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(Received for publication, September 5, 1995; and in revised form, December
20, 1995) From the
It has previously been shown that a human growth hormone (hGH)
analog, hGH-G120R, acts as a GH antagonist (Chen, W. Y., Wight, D. C.,
Wagner, T. E., and Kopchick, J. J.(1990) Proc. Natl. Acad. Sci. U.
S. A. 87, 5061-5065; Chen, W. Y., White, M. E., Wagner, T.
E., and Kopchick, J. J.(1991) Endocrinology 129,
1402-1408; Chen, W. Y., Chen, N-Y., Yun, J., Wang, X. Z., Wagner,
T. E., and Kopchick, J. J.(1994) J. Biol. Chem. 269,
15892-15897). In this study, we report the ability of hGH and
hGH-G120R to be internalized by GH receptor expressing cells.
Additionally, results of chemical cross-linking experiments revealed
that both native hGH and hGH-G120R form complexes similar in size to
that expected for hGH when bound to recombinant hGH-binding protein
(bp). The molecular mass of the complex was determined to be
approximately 280 kDa which is consistent with multiple receptors
interacting with the ligand. The predominant radiolabeled band detected
was a complex of approximately 140 kDa which probably represents one GH
molecule bound to one GH receptor. The cross-linked complexes were not
detected in the presence of excess unlabeled hGH or hGH-G120R and were
not observed in cells which do not express detectable levels of GH
receptors. Also, GH induced tyrosine phosphorylation of a complex of
proteins of approximately 95 kDa in these cells whereas hGH-G120R did
not. Thus, we have separated the hGH or hGH-G120R/GHR binding and
internalization capabilities from the ability to stimulate tyrosine
phosphorylation of intracellular proteins.
Growth hormone receptor (GHR) ( Recently,
the crystal structure of hGH with the extracellular domain of the
hGH-binding protein (hGHbp) was solved. It was found to exist as a
hGH It has clearly been demonstrated that the third Similarly, a hybrid receptor containing the extracellular binding
domain of the hGHR linked to the transmembrane and intracellular
domains of the murine granulocyte colony-stimulating factor receptor
was generated and expressed in a myeloid leukemia cell line, FDC-P1 (34) . Treatment of these cells with hGH resulted in cellular
proliferation. However, treatment of these cells with hGH-G120R, which
contains a functional site 1 but a sterically blocked site 2, failed to
promote cellular proliferation. Therefore, it was suggested that
hGH-G120R acted as a GH antagonist, presumably by its inability to
dimerize GHRs(34) . It has recently been reported that hGH
down-regulates GHRs in IM-9 lymphocytes whereas hGH-G120R does
not(30) . This result may imply that hGH and hGH-G120R are
``recognized'' differently by the cell and suggests that
hGH-G120R is not internalized following binding to GHRs. In order to
test the ability of hGH-G120R to dimerize and internalize, hGH-G120R
was purified and iodinated. The iodinated forms of hGH and hGH-G120R
were analyzed by binding and cross-linking to cells expressing the
GHRs. Additionally, the ability of the GH antagonist to be internalized
was determined.
Similarly, a pGHR truncation analog consisting of amino
acids 1-291, pGHR-TR1(41) , was chemically cross-linked
to
Figure 1:
Internalization time course of
Figure 2:
Cross-linking assay of hGH and hGH-G120R
to the pGHR. Cells were plated in 100-mm tissue culture dishes, grown
to confluence, depleted of GH in DMEM minus serum, and incubated with
A radiolabeled band with
a molecular mass of approximately 75 kDa was observed for pGHR-TR1 when
cross-linked to either
Figure 3:
Autoradiograph of receptor cross-linking
study on pGHR-TR1 cells. The pGHR-TR1 cells were grown and treated with
Figure 4:
pp95 induction assay on pGHR-W10 cells
that express the pGHR. Cells were plated in six-well culture plates. GH
in the medium was removed by incubating the cells in serum-free medium
overnight. Subsequently, cells were treated without or with hGH,
hGH-G120R, or pGH at 37 °C for 10 min and processed as described
under ``Materials and Methods.'' Lanes 1-4 represent no treatment, hGH, hGH-G120R, and pGH treatment,
respectively. The arrow on the left indicates the
position of pp95.
