From the Growth hormone receptor (GHR)-mediated activity
of ruminant placental lactogens (PLs) and ovine (o) GH was compared,
using cells transfected with full size human (h), rabbit (rb), and
oGHRs. All three PLs acted as agonists in heterologous bioassays,
whereas in homologous bioassays in cells transfected with oGHRs they
antagonized the oGH activity. Despite these differences, oGH and PLs
bound with similar affinity to the oGHR extracellular domain
(oGHR-ECD), indicating that the binding occurs through hormone site I. Gel filtration of complexes between oPL and oGHR-ECD showed a 1:1 stoichiometry, confirming this conclusion. The oPL T185D and bPL T188D,
which exhibited weak biological activity mediated through GHRs, behaved
as site I antagonists, whereas oPL G130R and bPL G133R formed a 1:1
complex with GHR-ECDs and bound to h/rb/oGHR-ECDs with affinity similar
to that of wild-type oPL. They had no agonistic activity in all models
transfected with h/rb and oGHRs, but were antagonistic to all of them.
In conclusion, ruminant PLs antagonize the activity of oGH in
homologous systems, because they cannot homodimerize oGHRs, whereas in
heterologous systems they act as agonists. The structural analysis
hints that minor differences in the sequence of the GHR-ECDs may
account for this difference. Since the initial step in the activity
transduced through cytokine/hemapoietic receptors family is receptor
homodimerization or heterodimerization, we suggest that the question of
homologous versus heterologous interactions should be reexamined.
Ruminant and other species' placentas synthesize and secrete
unique proteins belonging to the growth hormone/prolactin
(GH/PRL)1 family and are
termed placental lactogens (PLs). Ovine (o) (1, 2), bovine (b) (3) and
caprine (c) (4) PLs were isolated from placentas and found to be
22-23-kDa proteins that are structurally closer to the respective PRLs
than to GHs (5). Recombinant oPL (6, 7) and bPL (8), and recently cPL
as well (9), have been prepared, and the recombinant proteins can now
be produced in amounts that allow in vivo studies. Cloning
of cPL enabled us to compare its primary structure to that of oPL and
bPL. The similarity between cPL and oPL exceeds the one between bPL and oPL or cPL. In contrast to these, the similarity between the
corresponding GHs and PRLs in the three ruminant species is much
greater (5). It has been proposed that this finding suggests into
possible different physiological roles that PLs may play in the three
species, but this point has not yet been proven (5). Recently, we have determined the three-dimensional structure of the 1:2 complex between
oPL and the rPRLR-ECD, and have been able to identify the 25 residues
of oPL that participate in site I of the hormone and 24 residues that
participate in site II (10). This finding, along with our former direct
and indirect experiments using recombinant extracellular domains (ECDs)
of GH and PRL receptors, suggests that the initial step in PL signal
transduction consists of dimerization of the respective receptor, as is
well documented for GHs (11).
One early observed, unique property of ruminant PLs is their ability to
bind to both PRL and GH receptors, including receptors of hGH (for
review, see Refs. 12-14). Comparative binding studies of oPL and oGH
to fetal liver microsomes, along with demonstration of oGHR mRNA in
fetal liver, prompted several research groups to suggest that oGH and
oPL bind to identical or at least related proteins (15-17). Using a
similar approach, we have previously studied the biological activity of
the three ruminant PLs in several in vitro bioassays, in
which the signal was transduced through heterologous (mouse, rabbit,
and human) GHRs (7, 9, 18-22). In all cases the activity of bPL, oPL,
or cPL was equal to that of oGH, bGH, or hGH despite some differences
in affinity. Furthermore, mutagenesis of bPL allowed us to prepare
several bPL analogues with the selectively reduced or abolished
somatogenic activity, whereas the lactogenic activity (as judged by the
Nb2 rat lymphoma cell proliferation bioassay) was not
changed (20-22). These experiments were paralleled by
protein-interaction studies that showed that bPL, similarly to hGH, is
capable of forming a 1:2 complex with h- and rbGHR-ECDs. In contrast to
these results, Staten et al. (23) reported that bPL
interacts with bGHR-ECD in a 1:1 stoichiometry, whereas bGH forms a 1:2
complex. More recently, the same group briefly reported that bPL
antagonized bGH action in Baf/3 cells stably transfected with bGHRs and
was devoid of proliferative activity (24). These reports prompted us to
reexamine whether the somatogenic activity of ruminant PLs is relevant
in homologous species. To answer this question, we developed an
oGHR-mediated bioassay in 293 cells, prepared recombinant oGHR-ECD, and
used them in the present study.
