From Biologie Cellulaire et Moleculaire,
¶ Structure des Proteines et Biochimie, and
Virologie et
Immunologie Moleculaire, Institut National de la Recherche Agronomique,
78352 Jouy-en-Josas, France
Received for publication, June 15, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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Receptors for the luteotropin/human
chorionogonadotropin hormone belong to the G-protein-coupled
receptor family by their membrane-anchoring domains. They also possess
a large extracellular domain (ECD) responsible for most of the
hormone-receptor interactions. Structure-function studies identified
several contacts between hormone and receptor ECD, but the precise
topology of the complex is still unknown because of the lack of
suitable heterologous expression means. Receptor ECDs exhibit leucine
repeats and have been modelized on the basis of the three-dimensional
structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up
to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and
surface plasmon resonance data were in favor of a unique dimer with a
1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted
to circular dichroism characterization; CD spectra deconvolution
indicated more than 25% The LH/hCG1 receptor,
like the other members of the glycoprotein hormone receptor family
(follicle-stimulating hormone and thyreo-stimulating hormone
receptors), is composed of two domains of almost equal size. The
N-terminal part, thought to form an extracellular domain (ECD), has
been shown to bind ligands with high affinity (1-3) while being
structurally related to the leucine-rich repeats (LRR) superfamily of
proteins. The C-terminal moiety, including the seven transmembrane
helices and their connecting loops, interacts with hormones and ECDs on
one side, whereas it contacts the transducing G-proteins
intracellularly. Dimeric glycoprotein hormones bound by these
receptors, LH/hCG, follicle-stimulating hormone, and thyreo-stimulating
hormone, share a common A number of structure-function studies (6-9) identified several
contacts between hormones and receptors. For receptor ECDs, models have
been constructed based on the crystal structure of porcine ribonuclease
inhibitor (pRI), the first structurally defined member of the LRR
family (10, 11); according to pRI homology modeling, receptor ECDs
would form a partial horseshoe-like structure generated by a succession
of However, more than ten years after cloning of their cDNAs, little
information on the precise topology of the glycoprotein hormone
receptors is available. Expression have been reported in several
recombinant systems, as membrane full-length, free ECDs or as
membrane-anchored ECDs attached through fusion with a single
transmembrane domain or with glycophosphatidyl inositide (15-17),
achieving variable yields. However, high expression of a functional
receptor in any form has never been reported; this is mainly because of
intrinsic properties of this receptor family, which render their
production as recombinant functional proteins difficult and resulted in
the production of mostly unfolded species trapped intracellularly (18,
19). ECDs can indeed be secreted in culture media in an active
form when expressed at lower levels, though in highly unstable forms,
sensitive to proteolytic degradation.
However, when hCG subunits have been coexpressed with a complete
receptor within eucaryotic cells, constitutive receptor activation without addition of exogenous hormone was observed (20), whereas hormone coexpressed with receptor ECD leads to secretion of a stable
and soluble complex (21). When present during receptor synthesis, the
ligand seems to help folding and further stabilized and protected
receptor from hydrophobic aggregation, denaturation, and proteolytic
attacks. Hormone-ECD receptor coexpression could therefore be used as a
strategy for obtaining soluble and secreted complex stable enough to
allow biochemical purification, structural analysis, and crystallization.
In this paper, we demonstrate high expression (potentially 20 mg/liter)
of a secreted form of a complex formed by hCG and its receptor
extracellular domain by a stably cotransfected CHO cell line.
Biochemical analysis of the immunopurified complex suggests that it
exists as a unique 85-kDa assembly, corresponding, as confirmed by
surface plasmon resonance, to a 1:1 hormone-receptor stoichiometry.
Spectroscopic analysis by circular dichroism performed on the complex
indicates that receptor-ECD contributed for more than 25% Construction of Expression Vector and Generation of a CHO Cell
Line Expressing Soluble hCG-Receptor Complex--
A cDNA fragment
encoding the first N-terminal 277 amino acids of the porcine LH
receptor (pLHR; see Ref. 22) was obtained by polymerase chain reaction
using primer B (5'-GCTCTAGAATGAGACGGCGGTCCCTGG-3') and
primer S (5'-GGGTACCTGGGGTAAGTCAATGTGGC-3'), bearing a XbaI and a KpnI site, respectively. To add six histidines and a
stop codon after serine 277 of the pLHR, two complementary
oligonucleotides (5'-CATCATCATCATCATCATTAG-3' and
5'-AATTCTAATGATGATGATGATGATGGTAC-3') were annealed and ligated into the
PUC-18 vector opened by KpnI and EcoRI.