It has been established that E. coli-derived hGHbp
is able to form a dimer when incubated with
GH(4, 29) . This dimeric complex consists of one hGH
molecule bound to two hGHbp with hGH containing two binding sites for
the hGHbp. It was proposed that GH site 1 binds with one GHbp molecule
and then a second GHbp interacts with GH site 2(29) . De Vos et al.(4) have demonstrated that the hGH site 2 is
found on the exposed sides of helices 1 (amino acids Phe GH treatment of NIH
3T3-F442A fibroblasts resulted in tyrosine phosphorylation of GHRs as a
result of association with an associated tyrosine kinase, JAK2, a
non-receptor associated tyrosine
kinase(13, 14, 18, 20) . It has been
identified as a GH-activated tyrosine kinase responsible for
self-phosphorylation and tyrosine phosphorylation of GHRs and possibly
other intracellular proteins(20, 42) . A
95-96-kDa protein (pp95) of unknown identity has been shown to be
induced in MLCs stably transfected with the pGHR (pGHR-W10 cells) and
3T3-F442A cells upon treatment with physiological concentrations of GH (21, 38, 41, 43) . Additionally, the
GH antagonists, bGH-M8 and hGH-G120R, did not induce tyrosine
phosphorylation of pp95(3, 21) . These results suggest
that GH-induced tyrosine phosphorylation of pp95 plays an integral role
in GH signal transduction. GH treatment of a human lymphocyte cell
line (IM-9), which contain endogenous hGHRs, results in tyrosine
phosphorylation of two proteins with molecular masses of approximately
93 and 120 kDa(19) . Additionally, treatment of IM-9 cells with
hGH analogs (nM concentrations) directed toward site 1 (K172A, F176A)
and site 2 (G120R) were unable to stimulate tyrosine phosphorylation of
the 93-kDa protein but stimulated low level tyrosine phosphorylation of
the 120-kDa protein(19) . Incubation of IM-9 lymphocytes with
0.5 nM recombinant hGH and increasing amounts of hGH-G120R
antagonized the ability of hGH to stimulate tyrosine phosphorylation.
It was concluded that hGH-G120R antagonized tyrosine phosphorylation
due to its inability to dimerize because of a defective site 2. High
concentrations (µM) of hGH inhibited tyrosine
phosphorylation, presumably by inhibiting dimer formation and favoring
the monomeric form (e.g. GH It has been established that Our studies were directed at
determining the ability of a GH antagonist, hGH-G120R, to promote GHR
dimerization and to be internalized by use of cells which express GHRs.
We hypothesized that we would see receptor dimerization and
internalization with hGH but would not detect receptor dimerization and
internalization with hGH-G120R. Surprisingly, both Additionally, we report the ability of hGH and the hGH antagonist,
hGH-G120R, to dimerize in what appears to be a
(GH) Additionally, we detected a 70-kDa band, which may represent the
GH Together, our results indicate that the GH antagonist, hGH-G120R, is
able to bind, dimerize, and internalize GHRs in a similar manner as
hGH. However, the dimeric complex formed with hGH-G120R is not
functional as is the hGH:GHR dimer. Perhaps the hGH-G120R
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 6708-6712
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)activation is
mediated by the binding of GH. The GH/GHR interaction ultimately
results in alterations of lipid, nitrogen, mineral, and carbohydrate
metabolism as well as cellular
differentiation(5, 6, 7, 8, 9, 10, 11, 12) .
