Purification and Structural Analysis of a Soluble Human Chorionogonadotropin Hormone-Receptor Complex*

Jean-Jacques RemyDagger §, Claude Nespoulous, Jeanne Grosclaude||, Denise Grébert, Laurence Couture, Edith Pajot, and Roland Salesse

From Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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% alpha  helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  subunit non-covalently bound to an hormone-specific beta  subunit; the 3D structure of hCG has been resolved by x-ray crystallography (4, 5).

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 alpha  helices and beta  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 beta  sheets (14).

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% alpha  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

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 beta  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 beta  subunit loop II and alpha  subunit loop III) hormone (25), and mAb D1E8, directed against the C-terminal peptide of hCG beta  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).

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 2beta -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.

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-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



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Fig. 1.   Quantitative IRMA for hCG (LH05-FBT10; ) and soluble hCG-receptor complex (LHR38-FBT10; black-square) 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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Fig. 2.   Gel filtration chromatography. CHO-CGR cells were grown to confluency in low protein, serum-free, synthetic medium. Media were concentrated using Centricon 30 and dialyzed against high (100 mM Tris, 50 mM NaCl, pH 7.4) or low (15 mM NH4HCO3, 1 mM CaCl2, pH 7.4) ionic strength buffers in A or B, respectively. Superose 12 gel filtration columns were equilibrated with the same respective buffers and precalibrated with standard protein markers (Amersham Pharmacia Biotech); only the position of bovine serum albumin (66 kDa) is indicated. Volumes of 0.5 ml were deposited, and protein separations were monitored by UV (black-diamond , arbitrary units) and by the complex-specific IRMA LHR38-FBT10 (box-dot , bound cpm).

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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Fig. 3.   Immunoaffinity purification of hCG-receptor complex. Cell culture supernatants were loaded at 4 °C at a flow rate of 0.5 ml/min on a LHR38 HiTrap affinity Sepharose column (Amersham Pharmacia Biotech) pre-equilibrated with GMEM. After extensive washes, 15 acidic eluted (0.1 M glycine-HCl, pH 3) fractions were immediately neutralized and analyzed for the presence of complex by IRMA(s). Only odd-numbered IRMA-positive fractions were then separated by 10% SDS acrylamide gel electrophoresis in reducing conditions and analyzed for the presence of receptor-ECD at 35 kDa by LHR775 after electrotransfer on nitrocellulose.

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 alpha beta -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% beta -mercaptoethanol, the molecular mass of receptor did not change (LHR775 band), whereas hormone did dissociate, as D1E8 now revealed free beta -hCG subunit (Fig. 4, immunoblotting).



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Fig. 4.   Biochemical analysis of purified complex. Immunopurified complex was loaded on 10% SDS acrylamide gels under nonreducing or reducing (2% beta -mercaptoethanol) conditions. Proteins were either silver stained or blotted onto nitrocellulose membranes and then probed with anti-hormone mAb D1E8 or anti-receptor mAb LHR775.

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 beta  and alpha  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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Fig. 5.   Covalent cross-linking. Immunopurified products at 0.5 mg/ml were incubated in the presence of increasing concentrations of glutaraldehyde at 26 °C for 1 h, reduced by 2% beta -mercaptoethanol, and SDS-PAGE separated before silver staining.

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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Fig. 6.   Stoichiometric analysis of the complex by surface plasmon resonance assay. Immunopurified hCG-receptor complex was captured onto the biocaptor through interaction with anti-receptor LHR38 or with anti-hormone FBT10. Triangles on time bar indicate beginning and end of each injection; asterisks indicate the points where resonance unit levels are registered. The caption sums up the sequential injections. Sensorgram A, anti-receptor LHR38 was first installed on RAM followed by saturation of RAM with IgB. The complex was then captured, another IgB was performed, and LHR38 was injected again. No signal was detected, although injection of anti-hormone FBT10 revealed a significant amount of binding to the complex. Finally, injection of a pulse of 100 mM HCl regenerated the covalently linked RAM capacity (return to the initial baseline). Sensorgram B, anti-hormone FBT10 was installed onto RAM and then injection of complex was followed to saturation of antibody capacity; a second complex injection did not reveal any signal increase nor did the subsequent injection of free hormone. RAM regeneration was performed as above.

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 alpha  helices and beta  sheets in the hormone-receptor complex.



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Fig. 7.   Spectroscopic analysis by circular dichroism. Complexes were immunopurified and concentrated to 1 mg/ml in 50 mM phosphate buffer, pH 7. Far UV CD spectrum of hCG-ectodomain complex was recorded and analyzed according to published procedures (28, 29, 30).

The deconvolution of the measured spectrum revealed instead 17 ± 2% of alpha  helix and 33 ± 11% beta  sheet. The hCG hormone is known to be mostly composed of beta  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 alpha  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 alpha  helices and between 30 and 35% of beta  sheets.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  strand and an alpha  helix connected by a loop. Sixteen of such elements with beta  strand forming the inner and alpha  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 beta  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% alpha  helices in the soluble hormone-receptor ECD complex, and this might be more in favor of a pRI-like structure than an only beta  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.


    Aknowledgments

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.


    FOOTNOTES

* 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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Tsai-Morris, C. H., Buczko, E., Wang, W., and Dufau, M. L. (1990) J. Biol. Chem. 265, 19385-19388[Abstract/Free Full Text]
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