(Received for publication, June 27, 1994; and in revised form, November 17, 1994)
From the
It has become evident that intracellular protein phosphorylation
plays an important role in mediating signal transduction of hormones
and growth factors, including growth hormone (GH). We have previously
demonstrated that GH can stimulate tyrosine phosphorylation of cellular
proteins with approximate molecular masses of 95,000 daltons (pp95) in
GH-treated 3T3-F442A preadipocytes and in mouse L cells that express
recombinant porcine or bovine GH receptors. In present study, a series
of GH receptor (GHR) truncation analogs were constructed and examined
for their abilities to induce pp95. The results revealed that a region
of 40 amino acids in the porcine GHR cytoplasmic domain is
essential for induction of pp95. The results also established that the
115 amino acids(517-638) near the C terminus of porcine GHR are
not required for pp95 induction. Moreover, the basal levels of
GH-induced pp95 in parental mouse L cells was suppressed by expression
of these GHR truncation analogs. This suggests that pp95 induced by GH
may be mediated by GHR dimerization and can be inhibited by
overexpression of truncated porcine GHRs.
Growth hormone (GH) ()exerts its pleiotropic
biological functions by first binding with GH receptor(s) on the target
cell surface. GH treatment can elicit a variety of responses, such as
proto-oncogene induction, enhanced glucose utilization, accumulation of
lipid(1, 2, 3, 4) , and activation
of protein
kinases(5, 6, 7, 8, 9, 10, 11, 12) .
GH treatment also promotes conversion of the preadipocytes to
adipocytes(13, 14) . However, the signal transduction
pathway following the GH
GHR interaction remains undefined.
GHR belongs to a superfamily of growth factor and cytokine receptors, which includes prolactin, erythropoietin, interleukins, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, ciliary neurotrophic factor, and leukemia inhibitory factor (15, 16) . The family members share many structural features including a proline-rich sequence in the intracellular portion of the GHR located near the transmembrane region(15) . This proline-rich sequence has been termed ``Box 1''. The Box 1 is hypothesized to be the JAK2 association site and is critical for signal transduction by these receptors(17, 18, 19, 20) . Demonstration of involvement of Janus family kinases (JAK1, JAK2, and Tyk2) in interferon signal transduction pathway(21) , as well as identification of a GHR and erythropoietin receptor associated tyrosine kinase, JAK2, provides further insight into the signaling system of these family members(22, 23) . The possibility that other protein molecules may be involved in the signaling system has not been eliminated.
It has been demonstrated that the GHR itself along with several other cytosolic proteins with molecular masses of 121, 95, 43, and 40 kDa are tyrosine phosphorylated in mouse 3T3-F442A preadipocytes after GH treatment(24, 25) . The 121-kDa protein was identified as the JAK2 tyrosine kinase, and the 43- and 40-kDa proteins have been identified as mitogen-activated protein kinases based upon their co-migration with proteins identified by extracellular signal-regulated kinases 1 and 2 antibodies in GHR cDNA-transfected Chinese hamster ovary cells and 3T3-F442A cells(10, 11, 26) . Recently, interferon-stimulated 91-kDa transcription factor (also known as STAT 1) was found to be phosphorylated on tyrosine residues and shown to bind to the c-sis-inducible element of the c-fos promoter after GH treatment(27) . However, the identity of the 95-kDa proteins has not been reported.
In addition to mouse
3T3-F442A cells, we have previously demonstrated that a cytosolic
protein with molecular mass of 95 kDa (pp95) can be induced to be
tyrosine phosphorylated by GH treatment in a recombinant MLC which
expresses porcine GHR(28) . We have also shown that pp95
phosphorylation is GH-specific in this system. Tyrosine phosphorylation
of a protein with similar molecular mass (93 kDa) was also stimulated
in a GH-treated human lymphocyte line (IM-9) and was shown to be
GH-specific(29) . Therefore, we hypothesized that pp95 or the
equivalent protein(s) in other cells may be a mediator for GH signal
transduction. Also, it is possible that these molecules may dictate the
GH specificity in the GHGHR signal transduction pathway(s).