The mechanism(s) by which these biological signals are elicited are not
yet fully understood. However, it has been demonstrated that a GH/GHR
activated tyrosine
kinase(13, 14, 15, 16, 17, 18, 19, 20, 21) ,
protein kinase C(22, 23) , and mitogen-activated
protein or extracellular signal-regulated kinase (24, 25, 26, 27, 28) are
involved in the intracellular signaling mechanism of GH.
(hGHbp)
complex(4, 29) .
Additionally, GH dependent dimerization of this Escherichia coli expressed hGHbp was found to occur sequentially(29) . In
this model, hGH was shown to bind one hGHbp molecule through site 1 on
hGH and then a second hGHbp molecule binds through site two of hGH
subsequently establishing a hGH(hGHbp) complex. It
was suggested that formation of the 1-ligand2-receptor dimeric
complex may be important in the GH-induced signal transduction
system(29, 30) . This type of GH/GHR signal
transduction mechanism is analogous to that used by many tyrosine
kinase receptors, such as epidermal growth factor and platelet-derived
growth factor, in which binding of one hormone molecule to its receptor
is thought to induce formation of a dimer, through a
2-hormone2-receptor complex(31) . Binding of these growth
factors to their cognate receptors activate tyrosine kinases and
receptor autophosphorylation. Therefore, receptor dimerization may be a
critical step in mediating biological activities for a number of growth
factors.
-helix
of GH is critical for its biological
activity(1, 2, 3) . In a structure-function
study of this helical region, bGH-Gly and hGH-Gly
were identified as amino acids critical for growth
promotion(2, 3) . Transgenic mice expressing these GH
antagonist genes possessed a dwarf
phenotype(1, 32, 33) . In fact, substitution
of several amino acids (except alanine) of this Gly residue resulted in
GH antagonists which were found to be active both in vitro and in vivo(2, 3, 12, 33) .
Iodination of Hormones
Human GH and a GH
antagonist, hGH-G120R, were purified as described previously (35) and were labeled with NaI by the
lactoperoxidase method to a specific activity of 80-105
µCi/µg(36) . Briefly, 1.0 mCi of Na
I was
added to 1.0 mg of hGH or hGH-G120R. Lactoperoxidase (10 µg
dissolved in 10 µl of 0.4 mol/liter acetate buffer, pH 5.6) and
H
O (5 µl of 1.76 mmol/liter) were then
added. After 30 min, the reaction was terminated by the addition of 100
µl of transfer buffer (0.47 mol/liter sucrose, 0.06 mol/liter KI,
sodium azide 0/02%, pH 7.6). Radiolabeled hGH was then separated by
Sephadex G-100 chromatography.Radioimmunoassay
A radioimmunoassay hGH kit
(Hybritech, San Diego, CA) was used in order to determine the
concentrations of hGH and hGH-G120R. However, only hGH was detected by
the radioimmunoassay kit (3) . hGH-G120R was not detectable by
radioimmunoassay but was recognized by immunoblotting with a polyclonal
hGH antibody (kindly provided by Eli Lilly, IN). Therefore, the epitope
for the monoclonal antibody from the Hybritech radioimmunoassay kit may
be near Gly or in the third helical region. A second hGH
radioimmunoassay kit (Nichols Institute Diagnostics) was able to
recognize both hGH as well as hGH-G120R in order to determine GH
concentrations(3) .
GH/GHR Internalization
An acid extraction
procedure (37) was used to differentiate surface bound I-GH and internalized
I-GH. Briefly, mouse
L cells (MLCs), pGHR-W10 (MLCs which express the porcine GHR), and IM-9
cells were maintained in DMEM or RPMI 1640 plus 10% fetal bovine serum.