The resulting plasmid was then restricted by XbaI and KpnI into which the pLHR XbaI-KpnI
polymerase chain reaction fragment obtained by amplification with
primers B and S was ligated. This construct was entirely sequenced, and
the XbaI-EcoRI fragment encoding pLHR
1-277-polyHis was subcloned into the amplifiable glutamine
synthetase/cytomegalovirus expression vector pEE14 from Celltech (23).
This plasmid was used to transfect a CHO cell line secreting a high
level of hCG (a kind gift from Dr. E. Bos, Organon, The Netherlands)
by the calcium phosphate precipitation method. Transfectants were
submitted to increasing concentration of methionine sulfoximine in
glutamine-free GMEM medium containing dialyzed fetal calf serum.
CHO-CGR cell clones showing resistance to 25 mM methionine
sulfoximine were grown and tested individually for secretion of
hormone-receptor complex in supernatants. The best producing CHO-CGR
clone was further cultivated in selective medium, using regular culture
flasks (Falcon) or "concentrating" flasks (INTEGRA CELLine, Integra BioSciences).
Immunological Analysis--
Three different anti-hCG and two
different anti-receptor monoclonals were used according to the
different assays. For hormone detection or immobilization, mAb FBT10,
whose determinant on the hCG
Two-site sandwich monoclonal immunoradiometric assays (IRMA) for hCG
and for soluble hCG-receptor complexes have been previously established
(21); polystyrene beads (Precision Plastic Ball) were coated with the
capture monoclonal antibodies anti-hormone LH05 or anti-receptor LHR38
by incubation overnight at 20 °C with mAb concentrations of 4 mg/ml
in 10 mM phosphate buffer, 150 mM NaCl, pH 7.4. After washing with water, coated beads were used to capture antigens in
the different analyzed fractions. Incubation of beads and antigens
together in 50% calf serum for 2 h at room temperature was
sufficient for their association. After washing in phosphate buffer,
beads (LH05 or LHR38) were incubated with 100,000 cpm of
125I-labeled FBT10 for 1 h in 50% calf serum at
37 °C. Beads were then washed, and the associated radioactivity was counted.
Immunoaffinity Column Chromatography--
Purified LHR38
antibody was dialyzed against coupling buffer (0.2 M
NaHCO3, 0.5 M NaCl, pH 8.3) and loaded on a
HiTrap N-hydroxy-succinimide-activated Sepharose
column (Amersham Pharmacia Biotech). After three serial washes
with a deactivating buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) followed by an acidic buffer (0.1 M acetate, 0.5 M NaCl, pH 4), the gel was
equilibrated and stored in GMEM medium at neutral pH containing 0.4%
sodium azide. CHO-CGR cell supernatants were applied at 4 °C at a
flow rate of 0.5 ml/min.
After extensive washing with 100 column volumes of phosphate buffer (8 mM Na2HPO4, 2 mM
NaH2PO4) containing 0.15 M NaCl, pH 7.4, bound material was eluted in 0.5-ml fractions with two column volumes of 0.1 M glycine-HCl, pH 3, and immediately
neutralized by one-third volume of 1M Tris, pH 8.5. Fractions containing the complex were concentrated using Centricon 30 (Amicon, Beverly, MA) and dialyzed before further analysis.
Biochemical and Biophysical Analysis--
For Western blot
analysis, proteins were separated by SDS polyacrylamide gel
electrophoresis (PAGE) with or without boiling and reduction by
2
Gel filtration chromatography of CHO-CGR-secreted products was
performed by the use of a Superose 12 column, calibrated with standard
protein markers (Amersham Pharmacia Biotech). Serum-free supernatants
were concentrated approximately 20 times using Amicon 30 and dialyzed
against 15 mM NH4HCO3 and 1 mM CaCl2, pH 7.0, or against 100 mM
Tris, 50 mM NaCl, pH 7.4. Concentrated material (0.5 ml)
was deposited on the column equilibrated with the same buffer.
Chromatography was run at 0.5 ml/min. After collection, 0.5-ml
fractions were analyzed by complex-specific IRMA and immunoblotting.
Covalent cross-linking of the immunopurified complex was done as
follows: concentrated proteins to 0.5 mg/ml were incubated in the
presence of increasing concentrations of glutaraldehyde (5, 10, or 20 mM) at 26 °C for 30 min. Cross-linked products were then
analyzed by denaturing SDS-PAGE and silver staining.
