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(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
Growth hormone (GH) ( 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 GH 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). [
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(
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
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
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 GH
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 GH 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-TR
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 GH 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. ( 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
GH
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
X54429[GenBank].
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6261-6266
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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 signal transduction pathway(s).
-
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.Construction of pGHR cDNAs Which Encode pGHR Truncation
Analogs
The full-length pGHR cDNA coding sequence ((30) , EMBL X54429) was inserted into a mammalian expression
vector, pMet-IG7, at the XbaI and EcoRI restriction
sites. Transcription of pGHR cDNA was directed by the mouse
metallothionein I (mMet-I) transcription regulatory sequence and
employs the bovine GH (bGH) poly(A) addition signal. The parental
plasmid, pMet-IG7, possesses the bacteriophage f1 intergenic region,
which enables it to produce single-stranded DNA. Briefly, the
1.9-kilobase DNA fragment, which contains the full-length pGHR cDNA was
excised from plasmid pGHR15 (31) by XbaI and EcoRI partial digestion, separated by 1% agarose gel
electrophoresis, and purified by Elutip-D column (Schleicher &
Schuell). The pGHR cDNA fragment was ligated into XbaI- and EcoRI-digested pMet-IG7. The ligation mixture was transformed
into E. coli JM110. Bacterial colonies were grown on LB plates
containing 100 µg/ml ampicillin. Colonies containing the pGHR cDNA
inserts were screened by restriction digestion analysis. The resulting
plasmid containing the pGHR cDNA was termed pMet-IG-pGHR.
Single-stranded DNA from pMet-IG-pGHR was prepared by retransforming E. coli CJ236 with pMet-IG-pGHR followed by isolation of the
single-stranded DNA, aliquoting, and freezing at -20 °C as
described previously(32) .
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.Establishment of Stable Cell Lines That Express pGHR
Truncation Analogs
pMet-IG-pGHR:TR1, TR2, TR3, TR4, TR5, and TR6
DNAs were used to establish stable MLC lines that permanently express
mutated pGHR cDNA. MLC were maintained in Dulbecco's modified
Eagle's medium containing 10% Nu-serum I culture supplement
(Becton Dickinson), 10 µg/ml gentamicin (Life Technologies, Inc.)
at 37 °C in humidified atmosphere containing 5% CO
. A
previously described strategy was employed for
transfection(34, 35) . Briefly, MLC were
co-transfected with the mutated plasmid DNAs and pRSV-Neo DNA. The
transfected cells were cultured with Dulbecco's modified
Eagle's medium supplemented with 10% calf serum and G418 (400
µg/ml). G418-resistant colonies were propagated in 25-cm tissue culture flask. Expression of pGHR-TR analogs mRNA was
determined either by RNA slot blotting or ribonuclease protection
assays. Each cell line was named for the truncation analogs that it
expressed, e.g. pGHR-TR1 through pGHR-TR6.Receptor Binding Analyses of the pGHR-TR Stable Cell
Lines
To examine the total GHR binding sites existed in pGHR-TRs
cells, maximum displacement experiments (with or without excess amount
(2 µg/ml) of unlabeled pGH)) were employed. pGHR-WT cells and MLC
were plated in six-well tissue culture plates and propagated to
confluence (1 
10
cells). Approximately, 60,000
cpm of I-pGH (100 µCi/µg) was used in each assay.