To determine the relationships between the induction of pp95 after GH treatment and the elements or motifs within the GHR intracellular domain essential for or related to signal transduction, we have selectively generated a series of GHR truncation analogs via site-directed mutagenesis and expressed these mutated cDNAs in MLC. The truncation positions were selected near the 6 conserved tyrosine residues of porcine, bovine, ovine, mouse, rat, and human GHRs. Subsequently, the induction of pp95 by GH treatment was examined in cells that express GHR or these GHR truncation analogs.
All restriction endonucleases, T4 DNA ligase, T4 DNA
polymerase, and Escherichia coli DNA polymerase (Klenow
fragment) were obtained from commercial sources (New England Bio-Labs,
Berverly, MA). [-
P]UTP,
[
-
P]dATP, and
I-hGH (
100
µCi/µg) were obtained from Dupont NEN.
I-pGH
(
100 µCi/µg) was kindly provided by American Cyanamid
Company (Princeton, NJ). Purified pGH was a generous gift from Smith,
Kline & Beecham (Westchester, PA). All chemicals used were of
reagent grade. The sources of any specific reagents are indicated in
the text.
The pGHR truncation analogs were
generated by introducing three translational stop codons via
site-directed mutagenesis at the desired positions. Negative strand
oligonucleotides were used (National Biosciences, Annapolis, MN). All
of the mutagenesis oligonucleotides were 45-50 bases in length
with three stop codons (TAGGTAGGTAG) near
the middle of the oligonucleotides. The first stop codon in
oligonucleotide was used to replace Arg at position 293 for
pGHR-TR1(291-638), Ser at 388 for pGHR-TR3
(
387-638), Asn at 477 for pGHR-TR4 (
476-638), His
at 517 for pGHR-TR5, and Ser at 559 for pGHR-TR6. The mutagenesis
reactions were carried out as described previously using T4 DNA
polymerase(32) . Each reaction mix was used to transform E.
coli DH5
. Colonies that possess the mutated pGHR cDNA were
selected by restriction digestion analysis using XbaI. This XbaI site was engineered into each oligonucleotide following
the third stop codon. Each mutation was confirmed by DNA
sequencing(33) . The plasmids that contain the mutated pGHR
cDNAs are referred to as pMet-IG-pGHR:TR1 through TR6. pGHR-TR2 was
generated by deletion of the fragment from the EcoRI site
after the transmembrane domain to the EcoRI site located at
the 3` end of the intracellular domain, which resulted in deletion of
amino acids 337-638 (
337-638). pGHR-TR2 utilizes the
translational stop codon of bGH. This fusion gene adds 3 amino acids
(-Cys-Ala-Phe) derived from the C terminus of bGH to the pGHR-TR2
analog.
Figure 4:
A, schematic representation of
pGHR and pGHR truncation analogs structure. The numbers in parentheses
indicate the length of intracellular domain of pGHR or pGHR truncation
analogs. The dottedboxes in the pGHR or pGHR analogs
indicate the conserved regions among the GH/cytokines receptor family.
Also, the pp95 induction results are summarized in lowerpanel as follows filleddot,
positive; openeddot, negative. B, alignment
of Box 3 amino acid sequences of GHR from various animal species. The
positions of pGHR-TR4 and -TR5 are indicated in the sequence via arrows. The shaded amino acids represent the
conserved residues among the GHRs. Tyrosine residue at position 487 is
indicated by boldface.
Figure 1:
Maximum
displacement experiments on MLC, pGHR-WT, and cells that express pGHR
truncation analogs. Cells were incubated in serum-free medium overnight
before the assay was performed. Approximately 60,000 cpm of I-pGH was incubated with the cells in the presence or
absence of excess amount of unlabeled pGH at room temperature for 2 h.
The cells were washed and harvested. The radioactivity of each sample
was determined. Each value represents the results from three
experiments (also, see ``Materials and
Methods'').
Figure 2: Autoradiography of GH-GHR cross-linking experiments on MLC and MLC lines that express full-length pGHR or its truncation analogs. - and + indicate the cross-linking reactions were performed in the absence or presence of excess amount (2 µg/ml) of unlabeled pGH. Lanes1-4, 9 and 10 are prolonged exposures of the same gel.
