Domestic Animal Endocrinology 61 (2017) 3947

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Domestic Animal Endocrinology

journal homepage: www.domesticanimalendo.com

Growth hormone-specic induction of the nuclear

localization of porcine growth hormone receptor in porcine
hepatocytes
H.N. Lan a, *, y, P. Hong a, y, R.N. Li a, A.S. Shan b, X. Zheng a, *
a
College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, P. R. China
b
Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, P. R. China

a r t i c l e i n f o a b s t r a c t

Article history: The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human,
Received 20 January 2017 rat, and sh has been reported. To date, this phenomenon has not been described in a
Received in revised form 22 May 2017 domestic animal (such as pig). In addition, the molecular mechanisms of GHR nuclear
Accepted 31 May 2017
translocation have not been thoroughly elucidated. To this end, porcine hepatocytes were
isolated and used as a cell model. We observed that porcine growth hormone (pGH) can
Keywords:
induce porcine GHRs nuclear localization in porcine hepatocytes. Subsequently, the dy-
Porcine growth hormone
namics of pGH-induced pGHRs nuclear localization were analyzed and demonstrated that
Growth hormone receptor
Nuclear translocation pGHRs nuclear localization occurs in a time-dependent manner. Next, we explored the
Subcellular localization mechanism of pGHR nuclear localization using different pGHR ligands, and we demon-
strated that pGHRs nuclear translocation is GH(s)-dependent. We also observed that pGHR
translocates into cell nuclei in a pGH dimerization-dependent fashion, whereas further
experiments indicated that IMPa/b is involved in the nuclear translocation of the pGH-
pGHR dimer. The pGH-pGHR dimer may form a pGH-GHR-JAK2 multiple complex in cell
nuclei, which would suggest that similar to its function in the cell membrane, the nuclear-
localized pGH-pGHR dimer might still have the ability to signal.
2017 Elsevier Inc. All rights reserved.

1. Introduction (ERK1/2). These signaling molecules exert their transcrip-

Porcine growth hormone (pGH) has important physio-
logical functions in the regulation of pig growth and
development [13]. The biological roles of GH are mediated
initially through its binding to the growth hormone re-
ceptor (GHR) of target cells. GHR exists in the cell mem-
brane as a dimer [4]. Following GH binding to the
extracellular domain of GHR (GHR-ECD), the Janus kinase
(JAK2) is activated, triggering a downstream signaling
cascade, involving signal transducer and activator of tran-
scription (STAT) and extracellular regulated protein kinases
* Corresponding author. Tel./fax: 86- 0431-84533462.
E-mail addresses: tougao@jlau.edu.cn (H.N. Lan), zhengtougao@163.
com (X. Zheng).
y
Hainan Lan, and Hong Pan have contributed equally.
tional functions by their nuclear localization [5,6].
Traditionally, it has been thought that GH exhibits its
physiological functions by binding to GHR expressed on the
cell membrane. However, many studies have shown that
GHR is not only localized in the cell membrane but also is
found in the cell nuclei [713]. This nuclear localization
leads to a question regarding what functions are being
exhibited by nuclear-localized GHR. There had been no
answer regarding the function of the nuclear GHR, until
Waters et al [7] reported that nuclear localization of GHR is
strongly correlated with highly proliferative tissues. More
recently, Figueiredo et al [9] also reported a proliferative
action of nuclear-localized GHR in sh.
Until now, the mechanism(s) by which GHR translocates
into cell nuclei is not fully understood. Membrane-bound
GHRs nuclear localization can be divided into three

