DeVos et al.,
1992), presumably after ligand binding, could present a conformation
that allows docking of signaling molecules regardless of the
phosphorylation state. At present we do not know the nature of the
molecular interaction(s) that is potentially disrupted by the
Y333F,Y338F substitution. These tyrosines are not part of a known
sequence reported to serve as docking sites for signaling molecules
(Cantley et al., 1991).
The GH stimulation of protein
synthesis does not require RNA synthesis (Martin and Young, 1965).
Instead GH treatment of animals is associated with an apparent increase
in ribosomal efficiency (Kostyo and Rilemma, 1971). Increase in
cellular protein synthesis is thought to be mediated by phosphorylation
of the ribosomal protein S6. The phosphorylation of ribosomal protein
S6 is mediated by S6 kinase, which is itself phosphorylated by MAP
kinase (Maller, 1991). GH has been reported to activate both MAP kinase
(Winston and Bertics, 1992; Campbell et al., 1992;
Möller et al., 1992) and S6 kinase
(Anderson, 1993). That protein synthesis was deficient in the
Y333F,Y338F receptor indicates that simple MAP kinase activation is
also insufficient for the GH induction of protein synthesis. This is
interesting as S6 kinase is reported to only partially phosphorylate
ribosomal protein S6, with additional phosphorylation mediated by
cAMP-dependent protein kinase (Palen and Traugh, 1987). There are
reports that GH may interact with the cAMP pathway (Catalioto et
al., 1992; Singh and Thomas, 1993). Further activation of S6
kinase can be mediated by IRS-1 (Myers et al., 1994), and
IRS-1 has been demonstrated to be phosphorylated in response to GH
(Souza et al., 1994; Ridderstråle et al., 1995;
Argetsinger et al., 1995). It has been demonstrated recently
that IL-3 stimulation of protein synthesis is mediated by the
regulation of double-stranded RNA-dependent protein kinase (Ito et
al., 1994). This molecule inhibits protein synthesis by
phosphorylating the subunit of eucaryotic transcription factor 2.
Such may also be the case for GH. It is possible that MAP kinase is
only involved in a negative regulatory feed back loop. Indeed MAP
kinase has been demonstrated to decrease epidermal growth factor
receptor phosphorylation by activation of a tyrosine phosphatase
(Griswold-Prenner et al., 1993).
We have described a situation in which the Y333F,Y338F receptor results in activation of JAK2, but with deficient lipogenesis, mitogenesis, and protein synthesis. This may suggest some disjunction between activation of JAK2 and GH stimulation of these events. However, previous studies have demonstrated that cellular tyrosine phosphorylation is required for some of these functions (Möller et al., 1994), and a GH receptor with mutations in ``Box 1'' which is deficient in JAK2 activation is also deficient in these functions (for review see Lobie et al. (1994b)). Thus it is apparent that JAK2 activation (or tyrosine phosphorylation) is necessary but not sufficient for the response to GH. Such a situation also exists for GH stimulation of transcription in which a truncated receptor (at position 455) retains JAK2 activation (VanderKuur et al., 1994) but is unable to fully activate transcription (Goujon et al., 1994). Other signaling molecules which participate in and reconstitute the full cellular effect of GH remain to be identified. One of these is SHC, although this molecule apparently does not associate with the receptor and is not affected by Y333F,Y338F substitution (VanderKuur et al., 1995a).
In conclusion, we have demonstrated that tyrosine residues 333 and 338 of the GH receptor are required for selected cellular responses to GH. We therefore provide the first experimental evidence that tyrosine phosphorylation of the GH receptor is important for GH-stimulated function. In analogy to other receptor signaling systems (Cantley et al., 1991; Nishimura et al., 1993; Mayer and Baltimore, 1993), it is anticipated that these tyrosine residues participate in the recruitment of molecules containing SH2 domains which subsequently execute their cellular effect.