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https://www.abbs.info/ e-mail:[email protected] ISSN 0582-9879 |
Functional Expression of Guinea Pig Growth
Hormone Receptor
and Its Mutants in Mammalian Cells
LIAO Zhi-Yong, ZHANG Xin-Na,
JING Nai-He, ZHU Shang-Quan*
(
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology,
Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200031, China )
Abstract
The cDNA of guinea pig (Cavia porcellus) growth hormone receptor (gpGHR)
was cloned using RT-PCR in our laboratory. By sequence alignment, substitutions
of amino acids conserved in other mammalian GHRs were found. For example,
histidine-168 and tyrosine-332 equivalent to positions 170 and 333 in other
mammalian GHRs, which were considered to be necessary for the dimerization
of GHR and the specific GH-stimulated functions respectively, were replaced
by tyrosine and serine in gpGHR. Here, we report the functional expression
of gpGHR and its mutants, gpGHRY168H and gpGHRS332Y, in COS-7 cells and/or
Chinese hamster ovary (CHO) cells. It was shown that the COS-7 cells transfected
with pcDNA3-gpGHR possessed high affinity to bovine GH [K
= 1.3 × 109 (mol/L)－1] and a protein band with molecular
weight around 92 kD was detected by anti-mouse GHR monoclonal antibody (mAb263).
When CHO cells were transfected with the expression vectors, pcDNA3-gpGHR,
pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y, the gpGHR and its mutants were expressed
and the ligand binding, phosphorylation of JAK2, protein synthesis, and lipogenesis
were studied. It was found that the mutation of serine to tyrosine at position
332 greatly increased the GH-stimulated protein synthesis and the phosphorylation
of JAK2, while the mutation of tyrosine to histidine at position 168 increased
the protein synthesis and decreased the phosphorylation of JAK2 only weakly.
However, both mutations decreased the GH-stimulated lipogenesis. Thus, our
study provides the experimental evidence that gpGHR may mediate the metabolic
actions of GH and the substitutions of some conserved amino acids in gpGHR
result in the changes of post-binding signaling.
Key words guinea
pig; growth hormone receptor; mutagenesis; lipogenesis; protein synthesis
The evolutionary position
of guinea pig (Cavia porcellus) was not unequivocally clarified. Although
it was traditionally classified as a New World Hystricomorph rodent, some
demonstrations in phylogeny suggested that the guinea pig and myomorph rodents
diverged before the separation between myomorph rodents and a lineage leading
to primates and artiodactyls[1, 2]. In addition, guinea pig showed abnormality
in response to GH: neither hypophysectomy nor administration of guinea pig
pituitary extracts to normal or hypophysectomized guinea pig altered the growth
rate of guinea pig[3]. However, the guinea pig pituitary extracts did promote
growth in hypophysectomized rats[4]. Previous studies have shown that GH secretion
in guinea pig is pulsatile and under the control of GH releasing factor and
somatostatin[5].
The biological activities of GH include somatogenic, lactogenic, insulin-like
and diabetogenic effects, et al.[6－8]. GH initiates its diverse functions
in various target tissues by binding to its specific receptors located on
the cell plasma membranes[9]. GHR has been identified in guinea pig liver
and binding assay showed that ovine GH could bind to the liver membrane of
guinea pig[10]. In addition, a soluble GH-binding protein (GHBP) with high
affinity to ovine GH was detected in the plasma and liver cytosol of guinea
pig[11, 12]. However, neither ovine GH nor bovine GH can promote the growth
of guinea pig except that increased estrogen receptor concentration stimulated
by bovine GH in guinea pig uterus was observed[13].
Previously, we reported the cloning and sequencing of gpGHR cDNA[14, 15].
It was found that some amino acid residues that were considered to be important
for mammalian GHR function were substituted. In the present study, we describe
the characterization of gpGHR and its mutants, gpGHRY168H and gpGHRS332Y,
expressed in mammlian cells.