Materials--
Recombinant bPL, bPL G133R, bPL K73D, bPL T188D,
oPL, oGH, and non-glycosylated human GHR-ECD were prepared as described
previously (7, 8, 18, 20-22, 25). Recombinant caprine (c) PL was recently prepared in our lab (9). Rabbit (rb) non-glycosylated GHR-ECD
was prepared in our laboratory, and its preparation will be described
elsewhere. Carrier-free Na125I was purchased from NEN Life
Science Products. Molecular weight markers for SDS-PAGE, RPMI 1640 medium, lysozyme, nalidixic acid, Triton X-100, bovine serum albumin
(radioimmunoassay grade) were obtained from Sigma. SDS-PAGE reagents
and protein assay kit were purchased from Bio-Rad. Fetal calf serum and
horse serum were purchased from Labotal Co. (Jerusalem, Israel), and a
SuperdexTM 75 HR 10/30 column and Q-Sepharose (fast flow)
were obtained from Pharmacia LKB Biotechnology AB (Uppsala, Sweden).
All other chemicals were of analytical grade.
Construction of oPL Analogue T185D Expression
Vector--
Synthetic gene fragment for preparation of oPL analogue
T185D was constructed using polymerase chain reaction (PCR) technology. Oligonucleotides (primers) were used to generate a double-stranded DNA
from a template, pET-8-oPL (7), for subcloning. An NcoI site
(underlined) was created with a forward primer at the 5' end of the
gene, which also added an initiator methionine codon immediately
upstream to the first mature codon (alanine)
(5'-GGAGATATACCATGGCACAGCATCCACC-3') and a
AflII site (underlined), was included close to mutation area
at the 3' end of the gene, with a reverse mutant primer
(5'-GCACTTAAGTATCCGGAGGTAGTCGTAAATTTTAC-3'). The reverse
mutant primer encoded the mutation of interest. The PCR reaction was
conducted using Taq polymerase in a capillary PCR apparatus
(Idaho Technology, Idaho Falls, ID), with the following program:
15 s × 94 °C, 25 cycles of (0 s × 94 °C, 0 s × 55 °C, 25 s × 72 °C), 15 s × 72 °C. The PCR
product was gel-purified, subcloned into pGEM-T vector (Promega,
Madison, WI), and transfected to JM-109 Escherichia coli
cells. The NcoI/AflII insert was isolated and
ligated into the parental vector (pET8) encoding for the wild-type oPL
(7), from which the respective NcoI/AflII insert
was removed. Subsequently, the pET8/oPL T185D cloning vector was
isolated and used for transformation of BL21 E. coli cells.
One of the isolated colonies that expressed the protein after induction
by isopropyl-1-thio- Construction of oPL Analogue G130R Expression Vector--
The
oPL analogue expression vector was modified with the
QuickchangeTM mutagenesis kit (Stratagene, La Jolla, CA)
according to the manufacturer's instructions, using two
complementary primers: (5'-GGCCAAAGTACTTGTAGAACGTGTGGAAGTGATAC-3') and
(5'-GTATCACTTCACACGTTCTACAAGTACTTTGGCC-3'). These primers were
designed to contain a specific restriction site (AflIII), still conserving the same amino acid sequence, for colony screening. The procedure included 12 PCR cycles and the use of Pfu
polymerase enzyme for the reaction. The template used for mutant
construction was wild-type oPL in pMON3922 (26). The mutated construct
was then digested with DpnI restriction enzyme, which is
specific to methylated and hemimethylated DNA (target sequence:
5'-Gm6ATC-3'), in order to digest the template and to
select for mutation-containing synthesized DNA. The vector was then
transfected into XL1-competent cells. Ten colonies were then screened
for mutation, using the specific restriction site designed, and
revealed 80% efficiency. Two colonies were sequenced and confirmed to
contain the mutation and no undesired misincorporation of nucleotides.
Expression, Refolding, and Purification of oPL
Analogues--
E. coli MON105 cells transformed with the
expression plasmids containing the oPL G130R were incubated in 500 ml
of Terrific Broth (TB) medium (27) by shaking at 200 rpm at 37 °C in
2-liter flasks to an A600 of 0.9, after which
nalidixic acid (25 mg/flask) was added. The cells were incubated for an
additional period of 4 h, harvested by 5-min centrifugation at
10,000 × g, decanted, and then frozen at Construction of oGHR-ECD Expression Vectors and Preparation of
the Recombinant Protein--
Synthetic oligonucleotides (primers) were
used to generate a double-stranded DNA from a template of full-size
oGHR (35), in addition to restriction-enzyme sites for cloning.