Surface plasmon resonance experiments were performed on a Biacore 1000 according to described procedures (26, 27). Rabbit anti-mouse
IgG-purified immunoglobulins (RAM) were covalently linked to the matrix
gel and then capture antibodies, at a concentration of 100 µg/ml in
HBS buffer (25 mM Hepes, pH 7.4, containing 150 mM NaCl), were injected (flow rate, 5 ml/min for 5 min).
RAM capture ability was then saturated with a 10-min injection of
non-relevant purified mice immunoglobulins to prevent further direct
installation of any injected monoclonal (IgBlock, designated IgB). The
complex was injected either as a cell supernatant or after
immunopurification and captured by its hormone or receptor side,
depending of the first monoclonal injected. Potential receptor or
hormone homologous dimers (H-H or R-R) were assessed by a second
injection of the capture antibody; a null signal was indicative of
absence of these forms. Symmetrically, an injection of detection
antibody against the counterpart antigen within the complex allowed
us to confirm stoichiometry. Alternatively, once the complex was
installed until saturation of the capture antibody ability, a second
injection of the complex allowed us to check any further association
into the complex of unbound components or formation of higher order complexes. Resonance unit levels are compared before and after each
injection (asterisks), measuring the mass of material
immobilized or captured. Intrinsic refractive index of each solution
relative to the HBS buffer accounts for the sudden ups and downs in the sensorgrams and does not affect the resonance unit levels registered after the end of injection. After each run, matrix-bound RAM was regenerated by a pulse of 100 mM HCl taking off all bound immunoglobulins.
For circular dichroic analysis, the protein concentrations were
determined in 10 mM phosphate buffer, pH 7.4, using
extinction coefficients of 11745 and 22330 M Anti-hormone and anti-receptor antibodies were used according to
their specificity and previously known properties toward their
respective antigens. LH05 and LHR38 were used as "capture" antibodies, and D1E8 and LHR775 were used for immunodetection on blots,
whereas FBT10, as it can bind LH05-captured hCG or LHR38-captured complex, was used mostly as second antibody in free hormone and in
complex sandwich assays.
The concentration of hCG in medium was deduced from standard curves
using purified hCG as antigen into the sandwich assay LH05/FBT10,
whereas the concentration of receptor bound to hCG (soluble complex)
was evaluated by the use of an anti-receptor antibody followed by the
same iodinated anti-hormone antibody (sandwich assay LHR38/FBT10). Fig.
1A shows that the LH05/FBT10 IRMA assay can detect amounts of purified standard hGC lower than 1 ng/ml, whereas the LHR38/FBT10 IRMA assay did not detect free hormone
up to 2 mg/ml. This assay therefore can be considered as highly
specific for the soluble hCG-receptor complex. As shown by Fig.
1B, both assays indicated that the complexed hormone
represented about 10% of total hormone in supernatants (medium
dilutions for complex assay was ten times less than for hCG assay) at
any time during cell growth. From Fig. 1, A and
B, final concentrations of hCG-receptor complex in regular
flask monolayer culture conditions have been estimated to 2 mg/liter,
(for an amount of 20 mg/liter of free hormone) at 100% cell
confluence.
helices contributed by the receptor, in
agreement with the porcine ribonuclease inhibitor-based modelization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit non-covalently bound to an
hormone-specific
subunit; the 3D structure of hCG has been resolved
by x-ray crystallography (4, 5).
helices and
sheets (12, 13). Although excluding the two N-
and C-terminal cystein-rich clusters, the model allows hormone docking
inside the horseshoe inner circumference formed by the parallel
sheets (14).
helices,
consistent with the predicted 3D models based on the crystal structure
of the porcine ribonuclease inhibitor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit (loop I and loop III) is
accessible in free or receptor-bound hormone (24), mAb LH05, which
recognizes only dimeric (determinants were identified on
subunit
loop II and
subunit loop III) hormone (25), and mAb D1E8, directed
against the C-terminal peptide of hCG
subunit. To identify or
capture the receptor, we used mAb LHR38 and mAb LHR75, both selected
against the testis-purified porcine LH/hCG receptor (18).