1 Materials and Methods
1.1 Materials
Recombinant human growth hormone (hGH) and recombinant bream growth hormone
(brGH) were purchased from BresaGen (Adelaide, Australia). Recombinant bovine
growth hormone (bGH), recombinant ovine growth hormone (oGH), ovine prolactin
(oPRL), and anti-GHR antibody mAb263 were obtained from Dr. Chueng in Department
of Biochemistry, Chinese University of Hong Kong. Peroxidase-labeled anti-mouse
IgG was from Santa Cruz Biotechnology and anti-phosphotyrosine (PY20) peroxidase
conjugate from Calbiochem. Lipofectin and Altered sites-II in vitro mutagenesis
system were purchased from Gibco BRL and Promega respectively.
1.2 Mutagenesis, transfection and cell culture
An XbaI/KpnI fragment containing the gpGHR gene was cloned into the pAlter-1
vector containing the bacteriophage f1 intergenic region for the production
of single-stranded DNA (ssDNA). The ssDNA was prepared as described previously[16].
Site-directed mutagenesis of gpGHR cDNA was performed using established procedures[17],
and the mutations were verified by DNA sequencing. The mutated gpGHR cDNAs
were cloned into the CMV/neo expression plasmid pcDNA3 (Invitrogen, cat. No.V790-20).
COS-7 or CHO cells were cultured in DMEM/F12 medium containing 10 % fetal
bovine serum in 5 % CO2/ 95 % air at 37 °C. Transfection of COS-7 or CHO cells
with the constructs or pcDNA3 was performed using Lipofectin (Life Technologies,
cat. No.18292-011) as described by the manufacturer. The transfected cells
were incubated for 48 h and then deprived of serum for 12 h before being processed.
1.3 RT-PCR
Total RNA was isolated 24－48 h after COS-7 cell was transiently transfected
with or not with pcDNA3-gpGHR. With the mRNA in the obtained total RNA as
template, a pair of gpGHR-specific primers were used to synthesize and amplify
the gpGHR cDNA. The resulting RT-PCR product was electrophoresed on 1 % agarose
gel with labeled marker in parallel.
1.4 Receptor binding assay
After removing the medium, the confluent cells (≈106) grown in 35-mm culture
dishes were washed twice with PBS containing 0.1% BSA (PBS-BSA). The binding
assay was performed by adding [125I]-labeled GH (50 000－100 000 cpm) in PBS-BSA
to each dish with or without unlabeled GH in a final volume of 0.5 ml. The
cells were incubated for 4 h at 37 °C, rinsed three times with PBS-BSA and
then solubilized in 0.5 ml 0.1 mol/L NaOH containing 10 g/L SDS[18]. The bound
radioactivity was measured in a gamma counter. Nonspecific binding was determined
in the presence of 1 μg of unlabeled GH.
1.5 Immunoblot analysis
The transfected confluent cells grown in 100-mm culture dishes were stimulated
with or without oGH for 30 min and then lysed in 0.5 ml RIPA buffer (PBS with
1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) containing 0.1 μg/L phenylmethylsulfonyl
fluoride (PMSF), 30 ng/L aprotinin, 1 mmol/L sodium orthovanadate. After centrifugation
for 20 min at 4 °C, the supernatant was boiled for 2 min in an equal volume
of Laemmli sample buffer (Bio-Rad), and then analyzed by SDS polyacrylamide
gel electrophoresis. The proteins were transferred onto nitrocellulose membrane
and reacted with either anti-GHR antibody mAb263 followed by horseradish peroxidase-labeled
anti-mouse IgG or with anti-phosphotyrosine (PY20) peroxidase conjugate and
revealed by chemilumine-scence[19].
1.6 Lipid synthesis
Lipogenesis was estimated as described by Moody et al.[20] with minor modifications.