Moreover, a second band was observed in the cross-linking studies of
pGHR-TR1 and TR2 (Fig. 2, lanes15 and 13) and less distinct bands were seen in lanes3, 5, 7, 9, and 11 of Fig. 2. The molecular masses of these bands decreased,
concomitantly, with the size of GHGHR monomer and could be
competed by cold pGH specifically. The estimated molecular masses of
these bands are 156 ± 6 and 163 ± 6 kDa for pGHR-TR1 and
pGHR-TR2, respectively (summary of results from five cross-linking
gels).
Figure 3: pp95 induction assay on cell lines that express full-length GHR or its truncation analogs. Cells were plated in six-well tissue culture plates. GH in the medium was removed by incubating the cells in serum-free medium overnight. Subsequently, cells were treated with or without pGH (500 ng/ml) for 10 min at 37 °C and processed as described in ``Materials and Methods.'' The arrow on left indicates the position of pp95.
Interestingly, in addition to the lack of pp95 induction in pGHR-TR1, TR2, and TR3 cells, the basal levels of pp95 induction was also diminished (Fig. 3).
A tyrosine residue (Tyr-487) is conserved among the GHRs from all species in the identified 40-amino acid segment (Fig. 4B). We propose that Tyr-487 of pGHR or the corresponding tyrosine residue of GHRs from other species may be important for the induction of pp95 tyrosine phosphorylation. We hypothesize that Tyr-487 is phosphorylated by the GHR-associated tyrosine kinase, JAK2(22) . The phosphorylated Tyr-487 would provide a docking site for proteins that possess Src-homology (SH2) domains and may play important roles in GH signal transduction as described for the epidermal growth factor receptor or insulin receptors(37) .
In addition to the
proline-rich region, it has been shown that the 80 amino acids at the
C-terminal region of GHR are required for activation of serine protease
inhibitor promoter(38) . Also, it has been shown that deletion
of the C terminus of GHR results in loss of stimulation of insulin
production in insulinoma RIN-5AH cells(39) . Our data
demonstrate that the carboxyl-terminal 115 amino acids(517-638)
are not required for pp95 induction. Combining the present and
previously described data, it is reasonable to deduce that multiple
motifs within the GHR intracellular domain may mediate the pleiotropic
biological functions of GH. Thus, elucidation of GHGHR
interaction, as well as the linkage between the GHR (especially the
cytoplasmic domain of the GHR), and the intracellular elements becomes
very important in understanding GH
GHR signal transduction.
No pp95 induction was observed in pGHR-TR1 cells, i.e. cells that lack the intracellular domain. This result agrees with others that found that the cytoplasmic domain is essential for GH signal transduction(18, 19) . However, pGHR-TR2, TR3, and TR4 possess the intact Box 1 sequence and, presumably, provide sufficient elements for JAK2 kinase association but still lack the ability to induce pp95. This could be explained in the following two ways. 1) pp95 is not phosphorylated by the GHR-associated tyrosine kinase, JAK2, which would imply that pp95 and JAK2 kinase belong to different pathways of GH signal transduction. 2) pp95 phosphorylation is dependent on JAK2 activity. If the latter possibility is true, then truncation of the GHR intracellular domain or the 40-amino acid box 3 sequence would result in loss of a putative pp95 recruiting site(s) and would not lead to phosphorylation by JAK2. In this scenario, the pp95 protein requires access to the GHR (e.g. association with the 40-amino acid segment directly or through other anchor proteins) to be phosphorylated by the JAK2 kinase.
Interestingly, basal levels of
pp95 induction were not observed in pGHR-TR1, TR2, and TR3 cells. We
assume this phenomenon, a ``dominant negative effect,'' is
caused by the overexpression of nonfunctional pGHR-TRs in MLC.
Overexpressed pGHR-TRs may lead to the formation of a
pGHR-TRGH
mGHR heterodimer, which results in suppression of
basal levels pp95 induction in MLC. These heterodimers could not be
observed on the cross-linking gel probably because of the extremely low
levels of mGHR in MLC (Fig. 2, lane1)(31) . The formation of
pGHR-TR
GH
mGHR heterodimers would also not transduce the GH
signal. Together with the observations of pGHR
GH
pGHR
formation, we propose that the induction is not only dependent on a
functional motif(s) in the cytoplasmic domain but also on the
dimerization of the GHRs.