0739-7240/$ see front matter 2017 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.domaniend.2017.05.003
40 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947
steps: GHR internalization from the cell membrane, its
transport through the cytoplasm and its movement
through the nuclear membrane. At present, information
about GHR nuclear translocation is very limited. It has been
reported that the ubiquitin conjugating system may play an
important role in GHR internalization and that IMP a/b is
involved in growth hormone-binding protein (GHBP, also
named extracellular domain of GHR, ECD-GHR) nuclear
translocation, although GHR typically has no nuclear
localization signal [7,10,11].
In addition to the aforementioned cytoplasmic mole-
cules involved in GHR nuclear translocation, it has been
found that a ligand is also required for the GHR nuclear
localization [7,8,12], which proposes a scientic question:
what is relationship between ligand and GHRs nuclear
localization? Except for GH, other ligands exist for GHR that
could serve as an agonist or antagonist for GHR. For
example, Harding et al [14] found that G120R, a GHR
antagonist, induced GHR internalization in a similar
manner to that of GH. In addition, our previous study also
found that a pGHR antibody (B32) agonist could induce
similar pGHR internalization to that of pGH [15]. The
aforementioned ligands can be used as a tool to explore
relationship between ligand and GHRs nuclear localization.
Taken together, although the phenomenon of nuclear
translocation of GHR in human, rat, and sh has been re-
ported [79], to date, this phenomenon has not been
described in a domestic animal (such as pig). In addition,
the molecular mechanisms of GHR nuclear translocation
have not been thoroughly elucidated. The aim of present
study is to nd the possible answers to the questions
mentioned above.
2. Materials and methods
Animal protocols were approved by Jilin Agricultural
University Internal Animal Care and Use Committee.
2.1. Reagents and antibodies
Porcine growth hormone was obtained from Sigma-
Aldrich (St. Louis, USA). Porcine GHR antibody was ob-
tained from Abcam (Cambridge, UK). Cell lysis buffer and
BCA kits were obtained from Beyotime (Shanghai, China).
Nonfat milk, BSA and Enhanced chemiluminescence (ECL)
was obtained from Pierce (Rockford, USA). Glutaraldehyde
and paraformaldehyde were obtained from Hua-yi
biotechnology (Changchun, China). Polyvinylidene uoride
membranes were obtained from Millipore. Fetal calf serum
and cell culture media were from Gibco (Grand Island, USA).
Fluorescein isothiocyanate (FITC) was obtained from Sigma-
Aldrich (St. Louis, USA). FITC-conjugated second antibodies
were obtained from Abcam (Cambridge, UK). Nuclear and
Cytosol Fractionation Kit were purchased from BioVision
(San Francisco, USA). Pierce Cell Surface Protein Isolation Kit
was purchased from pierce (Rockford, USA).
2.2. Isolation of porcine hepatocytes
Porcine hepatocytes naturally express abundant pGHR,
and it has been demonstrated that porcine hepatocytes are
an ideal somatic model for studying the interaction be-
tween pGH and pGHR. In addition, pGH does not interact
with porcine prolactin receptor [16]. Porcine hepatocytes
used in this study were isolated and cultured according to
our previous methods [15]. In brief, the pigs (Landrace,
weighing w60 kg) were subjected to an electric shock, and
the pigs were exsanguinated. The porcine livers were then
immediately excised, and the left lateral lobe was removed,
after which, the porcine hepatocytes were isolated by a 2-
step collagenase perfusion method. The trypan blue dye
exclusion assay was used to analyze the cell viability.
2.3. Isolation of subcellular fractions
The freshly isolated porcine hepatocytes were adjusted
at density of 1  106/mL and seeded into cell culture plate.
The hepatocytes were serum starved for 6 h, after which,
the cells were washed 3 times with PBS. The cells were then
treated with different ligands as described below. After
treatment, the cells were harvested at selected time points
depending on the experiment. Nuclear and cytosol
extraction kit were used to isolate nuclear and cytosol
fractions of the cells according to manufacturers protocols.
The corresponding cytosol and nuclear extracts were used
as Immunoprecipitation and Western-blot experiments as
described as below.
2.4. Indirect immunouorescence assay (IFA)
The porcine hepatocytes were serum starved for 6 h.
After washing 3 times with PBS, the hepatocytes were
stimulated with the different ligands for different durations.
The cells were then rinsed and xed with 4% para-
formaldehyde for 10 min, after which, the cells were per-
meabilized with 0.2% TritonX-100 for 5 min at 4 C. Next, the
cell samples on the slides were blocked with 3% BSA for 2 h.
After washing, the cells were incubated sequentially with
anti-pGHR primary antibody and secondary antibody (FITC-
labeled). Propidium iodide (PI) was used to stain cell nuclei.
After washing with PBS, the cell samples were detected by
confocal laser scanning microscopy (Olympus FV1000).
2.5. Biotinylation of cell-surface proteins
The cell membrane proteins were biotinylated with
commercially available reagents according to manufac-
turers protocols. In brief, porcine hepatoyctes were labeled
for 30 min at 4 C with 0.3 mL of 0.5 mg/mL Sulpho-NHS-
Biotin solution. After washing 3 times with ice-cold PBS,
the cells were incubated with pGH for 60 min at 37 C. After
3 washes with ice-cold PBS, the hepatocyte nuclei fractions
were prepared as described above. Biotin-tagged proteins
from the nuclear fractions were isolated, and the isolated
samples were then analyzed with anti-pGHR antibody by
Western blotting as described below.
2.6. Immunoprecipitation and Western blotting
Following stimulation with different ligands, porcine
hepatocytes were washed 3 times with ice-cold PBS to
remove remnant ligands. The porcine hepatocytes were
 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947 41