The transfected cells were grown to confluence in six-well plates and incubated
in serum-free DMEM/F12 medium supplemented with 10 g/L BSA for 12 h. The assay
was initiated by adding 1 ml of Krebs-Ringer-HEPES buffer containing 10 g/L
BSA, 0.55 mmol/L glucose, 0.5 μCi of 140 Ci/mmol [3H]-glucose and 100 nmol/L
oGH. The incubation was continued for 2 h and terminated by washing with cold
phosphate-buffered saline followed by the addition of 0.5 ml of 0.5 mol/L
NaOH, 0.1% Triton X-100. Solubilized cells were transferred to scintillation
vials containing 3.5 ml of toluene scintillant, and lipid-incorporated radioactivity
was measured in the organic phase by scintillation counting. Results were
expressed as the percentage of induction compared to that assayed in cells
transfected with pcDNA3-gpGHR.
1.7 Protein synthesis
The transfected cells were grown to confluence in 60-mm cell culture dishes,
washed with phosphate-buffered saline and incubated in serum-free medium before
incubating with 100 nmol/L oGH for 12 h. Protein synthesis was estimated by
the incoporation of [3H]-leucine into proteins precipitated by trichloroacetic
acid[17]. The cells were pulse-labeled with 3 μCi [3H]-leucine for 2 h, washed
four times with cold phosphate-buffered saline, and solubilized in 2 ml of
1 g/L SDS. The proteins were precipitated by the addition of 200 μl of trichloroacetic
acid and incubated for 60 min at 4 °C. The precipitate was collected on glass
fiber filters and washed twice with 4 % trichloroacetic acid and 100 % ethanol
(2 ml). Radioactivity on the filters was determined by liquid scintillation
counting. Results were presented as the fold of stimulation relative to that
assayed in cells transfected with pcDNA3-gpGHR.
2 Results
The mutations of gpGHRY168H and gpGHRS332Y were verified by DNA sequencing.
The full open reading frames of cDNA of gpGHR or mutant gpGHRs were cloned
into the mammalian expression vector, pcDNA3 and the resulting vectors were
transiently transfected into COS-7 or CHO cells.
2.1 Expression of gpGHR in COS-7 cells and binding assay
From Fig.1, it was found that a specific band of around 2.1 kb was detected
in the total RNA from the COS-7 cells transfected with pcDNA3-gpGHR but no
such band was found in the COS-7 cells transfected with pcDNA3, indicating
that the pcDNA3-gpGHR was transcribed into its mRNA in the COS-7 cells.
To confirm the expression of the gpGHR in COS-7 cells, Western blot analysis
was performed using a monoclonal anti-GHR antibody(mAb263). A distinct band
with molecular weight of around 92 kD was detected in COS-7 cells transfected
with pcDNA3-gpGHR but not in normal COS-7 cells (Fig.2). Specific binding
of [125I]-labeled bovine GH to COS-7 cells transfected with pcDNA3-gpGHR
was inhibited by unlabeled bovine GH in a dose-dependent manner (Fig.3). When
the ratio of bound to free [125I]-labeled bovine GH was plot versus
bound bovine GH, a straight line was obtained (Fig.3), from which the association
constant (Ka) was calculated to be 1.3×109 (mol/L)－1. The specific binding
of [125I]-labeled human GH to COS-7 cells transfected with pcDNA3-gpGHR
was inhibited by unlabeled human GH and partially inhibited by bream GH and
ovine prolactin (data not shown here). These results are similar to those
obtained using the guinea pig liver membrane[21, 22].
Fig.1 RT-PCR analysis
of total RNA from the COS-7 cells
1, total RNA from COS-7 cells transfected with pcDNA3; 2, total RNA from COS-7
cells transfected with pcDNA3-gpGHR; 3, marker.
Fig.2 Western blot
analysis
1, extract of normal cells; 2, extract of cells transfected with pcDNA3-gpGHR.
Fig.3 Competitive binding
and Scatchard plot analysis
50 000－100 000 cpm of [125I]-bGH were incubated for 4 h at 37 °C
with about 106 COS-7 cells transfected with pcDNA3-gpGHR in the presence or
absence of increasing amounts of unlabeled bGH. Each point is the mean of
triplicate determinations.