The forward primer 5'-GTGGCAGGCTCCACCATGGCTTTTTCTGGGAGTG-3'
encoded an NcoI restriction-enzyme site (underlined) and an
initiator methionine codon immediately upstream to alanine codon and
the oGHR-ECD. The reverse primer 5'-
CCAAAGATAATAATTAAGAACCAAGCTTACTGGAAATC-3' encoded the
HindIII restriction site (underlined) and TAA termination codon immediately after the final codon (Gln).The PCR reaction was
conducted using the Taq polymerase in capillary PCR
apparatus (Idaho Technology), with the following program: 2 min × 94 °C, 30 cycles of (0 s × 94 °C, 0 s × 60 °C,
30 s × 72 °C), 2 min × 72 °C. The PCR product was
cleaned using a Promega PCR cleaning kit (Promega, Madison, WI),
digested with NcoI and HindIII restriction enzymes, and, after heat inactivation of the enzymes, ligated to
parental vector pMON3401 (26) linearized with the same enzymes. The
ligation product was transfected to JM-109 E. coli cells, prior to transformation of MON105 cells. Automatic DNA sequencing was
performed to confirm the proper sequence. One of the expressing clones
was chosen for large scale expression, which was performed as described
above for oPL G130R analogue. Preparation of inclusion bodies and the
refolding procedure was identical to that of oPL analogue G130R, except
that after the solubilization in 4.5 M urea, the solution
was stirred at 4 °C for 48 h, prior to dialysis against 10 mM Tris-HCl buffer pH 8.0 and subsequent purification on a
Q-Sepharose column (2.6 × 7 cm), preequilibrated with the same
buffer. The monomeric fraction was eluted with 150 mM NaCl at the same buffer, dialyzed, and lyophilized.
Binding Experiments--
Binding to soluble ovine, rabbit, and
human GHR-ECDs was carried out as described previously (7, 28). The
ligand was 125I-oGH or 125I-oPL, and the
competitors were oGH, bPL, cPL, oPL, and oPL analogues. Iodination of
oGH and oPL was performed according to the protocol described
previously (29).
Determination of Monomer Content and Complex Formation--
High
performance liquid chromatography gel-filtration chromatography on a
SuperdexTM 75 HR 10/30 column was performed with 200-µl
aliquots of Q-Sepharose-column-eluted fractions, freeze-dried
samples dissolved in H2O, or complexes between the soluble
recombinant GHR-ECDs and oGH, oPL, or oPL analogues, using
methods described previously (7, 30).
In Vitro Bioassays in Stably Transfected FDC Cells--
Two
in vitro bioassays, in which the signal was transduced
through somatogenic receptors, were based on the proliferation of
FDC-P1 cells transfected with rabbit (clone FDC-P1-3B9) or human (clone
FDC-P1-9D11) GHRs (31, 32) as described before (21). Cell growth was
determined by counting the cells with a Coulter counter (Coulter
Electronics Inc., Hialeah, FL), and the number of doublings was
calculated as described previously (33).
In Vitro Bioassays in Transiently Transfected 293 Cells--
Two
additional bioassays were carried out in a 293 cell line transiently
transfected with hGHR or oGHR and co-transfected with a plasmid that
carries the luciferase reporter gene under the control of a six-repeat
sequence of LHRE (lactogenic hormone response element with a Stat5
binding sequence) fused to a minimal thymidine kinase promoter. The
transfection and the bioassay were carried out as described previously
(34). The vector encoding for full-size oGHR in SP72 vector was
obtained from Dr. Tim Adams (35). It was first digested with
XbaI at 37 °C for 1 h and then with EcoRI
at room temperature for 5 min. The reaction products were then
separated on 1.0% agarose gel, and the insert corresponding to ~3200
bases was purified and ligated to pcDNA3 mammalian expression vector, linearized with XbaI and EcoRI. The
ligated plasmid was propagated, isolated, and sequenced to ensure the
proper ligation.
In Vitro Bioassays in Nb2 Cells--
An in
vitro bioassay, in which the signal was transduced through
lactogenic receptors, was performed in rat Nb2-11C lymphoma cell proliferation bioassay, in which the original protocol was slightly modified (33).
Purification of oGHR-ECD and oPL Analogues--
The profile of
oGHR-ECD elution from a Q-Sepharose column shows that over 60% of the
protein was eluted with 0.15 M NaCl (data not shown). Every
fifth tube was analyzed for monomer content, and fractions containing
>98% pure monomer were pooled, dialyzed against NaHCO3
(1:5 salt:protein ratio), and lyophilized. The overall yield was 110 mg
of monomeric protein obtained from a 5-liter fermentation culture. This
fraction was further used for binding and biological studies. Fractions
eluted with 0.4 M NaCl consisted mainly of oligomers (data
not shown). SDS-PAGE of the pooled monomer fraction, performed with and
without Gel-filtration Experiments--
The stoichiometry of the
interactions between soluble human, rabbit, and ovine GHR-ECDs and oPL,
oPL T185D, oPL G130R and, in the latter case, also with oGH was studied
by gel filtration. The complexes were prepared at a constant
concentration (1.75 µM) of the hormones and variable
concentrations (1.75-5.25 µM) of the respective receptor
ECDs. Ovine PL formed a 1:2 complex with hGHR-ECDs, which was eluted at
the retention time of 11.22 min (Fig.