transferred on ice and lysed in RIPA lysis buffer in the
presence of protease inhibitors. The samples were then
collected by centrifugation at 20,000  g for 15 min.
Immunoprecipitation was conducted by incubating cell
lysates with the indicated antibodies or control antibody
for 12 h at 4 C with constant rotation. The concentration of
immunoprecipitated proteins was measured by BCA kit.
The samples were boiled for 5 min, and the samples (30 mg/
lane) were loaded on to 10% polyacrylamide gels for sepa-
ration followed by a transfer onto polyvinylidene uoride
membrane. After washing with TBST, the membranes were
blocked with 2% BSA. Next, the membrane was incubated
sequentially with appropriate primary and secondary
antibody. After 3 washes with TBST, the membranes were
detected with ECL detection system.
2.7. Methods to describe observations
The data are presented as the mean values standard
error.
3. Results
3.1. pGHR localization in porcine hepatocytes without pGH
stimulation
To observe pGHR localization in porcine hepatocytes
under no pGH stimulation, the IFA experiments were per-
formed. The porcine hepatocytes were treated as described
in materials and methods. As indicated in Figure 1A, pGHR
mainly localized in the cell plasma in nonpermeabilized
porcine hepatocytes (green signal), whereas pGHR was
localized in the cell membrane and cytoplasm in the per-
meabilized porcine hepatocytes (the cell nuclei was stained
with PI, red signal and is difcult to visualize). To further
conrm these observations, the immunoprecipitation (IP)
and Western-blot experiments were performed. We sub-
jected cytoplasm and nuclear extracts from porcine hepa-
tocytes without pGH treatment to immunoprecipitation
with anti-pGHR followed by immunoblotting with anti-
GHR antibody, as shown in Figure 1B; pGHR was detect-
able in cytoplasmic extracts. No or little pGHR was
detectable in nuclear extracts. These observations are
similar with those of the IFA experiments.
3.2. GHR targeting to cell nucleus under pGH stimulation
Aforementioned experiments indicate that pGHR is
localized in cytoplasm and cell membrane. Here, we further
determined if the pGHR nuclear translocation needs pGH
stimulation in porcine hepatocytes. As indicated in
Figure 2A, after the treatment with 50-nM pGH (in our pre-
experiment, this dose of pGH exhibits maximal signaling
activities) for 30 min, both the cytoplasm and cell nuclei
show intense immunoreactivity (green signal), which in-
dicates that pGH stimulation is required for pGHR nuclear
localization. We then analyzed the kinetics of pGHR nuclear
localization under pGH stimulation. For this, the porcine
hepatocytes were stimulated with pGH for 090 min. As
shown Figure 2B, after pGH treatment for 0 min, the pGHR