2.2 The biological
functions of gpGHR and its mutants expressed in CHO cells
The receptor binding assay showed that gpGHRs expressed in CHO cells could
bind with [125I]-oGH and the mutation of gpGHR at position 168 or 332 did
not obviously affect the interaction between gpGHR and its ligand (Fig. 4)
Fig.4 Comparison of
binding capacity of [125I]-oGH to the wild gpGHR and mutant gpGHRs
expressed in CHO cells
50 000－100 000 cpm of [125I]-oGH were incubated for 4 h at 37°C
with 106 CHO cells transfected with pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y,
respectively. Results are presented as x ± s from triplicate assays
of the -fold binding of the cells transfected with pcDNA3-gpGHR.
It was known that GH increased
the protein synthesis[23]. This effect was determined in the CHO cells transfected
with pcDNA3-gpGHRs. The oGH stimulated protein synthesis is shown in Fig.5.
The tyrosine substitution of serine at position 332 in gpGHR resulted in a
259.4% increase and histidine substitution of tyrosine at position 168 a 56.8%
increase in the total cellular protein synthesis in comparison with that of
cells transfected with pcDNA3-gpGHR.
Fig.5 oGH-stimulated
protein synthesis in CHO cells transfected with pcDNA3-gpGHR, pcDNA3-gpGHRY168H
or pcDNA3-gpGHRS332Y
Protein synthesis was estimated by the incorporation of [3H]-leucine
into trichloroacetic acid-precipitable proteins collected on glass fiber filters.
Results are presented as x ± s from triplicate determinations of the
-fold stimulation of the cells transfected with pcDNA3-gpGHR.
GH has been demonstrated
to stimulate the lipogenesis in GHR cDNA-transfected cells[24]. We determined
the oGH-stimulated lipogenesis in CHO cells transfedcted with pcDNA3-gpGHRs
and found that both mutations of gpGHR decreased the lipogenesis, being about
20 % lower than that in CHO cells transfected with pcDNA3-gpGHR (Fig.6).
Fig.6 oGH-stimulated
lipogenensis in CHO cells transfected with pcDNA3-gpGHR, pcDNA3-gpGHRY168H
or pcDNA-gpGHRS332Y
Lipid synthesis was estimated by the incorporation of [3H]-glucose
into the lipid-soluble fraction of the cells. Results are presented as x
± s from triplicate determinations of the -fold stimulation of the
cells transfected with pcDNA3-gpGHR.
Fig.7 Western blot analysis of the phosphorylation of JAK2 co-expressed in
CHO cells with gpGHR or its mutants
1, pcDNA3-gpGHRS332Y; 2, pcDNA3-gpGHRY168H; 3, pcDNA3-gpGHR; 4, pcDNA3; 5,
normal cells.
GH binding to its receptor
is known to recruit and activate the receptor-associated JAK2 which in turn
phosphorylates tyrosines of itself and GHR[25, 26]. When the CHO
cells transiently transfected with JAK2 cDNA alone or with pcDNA3-gpGHR, pcDNA3-gpGHRY168H,
and pcDNA3-gpGHRS332Y, respectively, were treated with oGH, the phosphorylation
of JAK2 was detected by anti-phosphotyrosine antibody (PY20). The results
showed that mutation of serine residue at position 332 to tyrosine increased
the phosphorylation of JAK2, while mutation of tyrosine residue at position
168 to histidine decreased it (Fig.7), indicating that tyrosine residues at
168 and 332 positions are very important for JAK2 phosphorylation.
3 Discussion
The evolutionary position of guinea pig has not been unequivocally clarified.