Experiments were performed using methods previously
described(31) . Specific binding was calculated as the
difference between total binding and nonspecific binding. All
determinations were made based upon the mean value of three
experiments. Each experiment was carried out in triplicate.Cross-linking Studies
pGHR-TRs cells, pGHR-WT, and
parental MLC (31) were propagated in 25-cm tissue
culture flasks. A modification of a previously reported cross-linking
procedure was employed(31) . Briefly, the cells were incubated
with 100,000 cpm of I-hGH in phosphate-buffered saline,
0.1% bovine serum albumin at room temperature for 2 h and then washed
with phosphate-buffered saline, 0.1% bovine serum albumin. pGHR or
pGHR-TRs and I-hGH were then cross-linked at room
temperature for 60 min with BS
(Pierce, Rockford, IN). An
excess amount of unlabeled pGH (2 µg/ml) was used in competition
reactions. Cells were solubilized in SDS-polyacrylamide gel
electrophoresis sample buffer, sonicated for 15 seconds and subjected
to 4-12% gradient SDS-polyacrylamide gel
electrophoresis(36) . After electrophoresis, the gel was dried
and exposed to x-ray film.pp95 Induction Assay in Cell Lines That Express pGHR
Truncation Analogs
To evaluate the biological function of the
pGHR truncation analogs in regard to induction of pp95 following GH
treatment, pGHR-TRs cells as well as pGHR-WT cells were propagated in
six-well tissue culture plates to confluence. GH in the medium was
removed by incubating the cells in serum-free medium overnight. The
cells were treated with or without 500 ng/ml of pGH at 37 °C for 10
min. Subsequently, the pp95 assay was performed as described previously
using the phosphotyrosine antibody, PY20 (ICN, Costa Mesa,
CA)(28) .
Construction of pGHR Truncation
Analogs
Intracellular pGHR truncation analogs were generated by
oligonucleotide directed mutagenesis (see ``Materials and
Methods''). Four colonies from each transformation reaction were
propagated, and DNA was extracted and sequenced. One clone containing
the predicted nucleotide sequence was selected for each GHR truncation.
pGHR-TR2 was generated by deletion of the intracellular sequence
following the EcoRI sites. This deletion results in a loss of
302 amino acids of the pGHR. In summary, six intracellular domain
truncations of pGHR cDNA were generated, and clones were selected:
pGHR-TR1 contains pGHR amino acid residues 1-291 or has residues
292-638 deleted (); pGHR-TR2 (1-336 or
337-638); pGHR-TR3 (1-387 or 388-638);
pGHR-TR4 (1-476 or 477-638); pGHR-TR5 (1-516 or
517-638); and pGHR-TR6 (1-558 or 559-638)
and are shown schematically (see Fig. 4A).
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.
Expression of pGHR-TR Analogs in
MLC
G418-resistant MLC pools transfected individually with the
various pGHR-TR plasmids were grown in 25-cm tissues
culture flasks, and total cellular RNA was extracted for expression
analyses. Both RNA slot blot and ribonuclease protection analysis
revealed that all of the pGHR-TR cell lines express pGHR-TR analogs
mRNA at comparable levels with that of the pGHR-WT (data not shown). To
further determine the binding capacity of pGHR-TRs to GH, maximum
displacement GH binding experiments were performed. The results are
summarized in Fig. 1. All of the pGHR-TRs cells were able to
bind to I-pGH. The specific binding capacity correlated
with RNA expression levels in pGHR-WT and pGHR-TR1 through TR4.
However, the binding capacity of pGHR-TR1 and TR2 is much greater than
other pGHR-TR analogs (Fig. 1) despite similar levels of pGHR
RNA in the cell lines.
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'').
Cross-linking Studies of pGHR Truncation
Analogs
To assure that pGHR on the surface of pGHR-TRs cells
represents the truncated pGH receptors, cross-linking studies were
performed, and results are shown in Fig. 2. In Fig. 2, lanes 1-4 and 9 and 10 represent
prolonged exposures (48 h) of the gel. No signal was observed in the
parental MLC in either the absence or presence of cold pGH (Fig. 2, lanes 1 and 2). A radiolabeled band
with estimated molecular mass of 140 kDa was observed in pGHR-WT cells
that could be specifically competed by unlabeled pGH (Fig. 2, lanes3 and 4). This band apparently
represents the I-hGHpGHR complex. When the
molecular mass of hGH (22 kDa) was subtracted from the complex, the
molecular mass of the pGHR was found to be approximately 118 kDa, which
agrees with previous reports(31) . Radiolabeled bands were
observed in pGHR-TRs cell lines (Fig. 2, lanes5, 7, 9, 11, 13, and 15) and were competed by unlabeled pGH (Fig. 2, lanes6, 8, 10, 12, 14, and 16). The apparent molecular mass of the
observed bands in pGHR-TRs cells decreased sequentially as expected for
the deletion analogs. The estimated molecular masses of the various
pGHR-TR analogs were as follows: pGHR-TR6, 126 kDa; pGHR-TR5, 120 kDa;
pGHR-TR4, 117 kDa; pGHR-TR3, 98 kDa; pGHR-TR2, 81 kDa; and pGHR-TR1, 74
kDa. Subtraction of the molecular mass of hGH from these complexes
results in pGHR-TR1, TR2, TR3, TR4, TR5, and TR6 possessing approximate
molecular masses of 52, 59, 76, 95, 98, and 104 kDa, respectively.