Fig. 1. The localization of pGHR without pGH treatment. (A) The localization of pGHR by IFA experiments. The porcine hepatocytes were pre-treated as described
as in Materials and Methods. The cells were then rinsed, xed, and permeabilized with 0.2% TritonX-100. After blocking with BSA, the cells were incubated
sequentially with anti-pGHR primary and secondary antibody (FITC-labeled). Subsequently, the cell nuclei were stained with propidium iodide. After washing
with PBS, the cell samples were detected by confocal laser scanning microscopy (Olympus FV1000). Bar: 20 mm. (B). Analysis of pGHR localization by Western blot
experiments. The nuclear extracts from porcine hepatocytes without pGH treatment were subjected to immunoblotting with anti-pGHR antibody. The gure is
representative of 3 independent experiments. FITC, uorescein isothiocyanate; IFA, immunouorescence assay; pGH, porcine growth hormone; pGHR, porcine
growth hormone receptor.
42 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947

was mainly localized in the cytoplasm but not in the cell

nuclei. After pGH stimulation for 560 min, the pGHR stain-
ing in the porcine hepatocyte nucleus was increased with an
increasing stimulation time. In addition, to quantitatively
analyze pGHRs nuclear localization, the IP and Western blot
analysis were carried out in the similar experimental condi-
tions. As shown in Figure 2C, after pGH treatment for 0 min,
the pGHR could not be detectable in the cell nuclei. After pGH
stimulation for 5 min, pGHR could be detectable in the cell
nuclei, after which, the nuclear-localized pGHR was increased
with an increasing stimulation time and reached a maximum
in 60 min. After pGH treatment for 90 min, pGHRs nuclear
localization declined slightly.
To determine if the nuclear-localized pGHR is derived
from the cell membrane, we performed the following 2
experiments: First, the porcine hepatocytes were
treated with pGH at 4 C (low temperature), with little or
no pGHR detectable in the nuclei of cells. When cells
were treated with the same concentration of pGH at
37 C, the nuclear translocation of pGHR was recovered
(Fig. 3A). Second, before treatment with pGH, the pGHR
in the cell membrane of the porcine hepatocytes
were labeled with an impermeable biotin. After
pGH treatment, pGHR was detectable in biotin-tagged
proteins from the nuclear extracts, which indicated
that nuclear-localized pGHR, at least in part, was from
the cell membrane (Fig. 3B).

3.3. pGH-specic induction of pGHR nuclear localization

To determine if the nuclear translocation of GHR occurs

in a ligand-specicdependent manner, various ligands
were used to stimulate the porcine hepatocytes. As shown
in Figure 4A, except for pGH, the GHs from human and
bovine can also induce GHR nuclear localization and can
exhibit a similar nuclear staining pattern with that of pGH,
indicating that GHR nuclear localization is not GH-species
specic. Next, the G120R (an antagonist for human GHR,
we found that it is shown as an antagonist for pGHR in pre-
experiments) was used to further determine ligand effects
on pGHR nuclear translocation. As shown in Figure 4B,
G120R did not induce pGHR targeting to the cell nuclei,
with this observation possibly implying that GHR activation
induced by GH is required for the GHR nuclear trans-
location. However, when B32, a GHR antibody agonist

Fig. 2. (A) pGHR nuclear localization after pGH treatment. The hepatocytes
were treated with pGH (50 nM) for 30 min; the cells were then rinsed, xed,
and permeabilized with 0.2% TritonX-100. After blocking with BSA, the cells
were incubated sequentially with anti-pGHR primary and secondary anti-
body (FITC-labeled). (B) The kinetics of GHR nuclear localization. The porcine
hepatocytes were stimulated with pGH for 090 min, after which, the nu-
clear localization of pGHR were determined by CLSM as described as in
materials and methods. Bar: 10 mm. (C) Analysis of pGHR nuclear localization
by Western blot. The nuclear extracts from porcine hepatocytes with pGH
treatment were subjected to immunoprecipitation with anti-pGHR followed
by immunoblotting with anti-pGHR antibody. The gure is representative of
3 independent experiments. FITC, uorescein isothiocyanate; IFA, immu-
nouorescence assay; pGH, porcine growth hormone; pGHR, porcine growth
hormone receptor.
 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947 43