-mercaptoethanol and electroblotted onto nitrocellulose membranes
(Hybound C; Amersham Pharmacia Biotech). Membranes were then blocked
for 1 h in phosphate-buffered saline buffer containing 4% nonfat
dry milk (Régilait, Lyon, France) and incubated overnight at
4 °C in the same buffer in the presence of 4 mg/ml primary mAbs,
D1E8 for the hormone, LHR775 for the receptor. After being washed,
blots were incubated in the same buffer at 20 °C for 1 h with
biotinylated anti-mouse IgG and streptavidin conjugated to peroxidase
(Amersham Pharmacia Biotech). An enhance chemoluminescent substrate
(Pierce) for detection of peroxydase was then used for visualization of
the mAb-recognized bands.
1
cm
1 for hCG and hCG-receptor complex at 276 nm,
respectively, and calculated according to published procedures (28).
Protein samples (1 mg/ml) were then placed in a 0.01-cm path-length
cell. CD spectra were recorded and analyzed according to the method of
Yang et al. (29) as described previously in (30).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Quantitative IRMA for hCG (LH05-FBT10;
) and soluble hCG-receptor complex (LHR38-FBT10;
)
in CHO-CGR cell supernatants. In A, beads were coated
either with anti-hCG LH05 or anti-receptor LHR38 mAbs. Beads were
incubated with increasing concentrations of purified hCG from 0.01 ng/ml up to 2 mg/ml, and the iodinated anti-hCG FBT10 was used as
second antibody. The LH05-FBT10 curve was used as a
quantitative dosage of total (free and bound) hCG in supernatants. In
B, CHO-CGR supernatants were collected at different cell
densities. They were then diluted in serum-free GMEM, 2000 times for
LH05-FBT10 assays or 200 times for LHR38-FBT10 assays.
Free hCG and complex accumulated in medium even after cells reached confluence, so that supernatants were harvested only once a week. To facilitate purification, numerous attempts to decrease serum levels or to use serum-free synthetic media were made; without cell-growth rate alteration, all modifications of media resulted in a loss of product secretion. Several culture conditions (suspension cultures or hollow fibers) were also tested; higher yields of secreted complex (up to ten times monolayers yields) were only obtained by the use of cell line flasks.
Size Exclusion Chromatography--
Cell supernatants from no-serum
cultures were concentrated after dialysis against buffers of different
ionic strength and deposited on Sepharose 12 column equilibrated with
the same buffers; fractions were analyzed by the complex-specific
LHR38/FBT10 assay. When chromatography was done in 100 mM
Tris, 50 mM NaCl buffer, most of the IRMA-positive material
eluted as one specie at a molecular mass between monomeric
bovine serum albumin (66 kDa) and alcohol dehydrogenase (150 kDa)
markers, indicating little or no dissociation (Fig.
2A). By contrast, as shown on
Fig. 2B, at lower ionic strength (15 mM
NH4HCO3), positive fractions were found in two
peaks of molecular masses encompassing the bovine serum albumin marker. The low molecular mass species might be the result of partial dissociation of the complex resulting in free hCG and free receptor-ECD yet are reversible, because these fractions were also positive in the
complex-specific IRMA assay. According to the expected molecular size
of hCG-ECD complex (85 kDa), the absence of any higher molecular mass
species in non-dissociating conditions suggests that no high order
multimeric associations between hormone and receptor did occur.
Immunoblotting analysis of the 85-90-kDa peak fractions in denaturing
conditions (Fig. 2C) with antibody LHR775 revealed the
presence of a receptor-ECD specie of the expected molecular mass at 35 kDa.
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Affinity Purification of the hCG-hCGR Complex--
Receptor ECD
was fused to a poly-His tag in C-terminal, to allow metal ion
chromatography purification; although nickel or copper columns did
actually retain the complex, interaction between metal ions and
receptor appeared too weak for purification, because elution occurred
as soon as 50 mM imidazole was applied to the gels (not
shown). Immunoaffinity purification of the complex with immobilized
anti-receptor LHR38 mAb was therefore undertaken; fractions eluted by
acidic pH were analyzed by the complex-specific IRMA assay (Fig.
3). The presence of receptor ECD at 35 kDa in the IRMA-positive fractions was confirmed on blotted proteins after denaturing SDS-PAGE separation, using the anti-receptor antibody
LHR775, as shown on Fig. 3.
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Protein contents in immunopurified fractions were further
analyzed with different monoclonals; although LHR775 revealed the presence of ECD receptor at 35 kDa, D1E8 labeled the dimeric -hCG at 55 kDa when proteins were separated in non-reducing conditions. This
result indicated that SDS polyacrylamide electrophoresis indeed
dissociated receptor-ECD from hCG but not the two hormone subunits.