GHR 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).pp95 Induction Assay in pGHR-TRs Cells
To examine
the effects of truncations of pGHR intracellular domain in GHGHR
signal transduction, the ability of GH to induce
tyrosine-phosphorylated proteins with a molecular mass of 95,000
daltons (pp95) was examined. The results were shown in Fig. 3.
As described previously, basal levels of pp95 induction were observed
in GH-treated parental MLC (Fig. 3, lane2).
GH treatment induced pp95 in cells that express pGHR-WT (28) (Fig. 3, lane4). The pp95 signal
was induced to a similar level in pGHR-TR5 and TR6 cells. No pp95
induction was observed in pGHR-TR1, TR2, TR3, and TR4 cells after GH
treatment. These results are summarized in Fig. 4A.
GHR Intracellular Domain Structure and pp95
Induction
Employing a cDNA mutagenesis/protein truncation
approach for the intracellular region of the pGHR, our results reveal a
region of 40 amino acids (from 477 to 516) that is essential for GH
induction of pp95. Alignment of the amino acid sequence of the 40-amino
acid segment of pGHR with the corresponding regions of GHR from other
species shows a relatively high degree of amino acid identity (Fig. 4B). Importantly, this 40-amino acid domain is
distinct from the ``proline-rich'' region or Box 1 described
previously among all the GHRs as well as prolactin and other cytokine
receptors(17, 18, 19, 20) . It has
been demonstrated that the proline-rich region plays a critical role in
mediating GH signal transduction(19, 20) .GHR
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 GHGHR signal transduction.GHmGHR 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-TRGHmGHR heterodimers would also not transduce the GH
signal. Together with the observations of pGHRGHpGHR
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.GH and GHR Complex Dimerization and
Internalization
In the present study, we used an in vitro mutagenesis approach to generate pGHR cytoplasmic domain
truncation analogs. Transfection of MLC with these mutated pGHR cDNAs
resulted in at least a 10-100 fold increase of I-pGH binding at the cell surface (Fig. 1). The
truncations of the pGHR cytoplasmic domain did not affect the binding
ability of pGHR to pGH. We have previously shown that the binding
affinity of pGHR-TR1, which possesses the shortest (3 amino acids)
intracellular portion of pGHR, was found to be similar to wild-type
pGHR, i.e. 1.0 10
M (data not shown). All other pGHR truncations, which possess longer
intracellular domains than pGHR-TR1, did not change GH binding
affinity.GHR 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 GHRGHGHR 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-TR1pGHpGHR-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 GHGHR complex than a GHRGHGHR
complex. We hypothesize that these bands may represent three possible
GHGHR complexes. 1) The first is a GHRGHGHGHR
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
GHRGHGHR complex. 2) The second is a
GHRGHGHRX complex, whereas X represents an unknown protein molecule with a molecular mass of
25 kDa associated with the GHRGHGHR complex. 3) And
finally is a GHRGHY 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 GHBPGHGHBP
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 GHGHR
interaction(42) . However, it is possible that various forms of
GHGHR dimer exist and mediate different GH signals. More detailed
studies are required to test these hypothetical models. Regardless of
the GHGHR complex model, it was shown that the truncations of
pGHR cytoplasmic domain do not affect the ability of GHR to form
GHGHR complexes. The absence of the GHGHR dimer complex in
pGHR-TR6, TR5, TR4, and TR3 cells was probably due to fewer GH binding
sites on these cells.)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.GHR 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).