For the internalization studies the cells were grown to confluence in
six-well 35-mm culture plates and depleted of GH in DMEM for 16 h as
described previously(38, 39) . One ml of ice-cold
phosphate-buffered saline, 1% bovine serum albumin solution containing
100,000 cpm of
I-hGH or
I-hGH-G120R was
added to each culture dish which were pre-chilled on ice to prevent
internalization. We have previously established that the binding
affinity of hGH and hGH-G120R for the pGHR are approximately 1.0
nM. (
)The cells were allowed to reach equilibrium
at 4 °C for 24 h, after which the unbound ligand was removed by
rinsing (three times) with ice-cold phosphate-buffered saline/1% bovine
serum albumin. The cells were then incubated at 37 °C for various
time intervals (0, 5, 10, 15, 30, 45, 60, 90, and 120 min) and exposed
to 0.5 ml of ice-cold solution of 50 mM glycine, 150 mM NaCl, pH 3.0, for 5 min. This solution was collected and
represented the surface bound ligand. The cell monolayer was washed
with 0.5 ml of 50 mM glycine, 150 mM NaCl, pH 3.0,
and added to the previous collection. The cell monolayer was then
solubilized with 0.5 ml of 0.1 N NaOH, 1% SDS and represented
the internalized ligand. The radioactivity of the surface bound and
internalized ligand were measured using a -counter (Beckman 5500).
GH/GHR Cross-linking
I-hGH and
I-hGH-G120R were chemically cross-linked to MLCs,
pGHR-W10 and pGHR-TR1 cells. Briefly, cells were plated in 100-mm
culture dishes and grown to confluence. Cells were depleted of GH
overnight in DMEM minus serum. Cells were then incubated in
phosphate-buffered saline, 1% bovine serum albumin containing 300,000
cpm of either
I-hGH or
I-hGH-G120R for 2 h
at 25 °C with gentle agitation. Additionally, unlabeled hGH and
hGH-G120R (500 ng/ml) were each used to specifically compete with the
I-hGH and
I-hGH-G120R. The bound
radiolabeled ligand was chemically cross-linked to the pGHR with 2.5
mM bis(sulfosuccinimidyl)suberate, BS
(Pierce),
for 90 min at 4 °C. The cells were then scraped and homogenized in
0.25 M sucrose in 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, containing a mixture of the following protease inhibitors:
phenylmethylsulfonyl fluoride (Sigma), tosyl lysine chloromethyl ketone
(Sigma), and tosylamide phenylethylchloromethyl ketone (Sigma) at a
final concentration of 2 mM each. The homogenate was
centrifuged at 14,000 
g for 15 min. The membrane
fraction was suspended in loading buffer (625 mM Tris, 1% SDS,
15% glycerol, 1% 
-mercaptoethanol) and subjected to 5-15%
SDS-PAGE(40) . After electrophoresis, the gel was fixed in 10%
methanol and 7.5% glacial acetic acid for 30 min, soaked in
Enlightening (DuPont) for 30 min, dried, and exposed to XAR-II film
(Kodak).I-hGH or
I-hGH-G120R in order to obtain
a more precise determination of the M
of the
GHGHR complex as well as the dimeric complex.GH-induced Protein Tyrosine Phosphorylation
Assays
The cell lines described above were plated in 6-well
tissue culture plates and propagated to confluence. For the protein
tyrosine phosphorylation assays, which we have termed the pp95 assay,
GH in the medium was removed by incubating the cells in serum-free
medium (DMEM) overnight. Subsequently, the pp95 assay was performed as
described previously(21, 39, 41) . Briefly,
cells were treated with or without hGH or hGH-G120R (500 ng/ml) at 37
°C for 10 min. The cells were lysed in SDS-PAGE lysis buffer,
separated by 7% gel electrophoresis, and transferred to a Hybond-ECL
membrane (Amersham). The membrane was blocked with phosphate-buffered
saline, 4% bovine serum albumin solution for at least 6 h and incubated
with a phosphotyrosine antibody, PY20 (ICN, Costa Mesa, CA). The
membrane was developed by the conventional ECL procedure (Amersham).
The signal was visualized by exposure to x-ray film.