1A), confirming the previous
results (7). At the oPL:hGH-ECD ratio of 1:3, an excess of hGHR-ECDs
was observed. In contrast, both oPL T185D and G130R formed only 1:1
apparent complexes in both cases (Fig. 1A). This conclusion
was based on both a comparison of peak sizes, their retention times
(11.94 and 12.44 min, respectively), and the fact that, at 1:2
analogue:hGHR-ECD ratios, an excess of the latter could be seen. The
shape of the peaks of the oPL T185D:hGHR-ECD complexes and the fact
that the retention time values shift forward by increasing the
ECD:analogue ratio indicate that a very weak 1:1 complex that
dissociated in the course of chromatography was likely formed. Similar
results were also obtained with rbGHR-ECD (Fig. 1B). In
contrast, oPL was capable of forming only a 1:1 complex with oGHR-ECD,
whereas with oGH, a clear 2:1 complex was detected (Fig.
1C). The oPL analogue G130R acted similarly to oPL. The
results of the interaction of oPL T185D with oGHR-ECD do not, however,
indicate formation of either 1:1 or 1:2 complexes, which likely results
from the loss of binding capacity, as shown in the next section.
Binding Experiments--
As oPL is capable of binding to both
homologous and heterologous somatogenic receptors, several binding
assays were performed. The results of a comparative binding assay in
which the ability of oPL, oPL G130K, and oPL T185D to compete with
125I-oPL or 125I-oGH for binding to recombinant
human, rabbit, and ovine GHR-ECDs is shown in Fig.
2. The results (for binding of bPL and
cPL to human and rabbit GHR-ECD, see our previous articles Refs. 7, 9,
and 20-22) clearly demonstrate that all tested hormones (except oPL
T185D) have an almost identical capacity to compete with the ligand for
binding to h-, rb-, and oGHR-ECDs, respectively. The oPL analogue T185D
exhibited 90 and >500 times lower competitive capacity in the binding
to h- and rbGHR-ECDs, respectively, and no capacity at all in binding
to oGHR-ECD.
In all cases, results were calculated using both one-site and two-site
analysis (37). In the two-site analysis, calculations were based upon
an assumption that both sites contribute equally to the binding. In all
analyses, the quality of fit for the nonlinear correlation was very
high (R2 > 0.97), making the choice of an
appropriate model difficult. Therefore, additional arguments were taken
into consideration. (a) In all bioassays (see below), oPL
G130R exhibited antagonistic activity, strongly suggesting that its
ability to bind the receptor through site II was severely or completely
damaged. (b) The same argument holds for the activity of oPL
in 293 cells transiently transfected with oGHR. (c) In the
previously described gel-filtration experiments, oPL G130R formed only
a 1:1 complex with all three GHR-ECDs, whereas a 1:2 stoichiometry was
observed for the interaction of oPL with human and rabbit GHR-ECDs but
only a 1:1 stoichiometry with oGHR-ECD. (d) The displacement
curves oPL, oPL G130R, bPL, cPL, and oGH in the binding to oGHR-ECD
were quite similar (the respective IC50 values were 3.2, 1.9, 1.5, 4.0, and 1.1 × 10 Proliferative Activity of oPL and oPL Analogues G130R and T185D in
FDC-P1 Cells--
Ovine PL was an agonist in FDC-P1 cells, transfected
with either rabbit (Fig. 3A)
or human (Fig. 3C) receptors. The respective EC50 values of 2.5 × 10 Diverse Activity of oGH, oPL, cPL, bPL, oPL T185D, oPL G130R, bPL
T188D, bPL K73F, and bPL G133R in 293 Cells Transiently Transfected
with hGH and oGH Receptors--
Ovine PL stimulated the expression of
LHRE promoter-linked luciferase activity in cells transfected with
hGHR. The respective EC50 values for oPL and hGH were
5.3 × 10 The main results of the present study that were summarized in
Table I show that all three ruminant PLs
acted as antagonists in 293 cells transiently transfected with
full-size oGHR. The antagonistic activity cannot be attributed to an
improper refolding, as all three molecules were active in the
lactogenic receptor-mediated Nb2 rat lymphoma cell bioassay
and in FDC-P1 cells stably transfected with h- or rbGHRs (see above and
Refs. 7, 9, and 18-22). The finding that all three PLs were agonists
in both FDC-P1 and 293 cells transfected with hGHRs further emphasizes
that the activity is dependent upon the chosen type of receptor and not
upon the cell model. We also found that recombinant or native oPLs lack the ability of oGH to inhibit the insulin stimulation of lipogenesis in
homologous adipose tissue. Furthermore, oPL could not inhibit this oGH
activity.2 In contrast, all
three ruminant PLs exhibited PRL-like activity not only in a
heterologous Nb2 cell proliferation bioassay (7, 9, 18),
but also in ovine mammary gland explants or ovine acini culture
bioassays based on stimulation of The proposed notion is fully supported by the present and former
binding and interaction studies. Whereas oPL, bPL, and cPL were capable
of forming 1:2 complexes with hGHR-ECD (see Fig. 1, and Refs. 7, 9, 19,
and 21) or rbGHR (Fig. 1),5
oPL (Fig. 1) and bPL (23) formed only 1:1 complexes with homologous GHR-ECDs. Since GH-induced homodimerizations of GHRs are sequential (39), we suggest that the ability of oPL's site II for homologous interaction is compromised and they act as site II antagonists (40),
thus interacting with the receptors exclusively through site I. Binding
studies (Fig. 2) along with the results of Gluckman's group (16) and
ours (7, 9, 21, 22) suggest that oPL, cPL, and bPL bind with high
affinity to membrane-embedded somatogenic receptors and to GHR-ECDs. We
conclude that this binding occurs through oPL's site I only. More
detailed arguments for this notion were presented under "Results"
and in our recent paper describing the activity of hGH des(1-6,14)
analogue (34).