Fig. 3. Determination of source of pGHR. (A) The porcine hepatocytes were treated with pGH at 4 C or 37 C, after which, the localization of pGHR were detected
by IFA experiments as described in the materials and methods. (B) The porcine hepatocytes were unlabeled or labeled with sulpho-NHS biotin, and then treated
with pGH for 60 min at either 37 C (upper) or 4 C (lower), the porcine hepatocytes were fractionated, and biotin-labeled proteins from the nuclear and cyto-
plasmic fractions were precipitated with avidin-agarose. The precipitates were then analyzed by Western blot. The gure is representative of 3 independent
experiments. Bar: 10 mm. FITC, uorescein isothiocyanate; IFA, immunouorescence assay; pGH, porcine growth hormone; pGHR, porcine growth hormone
receptor.

developed in our lab, which can activate pGHR, was used to
treat the porcine hepatocytes it failed to induce GHR tar-
geting to cell nuclei. The results from Western blotting
conrmed these observations (Fig. 4C). These ndings
indicated that pGHR activation by ligands other than GH is
not sufcient for pGHR nuclear targeting. Subsequently, we
explored why both G120R and B32 could induce similar
GHR cytoplasmic localization with that of pGH, but not
nuclear localization. It has been demonstrated that IMPa/b
is involved in GHR nuclear translocation [7]. IP and WB
experiments were performed to detect the interactions
between IMPa/b and cytoplasmic pGHR under different
ligand treatments. As shown in Figure 4D, after pGH
treatment, IMP a/b was detectable in the immunoprecipi-
tated proteins using pGHR antibody. However, the in-
teractions could not be detected between IMP a/b and the
pGHR after G120R and B32 treatments. These ndings may
provide an explanation for why the pGHR with B32 and
G120R treatment cannot translocate into the nuclei,
although the exact reasons why pGHR under non-GH
ligand treatments cannot interact with IMP a/b has not
been thoroughly elucidated. These ndings suggest that
B32 and G120R can serve as important tools for exploring
the mechanism of pGHR nuclear translocation.
3.4. pGH-GHR dimer interacts with IMPa/b in the cytoplasm
To analyze how pGH is involved in pGHR nuclear
translocation, immunoprecipitation and Western blotting
experiments were performed. As shown in Figure 5A,
we subjected cytoplasmic extracts from porcine hepato-
cytes with pGH treatment to immunoprecipitation with
anti-pGHR followed by immunoblotting with the indicated
antibodies to detect the pGH, IMP a and b components in
immunoprecipitates. pGH and pGHR components were
also detected in immunoprecipitates using anti-IMPa/b
antibody (Fig. 5B). These observations suggest that
these components may form a pGH-pGHR-IMPa/b multiple
complex in cytoplasm and that IMPa/b mediates pGHRs
nuclear localization. In addition, we could not detect
the association between IMPa/b and pGHR without pGH
stimulation (Fig. 5C), which suggests that the association of
44 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947

Fig. 4. (A). The nuclear translocation of pGHR is GH(s) specic. The porcine hepotocytes were stimulated either with human and bovine GH for 60 min.
The following IFA experiments were performed as described as in materials and methods. (B). The porcine hepatocytes were stimulated with G120R or B32, and
subsequent experiments carried out as described above. (C). Analysis of the nuclear localization of pGHR with different ligands treatments by Western blot. (D).
The interactions within IMP a/b and pGHR. The porcine hepatocytes were stimulated with the indicated ligands, the immunoprecipitation experiments were then
performed by using pGHR antibody, subsequently analyzed by Western blot by the indicated antibodies. The gure is representative of 3 independent experi-
ments. Bar: 10 mm. IFA, immunouorescence assay; pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.