Internalization of
In order to determine
whether hGH-G120R was able to be internalized, experiments were
conducted using cell lines which express GHR, i.e. pGHR-W10,
and human IM-9 lymphocytes. Previous results have shown that pGHR-W10
cells were able to internalize approximately 85% of the specific bound I-hGH and
I-hGH-G120R
I-pGH(38, 39) . Results of the
internalization assay of hGH and the hGH antagonist are shown in Fig. 1(A and B). Both the
I-hGH
and
I-hGH-G120R were internalized by pGHR-W10 cells and
IM-9 lymphocytes with radioactivity increasing from 5 to 65% within 30
min at 37 °C. Approximately 85% of the bound radiolabeled ligand
was internalized by 1 h. MLCs did not significantly internalize hGH or
hGH-G120R (data not shown).
I-hGH and
I-hGH-G120R into pGHR-W10 cells (Panel A) and IM-9 lymphocytes (Panel B). Cells were
grown to confluence in six-well culture plates, depleted of serum, and
incubated with 100,000 cpm of
I-hGH or
I-hGH-G120R for 24 h at 4 °C. Subsequently, the cells
were incubated at 37 °C for 0, 5, 10, 15, 30, and 120 min during
which time GH was internalized. Each point on the graph represents the
mean value of three experiments performed in duplicate. See
``Materials and Methods'' for
details.
Cross-linking of
In the presence of I-hGH and
I-hGH-G120R
I-hGH or
I-hGH-G120R and a chemical
cross-linker, BS
, radiolabeled bands with molecular masses
of approximately 70, 140, and 280 kDa were observed in cells expressing
the pGHR (Fig. 2, lanes 5 and 11). These
radiolabeled complexes were not seen in MLCs which do not express pGHRs (Fig. 2, lanes 2-4 and 8-10).
Additionally, the observed complexes were able to be specifically
competed with unlabeled hGH and hGH-G120R (500 ng/ml) (Fig. 2, lanes 6, 7, 12, and 13).
I-hGH or
I-hGHG120R for 2 h. Cells were
cross-linked with BS
, homogenized, and separated by
SDS-PAGE (5-15%). MLC cross-linked with I-hGH
without competing GH (lane 2), with unlabeled hGH (500 ng/ml, lane 3), and with unlabeled hGH-G120R (500 ng/ml, lane
4) are shown. Lanes 5-7 represent the same
conditions as described previously but in pGHR-W10 cells. Lanes
8-10 represent MLC cross-linked with
I-hGH-G120R, without competing GH, with unlabeled hGH,
and with unlabeled hGH-G120R, respectively. Lanes 11-13 represent identical conditions as lanes 8-10, but
in pGHR-W10 cells. Lanes 1 and 14 represent molecular
weight markers. The arrows on the left represent
radiolabeled bands specifically competed with excess unlabeled hGH or
hGH-G120R.
I-hGH or
I-hGH-G120R (Fig. 3, lanes 5 and 11). Upon subtraction of
the molecular mass of hGH, 22 kDa, from the complex, pGHR-TR1 was found
to possess a mass of approximately 53 kDa. Additionally, a larger
radiolabeled complex of 150 kDa was observed with pGHR-TR1 upon
cross-linking to
I-hGH or
I-hGH-G120R. Both
the 75- and 150-kDa complexes were able to be specifically competed
with unlabeled hGH or hGH-G120R (Fig. 3, lanes 6 and 12). Also, a radiolabeled complex of approximately 140 kDa was
detected in pGHR-W10 cells (Fig. 3, lanes 3 and 9) that were specifically competed with excess hGH or
hGH-G120R (Fig. 3, lanes 4 and 10). No
complexes were seen in MLC (Fig. 3, lanes 1, 2, 7, and 8).