The results obtained with o/bPL analogues also support our hypothesis.
The oPL analogues T185D, which exhibited weak mitogenic activity in
FDC-P1 cells transfected hGHR or rbGHR, also had a reduced capacity
(Fig. 2, A and B) or no ability at all (Fig. 2C), to compete with the ligand for the binding to human,
rabbit, or ovine GHR-ECDc (Fig. 2). This analogue was a weak agonist in 293 cells transfected with human GHRs (Fig. 4A), but was
inactive and did not antagonize oGH activity in 293 cells transfected
with oGHR (Fig. 4, C and D). In contrast, the oPL
analogue G130R and bPL analogue G133R that form a 1:1 complex with
GHR-ECDs (Fig. 1, and data not shown) and bind to human, rabbit or
ovine GHR-ECDc with affinity similar to that of wild-type oPL (Fig. 2
and Ref. 22), were not active in cells transfected with human, rabbit, and bovine GHRs, but were antagonistic in all of them (Figs. 3 and 5,
and Ref. 22).
The key event that leads to biological response for GHR and PRLR is
receptor homodimerization (41), which initially occurs via binding of
the ligand to two extracellular domains of these receptors. The
extracellular domains of these receptors(42, 43) consist of two FBN-III
type domains (D1 and D2) with a short helical region connecting them.
The interdomain flexibility, as well as the variation of binding loop
conformation, contributes to the structural plasticity of the receptor
to form either the cognate or coincidental hormone- receptor complexes.
Receptor interdomain flexibility has already been observed in the
structures of hGH:hGHR (42), hGH:PRLR (43), and
oPL:rPRLR.6 The structural
and biochemical aspects of the hGH:hGHR complex have been extensively
studied (42, 44, 45), and the hGHR is activated via sequential
dimerization (11, 40), as was also shown for erythropoietin receptor
(46). The initial GH:GHR 1:1 complex must have the correct orientation
to allow binding of receptor 2 to both the GH site II and receptor 1, otherwise, dimerization would not occur, and signaling will be
abolished. Mutations on GH site II (40) or in the receptor-receptor
interface (47) lead to non-productive complexes. In the structure of
the active oPL-rPRLR 1:2 complex, a similar homodimerization has been observed,6 and we assume that a similar mode of
hormone-receptor assembly will occur in the PL, GH active receptor
complexes. The results that ruminant PLs act as agonists toward both
hGHR, and rbGHR, and as antagonists to oGHR (Table I) can clearly be
attributed to their capability of induce either 2:1 active or 1:1
inactive complexes. The sequence alignment (Fig.
6) shows that there are several notable
differences, especially in the binding loops, between human or rabbit,
and ovine or bovine GHRs. The main differences are as follows: a
four-residue deletion can be observed at the L2 loop (residues 77-80),
two substitutions at the L3 loop (I103V, I105T), and a single
substitution at L4 (E127D). The L2 binding loop in the hGHR contributes
very little to the binding of hGH, and a substantial segment of it does
not have clear electron density maps, which may indicate high
flexibility. The other three residues, Ile-103, Ile-105, and Glu-127,
are part of the hGHR functional epitope (48) and interact with both
sites of the hGH. This may lead to the assumption that these
differences in primary structures, especially in conserved binding
determinants, may attribute the different biological activities. It is
most probable that the overall orientation, in which the coincidental
1:1 PLs-oGHR complexes exist, does not permit the correct recognition
of receptor 2 that leads to the formation of active 2:1 complexes.
Institute of Biochemistry,
ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
-D-galactopyranoside was selected
for large scale preparation. Automatic DNA sequencing was performed to
confirm the proper sequence.
20 °C.