IMPa/b and pGHR is ligand dependent. However, these
ndings cannot fully exclude the possibility that pGHR
alone can interact with IMP a/b, as it has been reported that
immature GHR can also appear in the nuclei, whereas these
GHR should not interact with GH [7].
3.5. pGH-GHR complex exists in the nuclei of porcine
hepatocytes
The above experiments have indicated that pGH-GHR
interact with IMP a/b in cytoplasm. In this study, we
further explored the interactions between pGH, pGHR, and
JAK2 in cell nuclei. As shown in Figure 6A, we detected the
pGH and JAK2 components in nuclear extract immuno-
precipitated by using anti-GHR antibody. GHR and JAK2
components were also detectable in nuclear extract
immunoprecipitated by using anti-pGH antibody (Fig. 6B).
These observations suggest that it may form a pGH-GHR-
JAK2 multiple complex in cell nuclei, similar to the cell
membrane, which suggests that pGH-GHR-JAK2 complex
may still have the ability to transmit signaling. To explore
this possibility, we sought to determine if JAK2 and pGHR
 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947 45

Fig. 5. The interactions within pGH-GHR and IMPa/b. The porcine hepatocytes were treated with pGH for 30 min. Cytosolic fractions of the cells were then
isolated by nuclear and cytosol extraction kit. The nuclear extracts from porcine hepatocytes with pGH treatment were subjected to immunoprecipitation with
anti-pGHR antibody followed by immunoblotting with the indicated antibodies. The gure is representative of 3 independent experiments (AC). pGH, porcine
growth hormone; pGHR, porcine growth hormone receptor.

localized in cell nuclei were phosphorylated using anti-
tyrosine antibody (4G10). As shown in Figure 6C,D, pGHR
and JAK2 were phosphorylated in the cell nuclei.
4. Discussion
In the present study, porcine hepatocytes were used as a
somatic cell model. We rst analyzed pGHR nuclear
translocation under pGH stimulation and found that pGH
could induce GHR nuclear translocation in porcine hepa-
tocytes. Subsequently, different ligands were used to eval-
uate the ligands roles on GHR nuclear localization, and we
found that, except for pGH, human GH and bovine GH can
also induce pGHR nuclear localization, whereas GHR
antagonist (G120R) and porcine GHR antibody agonist
(B32) could not induce GHR nuclear localization. This
nding suggested that GHR nuclear translocation occurs in
a pGH-dependent manner. Further experiments found that
the pGH-GHR dimer interacts with IMPa/b in the cyto-
plasm, which indicated that pGH is a partner involved in
pGHR nuclear translocation. In the cell nuclei, we also
found that pGH and pGHR exist in a dimer (pGH-GHR), and
pGHR is phosphorylated, which suggests that similar to the
cell membrane, nuclear-localized pGH-GHR dimer may still
possess the ability to transmit signaling. To our knowledge,
this is the initial report that pGH-induces pGHR nuclear
localization in cells from a domestic animal model.
The mechanism(s) of pGHR nuclear translocation re-
mains to fully be revealed, and there are a number of
questions needing to be answered. One of these questions