After reduction with 2%
-mercaptoethanol, the molecular mass of
receptor did not change (LHR775 band), whereas hormone did dissociate,
as D1E8 now revealed free
-hCG subunit (Fig. 4, immunoblotting).
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Immunoblotting analysis was confirmed by direct visualization of the
eluted proteins from LHR38 affinity column by silver staining of the
gels; only two bands of 35 and 50-55 kDa, respectively, were seen in
non-reducing conditions, receptor ECD, and dimeric hCG, respectively,
whereas the 50-55-kDa band, once reduced, dissociated into two 32- and
20-kDa bands, corresponding to the apparent molecular masses of free
and
subunits of hCG, respectively (Fig. 4, silver
staining). Silver staining also indicated that a high degree of
purification has been achieved in a single immunoaffinity step.
Covalent Cross-linking of the Complex--
The immunopurified
complex was submitted to covalent cross-linking with different
concentrations of glutaraldehyde; whatever the concentration of
cross-linker used, complex IRMA counts were constant, indicating no
complex dissociation (not shown). Electrophoresis in reducing
conditions followed by silver staining of the covalently bound proteins
revealed a new protein band at 85 kDa, which could correspond to the
hormone-receptor complex. The relative proportion of the 85-kDa protein
increased with linker concentration (Fig. 5). Other bands generated during
cross-linking migrated at 45 and 65 kDa and could represent ECD
receptor covalently bound to either hCG subunits, as already suggested
in Ref. 31.
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Surface Plasmon Resonance Analysis-- Although biochemical characterization of the complex suggested a 1:1 stoichiometry between hormone and receptor, more questions regarding the complex stability and hormone-receptor (H-R) interactions have been addressed through surface plasmon resonance. They confirmed that all three monoclonals, LHR38, LH05, and FBT10, once bound to immobilized rabbit anti-mouse antibody, displayed capture abilities for the complex, either by the receptor or by the hormone side (not shown). As already shown, anti-receptor LHR38 and anti-hormone LH05 and FBT10 were not competitors for the complex, whereas FBT10 and LH05 were not competitors for free hCG nor for receptor-bound hCG (not shown).
Once bound to LHR38, the hormone-receptor complex was not able to bind
free hormone anymore, suggesting the absence of free soluble receptor
in samples. The possibility of having R-R, R-H-R, or R-H-H-R species
was also eliminated, because the LHR38-bound complex was unable to bind
more LHR38 or more complex, as shown by analysis of the first sequence
of injections shown on Fig. 6.
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The symmetrical situation, where the complex was bound by the anti-hormone antibodies, was then analyzed; no further binding of complex (eliminating the formation of H-R-H, H-R-R, or H-R-H-R species) and no further binding of free hormone (no formation of H-R-H or H-H) were observed, as shown for the second sequence of injections in Fig. 6. Moreover, free hCG bound to FBT10 was never able to bind the complex, confirming a high stability of hormone-receptor interactions and the absence of an H-R-H specie (not shown). Altogether, Biacore experiments strongly suggest the existence of only one stably associated 1:1 hormone-receptor complex.
Structural Analysis--
Fig. 7
shows the CD spectrum of the hCG-ectodomain complex in 10 mM phosphate buffer. The shape of the spectrum, with a
maximum at 191 and a minimum at 210 nm, clearly indicated the presence of helices and
sheets in the hormone-receptor complex.
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The deconvolution of the measured spectrum revealed instead 17 ± 2% of helix and 33 ± 11%
sheet. The hCG hormone is
known to be mostly composed of
sheet (4, 5) and contributes to 47%
of the complex-calculated molecular weight. If one assumed that hCG
does not undergo sizeable conformational changes upon binding to the
receptor, the calculated
helix proportion in the complex CD
spectrum was likely because of the receptor. Therefore, after deduction
of the hormone contribution from complex secondary structure values,
the ECD receptor structural contribution could be estimated between 25 and 30% of
helices and between 30 and 35% of
sheets.
