Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes

Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes

Domestic Animal Endocrinology 61 (2017) 39–47 Contents lists available at ScienceDirect Domestic Animal Endocrinology journal homepage: www.domestic...

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Domestic Animal Endocrinology 61 (2017) 39–47

Contents lists available at ScienceDirect

Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com

Growth hormone-specific 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 b

College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, P. R. China 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: Received 20 January 2017 Received in revised form 22 May 2017 Accepted 31 May 2017

The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human, rat, and fish has been reported. 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. To this end, porcine hepatocytes were isolated and used as a cell model. We observed that porcine growth hormone (pGH) can induce porcine GHR’s nuclear localization in porcine hepatocytes. Subsequently, the dynamics of pGH-induced pGHR’s nuclear localization were analyzed and demonstrated that pGHR’s nuclear localization occurs in a time-dependent manner. Next, we explored the mechanism of pGHR nuclear localization using different pGHR ligands, and we demonstrated that pGHR’s 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 pGHpGHR 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 nuclearlocalized pGH-pGHR dimer might still have the ability to signal. Ó 2017 Elsevier Inc. All rights reserved.

Keywords: Porcine growth hormone Growth hormone receptor Nuclear translocation Subcellular localization

1. Introduction Porcine growth hormone (pGH) has important physiological functions in the regulation of pig growth and development [1–3]. The biological roles of GH are mediated initially through its binding to the growth hormone receptor (GHR) of target cells. GHR exists in the cell membrane 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 transcription (STAT) and extracellular regulated protein kinases * Corresponding author. Tel./fax: þ86- 0431-84533462. E-mail addresses: [email protected] (H.N. Lan), [email protected] com (X. Zheng). y Hainan Lan, and Hong Pan have contributed equally. 0739-7240/$ – see front matter Ó 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2017.05.003

(ERK1/2). These signaling molecules exert their transcriptional 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 [7–13]. 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 fish. Until now, the mechanism(s) by which GHR translocates into cell nuclei is not fully understood. Membrane-bound GHR’s nuclear localization can be divided into three

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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 molecules 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 scientific question: what is relationship between ligand and GHR’s 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 GHR’s nuclear localization. Taken together, although the phenomenon of nuclear translocation of GHR in human, rat, and fish has been reported [7–9], 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 find 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 SigmaAldrich (St. Louis, USA). Porcine GHR antibody was obtained 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 fluoride membranes were obtained from Millipore. Fetal calf serum and cell culture media were from Gibco (Grand Island, USA). Fluorescein isothiocyanate (FITC) was obtained from SigmaAldrich (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).

an ideal somatic model for studying the interaction between 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 2step 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 manufacturer’s protocols. The corresponding cytosol and nuclear extracts were used as Immunoprecipitation and Western-blot experiments as described as below. 2.4. Indirect immunofluorescence 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 fixed with 4% paraformaldehyde for 10 min, after which, the cells were permeabilized 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 (FITClabeled). 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 manufacturer’s protocols. In brief, porcine hepatoyctes were labeled for 30 min at 4 C with 0.3 mL of 0.5 mg/mL Sulpho-NHSBiotin 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

2.2. Isolation of porcine hepatocytes Porcine hepatocytes naturally express abundant pGHR, and it has been demonstrated that porcine hepatocytes are

Following stimulation with different ligands, porcine hepatocytes were washed 3 times with ice-cold PBS to remove remnant ligands. The porcine hepatocytes were

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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 separation followed by a transfer onto polyvinylidene fluoride 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.

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mainly localized in the cell plasma in nonpermeabilized porcine hepatocytes (green signal), whereas pGHR was localized in the cell membrane and cytoplasm in the permeabilized porcine hepatocytes (the cell nuclei was stained with PI, red signal and is difficult to visualize). To further confirm these observations, the immunoprecipitation (IP) and Western-blot experiments were performed. We subjected cytoplasm and nuclear extracts from porcine hepatocytes without pGH treatment to immunoprecipitation with anti-pGHR followed by immunoblotting with antiGHR antibody, as shown in Figure 1B; pGHR was detectable 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

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 performed. The porcine hepatocytes were treated as described in materials and methods. As indicated in Figure 1A, pGHR

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 preexperiment, this dose of pGH exhibits maximal signaling activities) for 30 min, both the cytoplasm and cell nuclei show intense immunoreactivity (green signal), which indicates 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 0–90 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, fixed, 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 figure is representative of 3 independent experiments. FITC, fluorescein isothiocyanate; IFA, immunofluorescence assay; pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.