Fig. 6. (A, B) The interactions within pGH, pGHR, and JAK2 in cell nuclei. The porcine hepatocytes were treated with pGH for 60 min. The nuclear fractions of the
cells were then isolated by cytosol extraction kit. The nuclear extracts were subjected to immunoprecipitation with anti-pGHR or pGH followed by immuno-
blotting with the indicated antibodies. (C, D) pGHR and JAK2 were phosphorylated in cell nucleus. The nuclear extracts were subjected to immunoprecipitation
with the 4G10 antibody followed by immunoblotting with anti-pGHR or JAK2. The gure is representative of 3 independent experiments. JAK2, Janus kinase;
pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.
46 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947
relates to the many studies that have shown GH is involved
in GHR nuclear translocation but have not determined the
action(s) of GH in GHR nuclear translocation. Based on this,
we further explored if GHR could target to cell nuclei with
different ligand treatments and found that GHR nuclear
translocation behaves in a GH-specicdependent manner.
Further experiments indicated that the pGH-GHR dimer
interacts with IMPa/b in cytoplasm (Fig. 4), which suggests
that pGH may serve as a partner in the pGHR nuclear
translocation. We found that pGH and pGHR also exist in
dimer form in cell nuclei (pGH-pGHR), and pGHR is phos-
phorylated (Fig. 6), which suggests that pGH-GHR may still
have the ability to transmit signaling.
It has been reported that except for full-length GHR,
several components of the GHR could also translocate into
the cell nuclei. Graichen et al reported that the extracellular
domain of GHR (also called GHBP) localized in cell nuclei in
a ligand-dependent manner, and nuclear-localized GHBP is
associated with the enhancement of STAT5-mediated
transcription [17]. In addition, Frank et al [18] found that
the GHR cytoplasmic domain (intracellular domain of GHR,
termed as ICD) was localized in the nucleus dependent on
PS1/2 activity; however, the function of nuclear-localized
ICD remains unclear. Together, these ndings indicate cell
membrane full-length GHR, cytoplamic GHR (immature
GHR), and GHR components including the ECD and ICD all
can appear in cell nuclei.
However, to date, the physiological roles of GHR and
GHR components that are localized in cell nuclei remain to
be fully understood. Graichen et al [17] found that nuclear-
localized GHBP functions as an enhancer of STAT5-
mediated transcription. Furthermore, Conway-Campbell
et al [7] found that GHR nuclear localization was associ-
ated with constitutive STAT5 activation but not other
signaling proteins. However, the mechanism by which GHR
localized in cell nuclei results in constitutive STAT5
signaling remains unclear. Our present study may provide a
possible explanation, as we found that pGH and pGHR also
exist in dimer form in cell nuclei (pGH-GHR), and pGHR
and JAK2 are phosphorylated in the nuclei (Fig. 6), which
suggests that the pGH-GHR complex may still have the
ability to transmit signaling and form a nuclear signaling
system. Of course this is only a speculation and further
experiments are required. In addition, it cannot be
excluded that pGHR localized in cell nuclei can also serve as
a transcription factor, similar to EGFR [19].
The routing of pGHR nuclear translocation remains
unclear. The process of GHR nuclear localization can be
divided into 3 parts: (1) GHR internalization from cell
membrane; (2) GHR cytoplasmic transport; and (3) passing
through the nuclear membrane. It has been reported that
the ubiquitin conjugating system is required for GHR
internalization and that IMP a/b is involved in GHR nuclear
translocation, although GHR has no typical nuclear locali-
zation signal, and GHBP does interact with the importin a/b
dimer (IMPa/b) [7,11]. In the present study, we found that
pGH-GHR, but not other ligand/GHR complexes, interact
with IMPa/b (Fig. 4), which suggests that GH may play
important roles in GHR interacting with IMPa/b. We spec-
ulate that GH binding induces a specic GHR conformation
change, which is required for the interaction with IMPa/b.
However, there are many questions needing to be
answered. Where and how does the pGH-GHR dimer
internalized from plasma interact with importin a/b? In
addition, which cytoplasmic events do pGH-GHRs nuclear
translocation undergo?
Overall, this study obtained the following ndings: (1)
to the best of our knowledge, we presented the rst
description of the phenomenon of pGHR nuclear localiza-
tion in a domestic animal (pig). The nuclear localization of