Cross-linking studies demonstrated that the pGH-TRs
produced were of correct apparent molecular masses. Furthermore, in the
cell lines that expressed pGHR analogs, pGHR-TR1 and TR2, a second band
in addition to the band that represents the GHGHR monomer was
observed (Fig. 2, lanes13 and 15).
However, based on their calculated molecular masses, the size of these
bands do not correspond to the GHR
GH
GHR complex as
described previously(40) . We have performed statistical
analysis of the data collected from five different cross-linking gels.
For example, the molecular mass of pGHR-TR1 is 52 ± 5 kDa (n = 5). The molecular mass of pGHR-TR1
pGH
pGHR-TR1
complex should be
126 ± 10 kDa. However, the molecular mass
of the second band observed in pGHR-TR1 cells is
156 ± 6
kDa (n = 5). Therefore, the second band may represent a
different form of GH
GHR complex than a GHR
GH
GHR
complex. We hypothesize that these bands may represent three possible
GH
GHR complexes. 1) The first is a GHR
GH
GH
GHR
complex since the difference between the calculated molecular mass and
the observed molecular mass is
30 kDa for TR1 and 22 kDa for TR2.
Therefore, these differences might indicate a second GH molecule in the
GHR
GH
GHR complex. 2) The second is a
GHR
GH
GHR
X complex, whereas X represents an unknown protein molecule with a molecular mass of
25 kDa associated with the GHR
GH
GHR complex. 3) And
finally is a GHR
GH
Y complex, where Y represents an unknown protein molecule with a molecular mass of
104 kDa (a GH signal mediator) similar the gp130 molecule used in
interleukin 6 signal transduction(41) . This difference in
expected size obtained here relative to the published report may be due
to a difference in experimental systems. The GHBP
GH
GHBP
complex model was established by use of E. coli-produced GH
binding protein, which is not glycosylated or
membrane-bound(40) . Our results were obtained from intact
mammalian cells that express the glycosylated, membrane-bound form of
the GHR analogs. It has been proposed that the use of living cells
producing GHR rather than bacterially produced GH binding protein may
generate more realistic results related to the GH
GHR
interaction(42) . However, it is possible that various forms of
GH
GHR dimer exist and mediate different GH signals. More detailed
studies are required to test these hypothetical models. Regardless of
the GH
GHR complex model, it was shown that the truncations of
pGHR cytoplasmic domain do not affect the ability of GHR to form
GH
GHR complexes. The absence of the GH
GHR dimer complex in
pGHR-TR6, TR5, TR4, and TR3 cells was probably due to fewer GH binding
sites on these cells.
By comparing the expression levels of pGHR-TRs
in the pGHR-TRs cell lines to the pGHR binding sites, it was found that
the difference in expression levels was not as variable as the
difference in the number of binding sites. This may result from the
fact that TR1 and TR2 lack the GHR internalization signals that,
therefore, lead to accumulation of pGHR-TRs on the cell surface.
Receptor internalization assays revealed the internalization rates of
pGHR-TR1 and pGHR-TR2 were 60% lower than that of the wild-type pGHR in
MLC. ()Also, this explanation is supported by the results
reported by Möldrup and colleagues (39) in
which a GHR containing a deletion of the majority of the intracellular
domain (GHR
) was found to have a dramatically
decreased internalization rate compared with the wild-type GHR. In the
same study, another GHR deletion mutant (GHR
)
retains the same internalization rate. The GHR
is
similar to our pGHR-TR4 (1-476 or
477-638). Therefore,
our data suggests that the ``internalization signal'' of GHRs
is located between amino acid 337 and 387 (from pGHR-TR2 to TR3, 50
amino acids), which is adjacent to the proline-rich region.
In
conclusion, we have found a 40-amino acid region of the pGHR
cytoplasmic domain(477-516), which is essential for the induction
of pp95 tyrosine phosphorylation in cells that express pGHR following
GH treatment. The results suggest that the pp95 induction by GH is not
only dependent on the existence of this 40-amino acid segment in the
GHR cytoplasmic domain, but it also requires the formation of
GHGHR complex that may be a receptor dimer or a yet to be defined
complex. Following the terminology of Box 1 and Box 2 for other members
of the cytokine receptor superfamily(19, 20) , we
refer to this 40-amino acid region as Box 3 (Fig. 4B).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X54429[GenBank].