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was mainly localized in the cytoplasm but not in the cell nuclei. After pGH stimulation for 5–60 min, the pGHR staining in the porcine hepatocyte nucleus was increased with an increasing stimulation time. In addition, to quantitatively analyze pGHR’s nuclear localization, the IP and Western blot analysis were carried out in the similar experimental conditions. 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, pGHR’s 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-specific induction of pGHR nuclear localization To determine if the nuclear translocation of GHR occurs in a ligand-specific–dependent 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 specific. Next, the G120R (an antagonist for human GHR, we found that it is shown as an antagonist for pGHR in preexperiments) 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 translocation. However, when B32, a GHR antibody agonist developed in our lab, which can activate pGHR, was used to 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, fixed, 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). (B) The kinetics of GHR nuclear localization. The porcine hepatocytes were stimulated with pGH for 0–90 min, after which, the nuclear 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 figure is representative of 3 independent experiments. FITC, fluorescein isothiocyanate; IFA, immunofluorescence assay; pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.

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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 cytoplasmic fractions were precipitated with avidin-agarose. The precipitates were then analyzed by Western blot. The figure is representative of 3 independent experiments. Bar: 10 mm. FITC, fluorescein isothiocyanate; IFA, immunofluorescence assay; pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.

treat the porcine hepatocytes it failed to induce GHR targeting to cell nuclei. The results from Western blotting confirmed these observations (Fig. 4C). These findings indicated that pGHR activation by ligands other than GH is not sufficient 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 immunoprecipitated proteins using pGHR antibody. However, the interactions could not be detected between IMP a/b and the pGHR after G120R and B32 treatments. These findings 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 findings 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 hepatocytes 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 pGHR’s 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 IMPa/b and pGHR is

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Fig. 4. (A). The nuclear translocation of pGHR is GH(s) specific. 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 figure is representative of 3 independent experiments. Bar: 10 mm. IFA, immunofluorescence assay; pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.

ligand dependent. However, these findings 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 immunoprecipitated 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-GHRJAK2 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

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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 figure is representative of 3 independent experiments (A–C). pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.

localized in cell nuclei were phosphorylated using antityrosine 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 first analyzed pGHR nuclear translocation under pGH stimulation and found that pGH could induce GHR nuclear translocation in porcine hepatocytes. Subsequently, different ligands were used to evaluate the ligand’s 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 finding 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 cytoplasm, 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 remains 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 immunoblotting 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 figure is representative of 3 independent experiments. JAK2, Janus kinase; pGH, porcine growth hormone; pGHR, porcine growth hormone receptor.

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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-specific–dependent 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 phosphorylated (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 findings 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 nuclearlocalized GHBP functions as an enhancer of STAT5mediated transcription. Furthermore, Conway-Campbell et al [7] found that GHR nuclear localization was associated 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 localization 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 speculate that GH binding induces a specific 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-GHR’s nuclear translocation undergo? Overall, this study obtained the following findings: (1) to the best of our knowledge, we presented the first description of the phenomenon of pGHR nuclear localization 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 Science Foundation-Young investigator grant program (grant number: 31602022). This project was partially supported by the Scientific Research projects of the Thirteenth FiveYear 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: 6434–7. [2] Etherton TD, Bauman DE. Biology of somatotropin in growth and lactation of domestic animals. Physiol Rev 1998;78:745–61. [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:E29–37. [4] Brooks AJ, Dai W, O’Mara 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: 515–25. [6] Pang XD, Zhou HX. A common model for cytokine receptor activation: 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;104:13331–6. [8] Mertani HC, Raccurt M, Abbate A, Kindblom J, Tornell J, Billestrup N, Lobie PE. Nuclear translocation and retention of growth hormone. Endocrinology 2003;144:3182–95. [9] Figueiredo MA, Boyle RT, Sandrini JZ, Varela AS, Marins LF. High level of GHR nuclear translocation in skeletal muscle of a hyperplasic transgenic zebrafish. J Mol Endocrinol 2016;56:47–54. [10] Govers R, van Kerkhof P, Schwartz AL, Strous GJ. Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligandinduced internalization of the growth hormone receptor. EMBO J 1997;16:4851–8. [11] Strous GJ, van Kerkhof P, Govers R, Ciechanover A, Schwartz AL. The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J 1996;15:3806–12.

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