Over 95% of the expressed protein was found in the inclusion bodies,
which were prepared as described previously for bPL (18). The inclusion
body pellet obtained from 2.5 liters of bacterial culture was
solubilized in 200 ml of 4.5 M urea buffered with 10 mM Tris base. The pH was increased to 11.3 with NaOH,
cysteine was added to 0.1 mM, the clear solution was
stirred at 4 °C for 1 h, diluted with two volumes of cold water, and dialyzed for an additional period of 48 h against
5 × 10 liters of 10 mM Tris-HCl, pH 9. The solution
was subsequently loaded at 120 ml/h onto a Q-Sepharose column (2.6 × 7 cm), pre-equilibrated with 10 mM Tris-HCl, pH 9.0 at
4 °C. Elution was carried out using a discontinuous NaCl gradient in
the same buffer at a rate of 120 ml/h, and 5-ml fractions were
collected. Protein concentration was determined by absorbance at 280 nm, and monomer content by gel-filtration chromatography on a
SuperdexTM 75 column. The oPL analogue T185D was expressed,
according to the procedure described for the wild-type hormone (7).
Then it was refolded and purified as described above.
RESULTS
-mercaptoethanol according to Laemmli (36), revealed only
one band with a molecular mass of 28 kDa (data not shown). The
oligomeric fraction eluted with 0.4 M NaCl has also yielded
a main 28-kDa band, indicating that the oligomers were formed by a
non-covalent interactions. The preparation of oPL analogues (oPL G130R
and T185D) was carried out according to the protocol described for the
wild-type recombinant oPL (7). The monomeric fractions were eluted from
the Q-Sepharose column developed with 10 mM Tris-HCl buffer
at pH 9.0 by 0.05 M NaCl, dialyzed against
NaHCO3 at a 4:1 protein:salt ratio and freeze-dried. The
homogeneity of the purified proteins was also verified by SDS-PAGE
under reducing and non-reducing conditions (data not shown). The
biological activity of both analogues resulting from proper
renaturation was further evidenced by their ability to stimulate the
proliferation of the lactogenic receptor-mediated Nb2
bioassay (data not shown) and to bind to human, rabbit, and ovine
GHR-ECDs (Fig. 2) The relative activity of the G130R and T185D
analogues in Nb2 bioassay as compared with wild-type oPL was, respectively, 2.5% and 87%.
View larger version (22K):
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Fig. 1.
Gel filtration of oPL, oPL T185D, oPL G130R,
or oGH complexes with hGHR-ECD (A), rbGHR-ECD
(B), or oGHR-ECD (C) on a SuperdexTM
75 HR 10/30 column. Complex formation was carried out during a
20-30-min incubation at room temperature in TN buffer using various
ECD:hormone ratios. Aliquots (200 µl) of the incubation mixture were
then applied to the column, pre-equilibrated with the same buffer. The
initial hormone concentration (1.75 µM) was constant in
all cases. The column was developed at 0.8 ml/min, and protein
concentration in the eluate was monitored by absorbance at 280 nm.
View larger version (17K):
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Fig. 2.
Competition of unlabeled oPL ( ), oPL G130R
(
), oPL T185D (
), bPL (
), cPL (
), and oGH (*) with the
binding of 125I-oPL to rbGH-ECD (A) and
hGHR-ECD (B) and with the binding of
125I-oGH to oGHR-ECD (C). The results
of all specific bindings in the absence of competitor were normalized.
The actual specific bindings in panels A-C were,
respectively, 44%, 37%, and 25%.
9 M),
despite the fact that only the latter is an agonist in 293 cells
transiently transfected with oGHR. Taken together, it seems that under
the conditions in which the binding experiments were performed, the
radiolabeled ligand binds to site I only, giving the one-site model an advantage.
11 M
and 8.3 × 10
12 M were similar to those
previously reported for oPL (7) bPL, hGH (21) and cPL (9), and the
maximal activity of those three hormones was equal. The oPL analogue
T185D acted as a partial agonist in both types, and the respective
EC50 values were 1000-fold and 60-fold higher as compared
with oPL. In contrast, the oPL G130R was devoid of any agonistic
activity (Fig. 3, A and C). In both cell lines,
stimulated for proliferation with 1.8 × 10
10
M oPL, the oPL G130R analogue acted as a weak antagonist
with an IC50 of 4.3 × 10
8 M
in the case of FDC-P1-3B9 cells (Fig. 3B) and 3.0 × 10
8 M in the case of FDC-P1-9D11 cells (Fig.
3D).
View larger version (24K):
[in a new window]
Fig. 3.
Mitogenic activity of oPL ( ), oPL G130R
(
), oPL T185D (
) in FDC-P1-3B9 cells stably transfected with
rbGHR (A) and in FDC-P1-9D11 cells stably transfected
with hGHR (C) and antagonistic activity oPL G130R in
FDC-P1-3B9 cells (B) and in FDC-P1-9D11 cells
(D) cultured in the presence of 1.8 × 10
10 M oPL.
10 M and 2.7 × 10
10 M, and the maximal activities of both
hormones were equal (Fig. 4A).