))
We thank Dr. Paul Harding for the valuable suggestions
and discussion about the results of present work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 J. J. Kopchick, C. Parkinson, E. C. Stevens, and P. J. Trainer Growth Hormone Receptor Antagonists: Discovery, Development, and Use in Patients with Acromegaly Endocr. Rev., October 1, 2002; 23(5): 623 - 646. [Abstract] [Full Text] [PDF]
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 Y. Li, B. Kelder, and J. J. Kopchick Identification, Isolation, and Cloning of Growth Hormone (GH)-Inducible Interscapular Brown Adipose Complementary Deoxyribonucleic Acid from GH Antagonist Mice Endocrinology, July 1, 2001; 142(7): 2937 - 2945. [Abstract] [Full Text] [PDF]
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 K. Iida, Y. Takahashi, H. Kaji, M. O. Takahashi, Y. Okimura, O. Nose, H. Abe, and K. Chihara Functional Characterization of Truncated Growth Hormone (GH) Receptor-(1-277) Causing Partial GH Insensitivity Syndrome with High GH-Binding Protein J. Clin. Endocrinol. Metab., March 1, 1999; 84(3): 1011 - 1016. [Abstract] [Full Text]
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T.-W. L. Gong, D. J. Meyer, J. Liao, C. L. Hodge, G. S. Campbell, X. Wang, N. Billestrup, C. Carter-Su, and J. Schwartz Regulation of Glucose Transport and c-fos and egr-1 Expression in Cells with Mutated or Endogenous Growth Hormone Receptors Endocrinology, April 1, 1998; 139(4): 1863 - 1871. [Abstract] [Full Text] [PDF]
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H. Kaji, O. Nose, H. Tajiri, Y. Takahashi, K. Iida, T. Takahashi, Y. Okimura, H. Abe, and K. Chihara Novel Compound Heterozygous Mutations of Growth Hormone (GH) Receptor Gene in a Patient with GH Insensitivity Syndrome J. Clin. Endocrinol. Metab., November 1, 1997; 82(11): 3705 - 3709. [Abstract] [Full Text] [PDF]
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 P. Domanski, E. Fish, O. W. Nadeau, M. Witte, L. C. Platanias, H. Yan, J. Krolewski, P. Pitha, and O. R. Colamonici A Region of the beta Subunit of the Interferon alpha Receptor Different from Box 1 Interacts with Jak1 and Is Sufficient to Activate the Jak-Stat Pathway and Induce an Antiviral State J. Biol. Chem., October 17, 1997; 272(42): 26388 - 26393. [Abstract] [Full Text] [PDF]
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B. C. Xu, X. Wang, C. J. Darus, and J. J. Kopchick Growth Hormone Promotes the Association of Transcription Factor STAT5 with the Growth Hormone Receptor J. Biol. Chem., August 16, 1996; 271(33): 19768 - 19773. [Abstract] [Full Text] [PDF]
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L. H. Hansen, X. Wang, J. J. Kopchick, P. Bouchelouche, J. H. Nielsen, E. D. Galsgaard, and N. Billestrup Identification of Tyrosine Residues in the Intracellular Domain of the Growth Hormone Receptor Required for Transcriptional Signaling and Stat5 Activation J. Biol. Chem., May 24, 1996; 271(21): 12669 - 12673. [Abstract] [Full Text] [PDF]
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P. A. Harding, X. Wang, S. Okada, W. Y. Chen, W. Wan, and J. J. Kopchick Growth Hormone (GH) and a GH Antagonist Promote GH Receptor Dimerization and Internalization J. Biol. Chem., March 22, 1996; 271(12): 6708 - 6712. [Abstract] [Full Text] [PDF]
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P. A. Ram, S.-H. Park, H. K. Choi, and D. J. Waxman Growth Hormone Activation of Stat 1, Stat 3, and Stat 5 in Rat Liver J. Biol. Chem., March 8, 1996; 271(10): 5929 - 5940. [Abstract] [Full Text] [PDF]
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