I-hGH and
I-hGH-G120R (lanes 5 and 11) and in the presence of excess hGH or hGH-G120R (lanes
6 and 12). Additionally, pGHR-W10 cells were treated with
I-hGH or
I-hGH-G120R (lanes 3 and 9) and in the presence of excess hGH or hGH-G120R (lanes 4 and 10). MLCs were also incubated with
I-hGH or
I-hGH-G120R l (lanes 1 and 7) and in the presence of excess hGH or hGH-G120R (lanes 2 and 8). Lower and upper arrows on the left indicate the migration of the putative GH:GHR
monomer and dimer, respectively.
pp95 Induction Assay
The ability of hGH or
hGH-G120R to induce tyrosine phosphorylated proteins of approximately
95 kDa in pGHR-W10 cells was examined. hGH and pGH were able to induce
pp95 in pGHR-W10 cells (Fig. 4, lanes 2 and 4,
respectively). No pp95 induction was observed when pGHR-W10 cells were
incubated with hGH-G120R (Fig. 4, lane 3).
,
Ile
, and Arg
) and 3
(Asp)(4) . Additionally, hGH-G120R, which retains
a functional site 1 but a sterically blocked site 2, has been shown to
inhibit proliferation of FDC-P1 cells transfected with a hybrid
receptor that contained the extracellular domain of the hGHR linked to
the transmembrane and intracellular domain of the murine granulocyte
macrophage colony-stimulating factor receptor(34) . It was
hypothesized that once hGH-G120R is bound to its receptor, it could not
dimerize and induce a GH signal. Therefore, these results suggest that
GHR dimerization is critical for GH-induced signal
transduction(19, 34) .
GHR
complex)(19, 34) . It should be noted that hGH-G120R
treatment of IM-9 lymphocytes was able to stimulate tyrosine
phosphorylation of the 120-kDa protein(19) . This protein may
possibly be JAK2 (20) which would imply that a GH antagonist
can bind and activate JAK2 while not activating subsequent signal
transduction events.I-hGH is internalized by IM-9 cells at physiological
temperatures(44, 45) . The fate of the internalized
hormone is not well understood; however, it has been shown that 25% of
the hormone is released into the extracellular environment (46) . The fate of the GHR is either lysosomal degradation or
exocytosis into the medium as a soluble GHbp. Ilondo et al.(30) proposed a model of GH:GHR internalization followed by
exocytosis of the extracellular domain of the full-length GHR, which is
the equivalent of the GHbp. Therefore, internalization may be a key
pathway in generation of GHbp.
I-labeled hGH and hGH-G120R were able to be internalized
following binding to GHR. Our results indicated that 75% of the bound
hormone was internalized within 40 min. Therefore, it appears that
there is no significant difference in the internalization process
between hGH and hGH-G120R (refer to Fig. 1). It may be that 1)
GHR internalization is constitutive independent of GH binding or 2)
once GH is bound to its receptor, the GH
GHR complex is
internalized. Regardless of the internalization pathway, we found that
hGH-G120R was internalized in a manner similar to that of hGH. (GHR) complex, unlike the
GH(GHR) complex previously described(34) .
Three major bands were observed upon cross-linking analysis of I-hGH or
I-hGH-G120R with the pGHR with
molecular masses corresponding to 70, 140, and 280 kDa (refer to Fig. 2). These radiolabeled complexes were specifically competed
with excess unlabeled hGH or hGH-G120R. The predominant radiolabeled
band was the 140 kDa protein, which may correspond to the GH
GHR
monomeric complex. It has been determined that the molecular mass of
the pGHR was approximately 118 kDa(38, 39) .
Cross-linking of this protein with either I-hGH or
I-hGH-G120R (molecular mass of 22 kDa) would corresponded
to the monomeric complex (GH
GHR) of 138 kDa. The less abundant,
higher molecular mass form of the cross-linked complex is approximately
280 kDa which is consistent with a dimeric complex of two GH molecules
bound to two GHR molecules (GH)(GHR).