The analogue oPL T185D exhibited agonistic activity 18 times weaker,
and oPL G130R was not active at all (Fig. 4A). The activity of oGH had in this model was very low (EC50 = 6.6 × 10
8 M; data not shown), consistent with the
inability of ruminant GHs to recognize primate GHR (38). In cells
stimulated for growth with 4.35 × 10
9 M
oPL, the oPL G130R analogue acted as an antagonist with an IC50 value of 3.7 × 10
8 M
(Fig. 4B), similarly to its action in FDC-P1 cells stably
transfected with hGHR (Fig. 3D). On the other hand, in cells
transfected with oGHR, only oGH acted as an agonist (EC50 = 1.3 × 10
9 M), whereas oPL, oPL T185D,
and oPL G130R were not active at all (Fig. 4C). The 5-fold
difference in the maximal induction of cells transfected with hGHR and
oGHR does not likely indicate a difference in signaling activity, but
rather results from the fact that in the transfection of the latter,
25-fold higher amount of cDNA (2.5 µg versus 0.1 µg)
was used. In 293 cells stimulated with 4.35 × 10
9
M oGH, both oPL and oPL G130R (but not oPL T185D) exhibited
antagonistic activity, and the respective IC50 values were
6.1 × 10
8 M and 3.5 × 10
8 M. The activity of bPL and cPL was
similar to that of oPL. In 293 cells transiently transfected with hGHR,
they acted as agonists (Fig.
5A) with the respective
EC50 values of 4.9 × 10
10 M
and 3.2 × 10
10 M, whereas in 293 cells
transfected with oGHR, they were not active (Fig. 5B) and
acted as antagonists (Fig. 5C). Interestingly, the
antagonistic activity of bPL in this experiment (IC50 = of 5.3 × 10
9 M), was higher than that of
cPL (IC50 = of 1.2 × 10
7
M), and even than that of oPL (IC50 = of
1.9 × 10
8 M; data not shown). Bovine PL
T188D, K73F, and G133R analogues were not active in 293 cells
transfected with oGHR, similarly to bPL (data not shown). However, bPL
G133R analogue was a potent antagonist, K73F was a weak antagonist, and
T188D analogue, similarly to oPL T185D, has no antagonistic activity
(Fig. 5D).
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[in a new window]
Fig. 4.
Agonistic activity of oPL ( ), hGH (
),
oPL G130R (
), oPL T185D (
), and oGH (*) in 293 cells transiently
transfected with full-size human (A) or ovine
(C) GHRs and antagonistic activity of oPL G130R (
)
in 293 cells transfected with hGHR (B) and oPL (
),
oPL T185D (
), and oPL G130R (
) in 293 cells transfected with oGHR
(D) cultured in the presence of 4.35 × 10
9 M oPL
(B) or oGH (D). The results are
presented as mean ± S.D.
View larger version (23K):
[in a new window]
Fig. 5.
Activity of bPL ( ) and cPL (
) in 293 cells transiently transfected with full-size hGHR (A),
and activity of bPL (
), cPL (
), and oGH (*) in 293 cells
transiently transfected with full-size oGHR (B);
antagonistic activity of bPL (
) and cPL (
) (C)
and of bPL (
), bPL T188D (
), bPL K73F (
), and bPL G133R (
)
in 293 cells transfected with oGHR (D) cultured in the
presence of 4.35 × 10
9
M oGH. The results are presented as mean ± S.D.
DISCUSSION
-casein synthesis (9)3 and in 293 cells
transiently transfected with ovine and bovine full-size
PRLR.4 Taken together, these
observations along with our previously presented findings provide a new
paradigm. In heterologous systems ruminant PLs act as GHRs' agonists,
whereas in homologous systems they are either inactive or they act as
antagonists.
Biological activity of oPL, bPL, cPL, oGH, and o/bPL analogues in vitro
and stoichiometry of their complexes with oGHR-ECD or bGHR-ECD (23)
View larger version (75K):
[in a new window]
Fig. 6.
Comparison of the primary structures of
human, rabbit, ovine, and bovine GHR-ECDs. strands in domain 1 are marked a-g, and, in domain 2, a-g;
?, disordered x-tal structure. hGH-(hGHR-ECD)2
contacts (L1-L6) were marked according to Refs. 52 and 53:
@@, sites I and II; $$, site I only. ECD-ECD contacts: ++, ECD 1;
xx, ECD 2; **, both ECDs. Amino acids that are identical in
ovine and bovine GHR-ECDs but different than in human and rabbit
GHR-ECDs are marked with bold letters. Missing
amino acids are marked by dots.