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DISCUSSION |
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Despite the fundamental physiological functions displayed by the gonadotropin hormones LH-hCG and follicle-stimulating hormone for the control of steroid production and gametes synthesis, the design of agonists or antagonists, although of obvious interest for clinical use in fertility control (32), has never been achieved. Because of the very high complexity of hormone and receptor structures, gonadotropin pharmacology will probably never be possible without spatial knowledge of hormone-receptor interactions. Gonadotropin receptors are amphipathic seven transmembrane proteins for which no crystallization strategy seems available yet. Fortunately, 3D structure of a soluble form of these receptors might bring the most important structure-function and docking information, because extracellular domains bear nearly all hormone-receptor contact sites.
In this paper, we were able to demonstrate for the first time a quantitative production of a purifiable extracellular domain of one of the members of the glycoprotein hormone receptor family. Transfection of a cell line overproducing the human chorionogonadotropin hormone with a plasmid encoding the extracellular domain of its receptor resulted in secretion in medium of a mixture of 9/10 free and 1/10 receptor-bound hormone, as measured by quantitative immunoradiometric assays. This soluble form of hormone-receptor complex did accumulate in media, and the free/bound hormone ratio found a constant over time. Previous attempts of high yield receptor-ECD secretion by different eucaryotic cell types always failed because of important intracellular retention and fast proteolytic degradation in media. By contrast, soluble hormone-receptor complexes were indeed secreted in cell culture media and displayed very high stability even after a year of storage at 4 °C (not shown). Moreover, from producing cell supernatants (potentially 20 mg per liter), complex was purified at high yield to near homogeneity by one affinity chromatography step with the anti-receptor monoclonal LHR38.
Extracellular domains of glycoprotein hormone receptors are
structurally related to the LRR protein family, a group of proteins involved in a variety of functions through molecular interactions. The
pRI has been the first LRR protein whose 3D structure was solved; it displays an overall horseshoe shape able to accommodate ribonuclease within its central hole. Each repeat of 25 to 28 amino
acids contributes to a structural brick made of a strand and an
helix connected by a loop. Sixteen of such elements with
strand
forming the inner and
helices the outer circumference of the
horseshoe forms the overall structure. Several models have been built
up based on this template for gonadoreceptors (12-14). Such models
must however be taken with some caution, because pertactin, another
member of the LRR family, displays a triangular prism structure only
made of
strands (33). LRR proteins therefore could adopt very
different overall topologies. Experiments displayed in this paper
allowed us to obtain for the first time some structural information
about one member of the glycoprotein hormone receptor family; circular
dichroism analysis suggested the presence of more than 25%
helices
in the soluble hormone-receptor ECD complex, and this might be more in
favor of a pRI-like structure than an only
strand structure for the
hCG/LH receptor N-terminal domain.
A few number of G-protein-coupled receptors have been shown to be able to form homologous or heterologous dimers (34, 35); although cooperative binding of glycoprotein hormones might be possible, receptor dimerization has not yet been demonstrated. In this work, multimers of higher order than one hormone for one receptor were never observed either by non-denaturing size separation chromatography or by surface plasmon resonance studies with well defined monoclonals. The possibility of multimerization cannot however be excluded for larger forms of ECDs (containing the C-terminal cysteine clusters) or for the membrane receptors.
According to the data presented here, mass production of
ectodomain-ligand complexes seems to be a realistic strategy if one aims at structural study of glycoprotein hormone receptors, as it has
been shown for other receptors (36-38). Biochemical characterization suggested that CHO-CGR produced a soluble and stable complex that can
be easily purified to homogeneity and seems to behave as a unique
monodisperse protein entity. This strategy might therefore meet all
quantitative and qualitative requirements for biophysical studies and
can be implemented to aim at solving a glycoprotein hormone-receptor
complex 3D structure.
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Aknowledgments |
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We are grateful to Dr. A. Pauloin for precious help with FPLC, to L. Cabanié for advice in cell culture upscalling, and to Dr. Ma Er Jin for continuous encouragement.
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FOOTNOTES |
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* 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. Tel.: 33 1 34 65 25 66; Fax: 33 1 34 65 22 41; E-mail: remy@biotec.jouy.inra.fr.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M005206200
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ABBREVIATIONS |
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The abbreviations used are: LH, luteotropin hormone; hCG, human chorionogonadotropin hormone; GMEM, Glasgow's modified Eagle's medium; CHO, Chinese hamster ovary; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; ECD, extracellular domain; LRR, leucine-rich repeats; 3D, three-dimensional; pRI, porcine ribonuclease inhibitor; pLHR, porcine LH receptor; CGR, chorionogonadotropin receptor; IRMA, immunoradiometric assays; RAM, rabbit anti-mouse; H, hormone; R, receptor.
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