Evidence leading to this conclusion is based on cross-linking of I-hGH to a truncated form of the pGHR, pGHR-TR1, which is
composed of the pGHR extracellular domain, transmembrane domain, and 3
amino acids of the intracellular domain. Upon cross-linking of GH to
these cells, radiolabeled complexes of 75 and 150 kDa were observed
(refer to Fig. 3). We believe that the 75-kDa complex is the
monomeric complex, one GH molecule and one truncated GHR molecule.
Additionally, the 150-kDa complex is consistent with a complex of two
GH molecules and two truncated GHR molecules (2GH:2GHR). If the complex
were in the ratio of 1GH:2GHR, based on the hypothesis of Fuh et
al.(34) , the expected molecular mass would be 130 kDa.
Similarly, if the full-length pGHR cross-linked to hGH were in the
ratio of one ligand and two receptors, the expected molecular mass
would be 260 kDa and not the 280 kDa complex observed in Fig. 2.
An alternative explanation for the unexpected molecular masses of the
cross-linked complexes may be that the presence of 1%
-mercaptoethanol in the SDS-PAGE buffer is affecting migration.
Previous results have demonstrated that cross-linking of I-hGH to rat adipocytes GHRs resulted in a complex of
127-135 kDa when in the presence of 0.8-1.2 mM dithiothreitol(47) . However, when dithiothreitol
concentrations were lowered, (0-0.4 mM), the GH
GHR
complex migrated at approximately 116-125 kDa(47) .
Therefore, the reduction of GHR disulfide bonds may possibly be
responsible for the larger cross-linked complexes seen in our results. GHbp complex. The GHbp may be the extracellular domain of the
full-length pGHR generated by proteolytic cleavage. The 246-amino acid
extracellular domain would produce a protein of approximately 25 kDa.
We and others have shown that the GHR is heavily glycosylated on
asparagine residues(39, 48) . N-Linked
glycosylation comprised approximately 24 kDa of the total molecular
mass of the pGHR (39) . Therefore, cross-linking of GH or G120R
to the extracellular domain (i.e. GHbp) would result in a
complex of approximately 70 kDa which can be seen in Fig. 2. GHR
dimeric complex is not biologically equivalent to the native GH:GHR
dimer and may be unable to associate with yet to be discovered
intracellular elements in order to elicit GH's signal
transduction system. Another possible explanation for the separation of
GH binding and internalization from subsequent intracellular signaling
is the hypothesis that the intracellular ``trafficking''
pattern is different for hGHreceptor versus hGH-G120Rreceptor complexes. Ultimately, the data suggests
that GHR internalization and dimer formation are not positively
correlated with GH induced intracellular signaling.
))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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J.-C. Lu, P. Scott, G. J. Strous, and L. A. Schuler Multiple Internalization Motifs Differentially Used by Prolactin Receptor Isoforms Mediate Similar Endocytic Pathways Mol. Endocrinol., November 1, 2002; 16(11): 2515 - 2527. [Abstract] [Full Text] [PDF]
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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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 J. Gent, P. van Kerkhof, M. Roza, G. Bu, and G. J. Strous Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis PNAS, July 23, 2002; 99(15): 9858 - 9863. [Abstract] [Full Text] [PDF]
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 S. J. Frank Minireview: Receptor Dimerization in GH and Erythropoietin Action--It Takes Two to Tango, But How? Endocrinology, January 1, 2002; 143(1): 2 - 10. [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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R. J. M. Ross, K. C. Leung, M. Maamra, W. Bennett, N. Doyle, M. J. Waters, and K. K. Y. Ho Binding and Functional Studies with the Growth Hormone Receptor Antagonist, B2036-PEG (Pegvisomant), Reveal Effects of Pegylation and Evidence That It Binds to a Receptor Dimer J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1716 - 1723. [Abstract] [Full Text]