Our present results raise again the question about the physiological role of oPL and bPL in vivo and the way ruminant PL signal is transduced. Several studies aimed at the elucidation of oPL and bPL binding sites in homologous maternal and fetal liver have been extensively reviewed (13, 14). In fetal liver, specific PL binding sites were detected (16, 17, 49, 50), but their molecular nature has not yet been identified and an attempt to purify a unique oPL receptor was also not successful. We (51) and others (52) have demonstrated that bovine endometrium microsomes contain high affinity binding sites for bPL, which have low affinity for either bGH or bPRL. However, the interaction of homologous PRLs with their receptors is very transient and may be overlooked in classical binding studies (53). Therefore, the physiological relevance of the findings that suggested that oGH and oPL bind to a common receptor (16) and the demonstration of oGHR mRNA in fetal liver (15, 17, 49) is not yet clear. The in vivo activity of ruminant PLs in homologous species was also extensively reviewed (13, 14). It was suggested that PLs are involved in the mammotropic action and in maternal and fetal metabolism, but the receptors that transduce this effect have not yet been identified. Recent studies (54-57)7 indicate that oPL in vivo exhibits oGH-like mammogenic and growth-promoting activity in pseudo-pregnant ewes and lambs, but completely lacks the GH-like galactopietic properties. Similar result were obtained in cows, although in that case weak galactopietic activity was reported (58).
Staten et al. (23) reported that bPL forms only a 1:1
complex with the bGHR-ECD, raising the question of whether bPL
activates homologous somatogenic receptors. The same group has recently reported that bPL was not active and antagonized the proliferative activity of bGH in Baf/3 cells stably transfected with bGHR (24). These
results support our hyphothesis that all three ruminant PLs do not act
in vivo by activating homologous GHRs, unless they act as
antagonists, but the question how the homologous PL signal in ruminants
is transduced remains unanswered, since it seems unlikely that the PL
signal may be transduced without dimerizing the GH (or other) receptor.
Other alternatives must be considered. (a) The PL signal is
transduced through homologous PRLRs; (b) PL is capable of
heterodimerization of homologous GHR, through site I and PRLR through
site II; (c) a unique unidentified PLR exists; and
(d) a yet unknown variant of GHR, mutated at its ECD in a
way that allows dimerization of GHR is expressed in specific tissues or
under unique physiological conditions as suggested (14). Our present
state of art does not allow an educated choice. The findings showing
that ruminant PLs are capable of signaling through oPRLR show that
alternative a is feasible. Preliminary results based on
kinetic analysis by surface plasmon resonance hint that bPL and oPL are
capable of heterodimerizing bGHR- and bPRLR-ECDs,8 but obviously do
not prove that heterodimerization occurs in vivo. As
mentioned above, a specific PLR was neither cloned nor purified.
Therefore, despite the novel findings described in the present paper
the mode of signal transduction by ruminant PLs remains to be an
enigma. Since the initial step in cytokine/hemapoietic receptor
activation by hormones and growth factors is homo or heterooligomerization (59), we suggest that the question of homologous
versus heterologous interactions be reexamined. This is
particularly important, as in many cases human systems were studied
with non-primate agonists.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. A. M. de Vos and Dr. P. A Elkins from Genentech Inc. (South San Francisco, CA), for the information concerning the structure of the oPL:(rPRLR-ECD)2 complex and for reading and commenting this manuscript. We also thank Prof. P. A. Kelly and Dr. A. Tchelet from INSERM U366 (Paris, France) for providing us the 293 cells and the vectors encoding for luciferase and receptors, and Dr. Tchelet for close guidance. We acknowledge Dr. T. E. Adams from the University of Melbourne (Melbourne, Australia) for providing us the vector encoding for full-size oGHR, Nick R. Staten from Monsanto Co. (St. Louis, MO) for providing the expression vector and E. coli MON105 cells, and Dr. A. Levanon from Biotechnology General (Ness-Ziona, Israel) for recombinant hGH.
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FOOTNOTES |
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* This work was supported by Grant US-2643-95 from the USA-Israel Binational Agricultural and Development Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, P. O. Box 12, Rehovot 76100, Israel. Tel.: 972-8-948-9006; Fax: 972-8-947-6189; E-mail: gertler{at}agri.huji.ac.il.
2 A. Finlay, I. A. Forsyth, A. Gertler, and R. Vernon, unpublished results.
3 N. Daniel, J. Djiane, and A. Gertler, unpublished results.
4 D. Helman, A. Herman, and A. Gertler, unpublished results.
5 E. Sakal and A. Gertler, unpublished results.
6 A. M. de Vos, P. A. Elkins, and H. W. Christinger, unpublished results.
7 H. Leibovitch, E. Gootwine, and A. Gertler, unpublished results.
8 N. R. Staten, J. C. Byatt, and W. C. Warren, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: GH, growth hormone; PRL, prolactin; PRLR, prolactin receptor; GHR, growth hormone receptor; ECD, extracellular domain; PL, placental lactogen; h, human; b, bovine; o, ovine; r, rat; rb, rabbit; c, caprine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; LHRE, lactogenic hormone response element with a Stat5 binding sequence.
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REFERENCES |
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