Although it was traditionally classified as a New World hystricomporph rodent,
it often showed anomalous morphological and molecular features in comparison
with other eutherian mammals. In 1991, Graur et al.[2] raised the question
of whether the guinea pig was a rodent. Some phylogenetic analyses of amino
acid sequence data imply that the guinea pig diverged before the separation
of the primates and the artiodactyls from the rodentia. In 1996, D’Erchia
et al.[1] presented findings based on the sequence of complete mitochondria
genome of the guinea pig using three different analytical methods, which strongly
contradicted rodent monophyly.
GHR is a receptor protein evolved rapidly, so GHRs among different species
have significant differences, reflecting the evolutionary process of GHR gene.
In our previous study, we observed that gpGHR presented over 72 % identity
with GHRs of human, pig and bovine, but much lower identity (63%) with GHRs
of rat and mouse, supporting the opinion that the guinea pig is not a rodent.
The abnormalities of guinea pig growth in response to GH evoked our interest
to study the cloning and functional expression of gpGHR cDNA. COS-7 and CHO
cells were used as host cells. In COS-7 cells transfected with pcDNA3-gpGHR,
gpGHR was identified. The Western blot analysis showed that a band of 92 kD,
corresponding to gpGHR, was detected by anti-GHR antibody mAb263. Binding
assay showed that gpGHR bound with bGH and the equilibrium association constant
(Ka) was 1.3×109 (mol/L)－1, similar to the report that oGH and bGH showed
high affinities to both gpGHR and guinea pig GH binding protein[12, 21, 22].
These data confirmed that the gpGHR we obtained was bioactive.
By amino acid sequence alignment, substitutions of some conserved amino acids
were found in gpGHR, including 168 and 332 that were considered to be important
for GHR functions[27, 28]. In order to understand the effect of these substitutions
in gpGHR on its biological functions, we prepared two mutants of gpGHR in
which tyrosine168 and serine332 were replaced by the conserved amino acids
in mammalian GHR, i.e. by histidine and tyrosine respectively. When CHO cells
were transfected with pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y,
the ligand binding assay showed that site-directed mutations of gpGHR at 168
and 332 did not obviously affect the ligand binding. However, the protein
synthesis stimulated by oGH in these cells was greatly increased to 359.4
% for gpGHRS332Y and to 156.8% for gpGHRY168H as compared with gpGHR, and
the oGH-stimulated lipogenesis was decreased. Our results indicated that the
substitutions of tyrosine with serine at position 332 and histidine with tyrosine
at position 168 in gpGHR significantly weakened its role in GH-stimulated
protein synthesis and slightly strengthened its role in GH-stimulated lipogenesis.
Our experimental data also supported the existence of distinct receptor domains
regulating GH-stimulated actions.
It was known that GH binding to its receptor recruits and activates the receptor-associated
JAK2 which in turn phosphorylates tyrosines of itself and the GHR. These phosphorylated
tyrosines form binding sites for a number of downstream signaling proteins[29].
We found that the phosphorylation of JAK2 was increased in the presence of
gpGHR and its mutants, when CHO cells co-transfected with JAK2 cDNA and pcDNA3-gpGHRs
were treated with oGH. The ability of gpGHRs to increase tyrosine phosphorylation
of JAK2 under oGH stimulation was found to be: gpGHRS332Y > gpGHR >
gpGHRY168H, which was not consistent with GH-stimulated protein synthesis
or lipogenesis, suggesting some disjunction between activation of JAK2 and
GH-stimulated actions. Thus, our study provides the experimental evidence
in molecular level that gpGHR may mediate the GH-stimulated metabolic actions
and the substitutions of some conserved amino acids in gpGHR also change the
post-binding signaling. In addition, our data further demonstrated that tyrosine
332 in gpGHR was very important for GH-stimulated protein synthesis and that
distinct receptor domains regulating GH-stimulated actions may exist in gpGHR.
Acknowledgement We are grateful to Dr. CHENG Christopher H.K. of the
Department of Biochemistry, Chinese University of Hong Kong for generously
providing experimental reagents and valuable suggestions during this work.
We also thank professor ZHANG You-Shang for his critical reading of the manuscript
and Ms. LIU Li for her technical assistance.
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