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K. K. Kaulsay, T. Zhu, W. F. Bennett, K.-O. Lee, and P. E. Lobie The Effects of Autocrine Human Growth Hormone (hGH) on Human Mammary Carcinoma Cell Behavior Are Mediated via the hGH Receptor Endocrinology, February 1, 2001; 142(2): 767 - 777. [Abstract] [Full Text] [PDF]
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 A. Bartke, V. Chandrashekar, D. Turyn, R. W. Steger, L. Debeljuk, T. A. Winters, J. A. Mattison, N. A. Danilovich, W. Croson, D. R. Wernsing, et al. Effects of Growth Hormone Overexpression and Growth Hormone Resistance on Neuroendocrine and Reproductive Functions in Transgenic and Knock-Out Mice Experimental Biology and Medicine, November 1, 1999; 222(2): 113 - 123. [Abstract] [Full Text]
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V. A. Barr, K. Lane, and S. I. Taylor Subcellular Localization and Internalization of the Four Human Leptin Receptor Isoforms J. Biol. Chem., July 23, 1999; 274(30): 21416 - 21424. [Abstract] [Full Text] [PDF]
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M. Maamra, J. Finidori, S. Von Laue, S. Simon, S. Justice, J. Webster, S. Dower, and R. Ross Studies with a Growth Hormone Antagonist and Dual-fluorescent Confocal Microscopy Demonstrate that the Full-length Human Growth Hormone Receptor, but Not the Truncated Isoform, Is Very Rapidly Internalized Independent of Jak2-Stat5 Signaling J. Biol. Chem., May 21, 1999; 274(21): 14791 - 14798. [Abstract] [Full Text] [PDF]
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W. Ruan, L. Powell-Braxton, J. J. Kopchick, and D. L. Kleinberg Evidence That Insulin-Like Growth Factor I and Growth Hormone Are Required for Prostate Gland Development Endocrinology, May 1, 1999; 140(5): 1984 - 1989. [Abstract] [Full Text]
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 E Juarez-Aguilar, F Castro-Munozledo, N. Guerra-Rodriguez, D Resendez-Perez, H. Martinez-Rodriguez, H. Barrera-Saldana, and W Kuri-Harcuch Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules J. Cell Sci., January 9, 1999; 112(18): 3127 - 3135. [Abstract] [PDF]
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J. J. E. Dore Jr., M. Edens, N. Garamszegi, and E. B. Leof Heteromeric and Homomeric Transforming Growth Factor-beta Receptors Show Distinct Signaling and Endocytic Responses in Epithelial Cells J. Biol. Chem., November 27, 1998; 273(48): 31770 - 31777. [Abstract] [Full Text] [PDF]
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H. Qiu, A. Belanger, H.-W. P. Yoon, and H. F. Bunn Homodimerization Restores Biological Activity to an Inactive Erythropoietin Mutant J. Biol. Chem., May 1, 1998; 273(18): 11173 - 11176. [Abstract] [Full Text] [PDF]
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 R. A. Anders, S. L. Arline, J. J.E. Doré, and E. B. Leof Distinct Endocytic Responses of Heteromeric and Homomeric Transforming Growth Factor beta Receptors Mol. Biol. Cell, November 1, 1997; 8(11): 2133 - 2143. [Abstract] [Full Text]
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M. Mellado, J. M. Rodriguez-Frade, L. Kremer, C. von Kobbe, A. M. de Ana, I. Merida, and C. Martinez-A Conformational Changes Required in the Human Growth Hormone Receptor for Growth Hormone Signaling J. Biol. Chem., April 4, 1997; 272(14): 9189 - 9196. [Abstract] [Full Text] [PDF]
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M. Sundstrom, T. Lundqvist, J. Rodin, LutzB. Giebel, D. Milligan, and G. Norstedt Crystal Structure of an Antagonist Mutant of Human Growth Hormone, G120R, in Complex with Its Receptor at 2.9AResolution J. Biol. Chem., December 13, 1996; 271(50): 32197 - 32203. [Abstract] [Full Text] [PDF]
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