pGHR that is induced by pGH exhibits a time-dependent
manner; (2) pGHR nuclear localization responds in a
pGH-dependent manner; and (3) The pGH-GHR dimer can
interact with an IMPa/b dimer, and pGH, pGHR, and JAK2
also exist in multiple forms in cell nuclei (pGH-GHR), which
suggests that similar to the cell membrane, the pGH-pGHR
complex may still have the ability to transmit signaling.
Acknowledgments
This work was supported by the National Natural Sci-
ence Foundation-Young investigator grant program (grant
number: 31602022). This project was partially supported
by the Scientic Research projects of the Thirteenth Five-
Year plan of Jilin Province Department of Education (grant
number: 2016178).
H. N. Lan designed the experiments; P. Hong and R. N. Li
performed the experiments; and A.S. Shan analyzed the
data and assisted with discussion; X. Zheng contributed
reagents and materials. H. N. Lan wrote the article.
References
[1] Abdel-Meguid SS, Shieh HS, Smith WW, Dayriner HE, Violand BN,
Bentle LA. Three dimensional structure of a genetically engineered
variant of porcine growth hormone. Proc Natl Acad Sci 1987;84:
64347.
[2] Etherton TD, Bauman DE. Biology of somatotropin in growth and
lactation of domestic animals. Physiol Rev 1998;78:74561.
[3] Wester TJ, Davis TA, Fiorotto ML, Burrin DG. Exogenous growth
hormone stimulates somatotropic axis function and growth in
neonatal pigs. Am J Physiol Endocrinol Metab 1998;274:E2937.
[4] Brooks AJ, Dai W, OMara ML, Abankwa D, Chhabra Y, Pelekanos RA.
Mechanism of activation of protein kinase JAK2 by the growth
hormone receptor. Science 2014;344:1249783.
[5] Brooks AJ, Waters MJ. The growth hormone receptor: mechanism of
activation and clinical implications. Nat Rev Endocrinol 2010;6:
51525.
[6] Pang XD, Zhou HX. A common model for cytokine receptor activa-
tion: combined scissor-like rotation and self-rotation of receptor
dimer induced by class I cytokine. Plos Comput Biol 2012;8:
e1002427.
[7] Conway-Campbell BL, Wooh JW, Brooks AJ, Gordon D, Brown RJ,
Lichanska AM, Jans DA, Waters MJ. Nuclear targeting of the growth
hormone receptor results in dysregulation of cell proliferation and
tumorigenesis. Proc Natl Acad Sci 2007;104:133316.
[8] Mertani HC, Raccurt M, Abbate A, Kindblom J, Tornell J, Billestrup N,
Lobie PE. Nuclear translocation and retention of growth hormone.
Endocrinology 2003;144:318295.
[9] Figueiredo MA, Boyle RT, Sandrini JZ, Varela AS, Marins LF. High
level of GHR nuclear translocation in skeletal muscle of a hyper-
plasic transgenic zebrash. J Mol Endocrinol 2016;56:4754.
[10] Govers R, van Kerkhof P, Schwartz AL, Strous GJ. Linkage of the
ubiquitin-conjugating system and the endocytic pathway in ligand-
induced internalization of the growth hormone receptor. EMBO J
1997;16:48518.
[11] Strous GJ, van Kerkhof P, Govers R, Ciechanover A, Schwartz AL. The
ubiquitin conjugation system is required for ligand-induced endo-
cytosis and degradation of the growth hormone receptor. EMBO J
1996;15:380612.
 H.N. Lan et al. / Domestic Animal Endocrinology 61 (2017) 3947
[12] Lobie PE, Mertani H, Morel G, Morales-Bustos O, Norstedt G,
Waters MJ. Receptor-mediated nuclear translocation of growth
hormone. J Biol Chem 1994;269:213309.
[13] Lobie PE, Wood TJ, Chen CM, Waters MJ, Norstedt G. Nuclear
translocation and anchorage of the growth hormone receptor. J Biol
Chem 1994;269:3173546.
[14] Harding PA, Wang X, Okada S, Chen WY, Wan W, Kopchick JJ.
Growth hormone (GH) and a GH antagonist promote GH receptor
dimerization and internalization. J Biol Chem 1996;271:670812.
[15] Lan HN, Jiang HL, Li W, Wu TC, Hong P, Li YM, Zheng X. Develop-
ment and characterization of a novel anti-idiotypic monoclonal
antibody to growth hormone, which can mimic physiological
functions of growth hormone in primary porcine hepatocytes. Asian
Austral J Anim 2015;28:57380.
47
[16] Chung CS, Etherton TD. Characterization of Porcine growth hor-
mone (pGH) binding to porcine liver microsomes: chronic admin-
istration of pGH induces pGH binding. Endocrinology 1986;119:
7806.
[17] Graichen R, Sandstedt J, Goh EL, Isaksson OG, Trnell J, Lobie PE.
The growth hormone-binding protein is a location-dependent
cytokine receptor transcriptional enhancer. J Biol Chem 2003;
278:634654.
[18] Cowan JW, Wang X, Guan R, He K, Jiang J, Baumann G, Frank SJ.
Growth hormone receptor is a target for presenilin-dependent g-
secretase cleavage. J Biol Chem 2005;280:1933142.
[19] Wang SC, Hung MC. Nuclear translocation of the epidermal growth
factor receptor family membrane tyrosine kinase receptors. Clin
Cancer Res 2009;15:64849.