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Endocrinology Vol. 142, No. 4 1517-1524
Copyright © 2001 by The Endocrine Society

ARTICLES

High-Level Expression of a Functional Single-Chain Human Chorionic Gonadotropin-Luteinizing Hormone Receptor Ectodomain Complex in Insect Cells1

Gregory B. Fralish, Prema Narayan and David Puett

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, Green Street, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bmb.uga.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reproductive capacity in primates is dependent on the high-affinity binding of the glycoprotein hormones LH and human (h)CG to the large ectodomain (ECD) of their common receptor (LHR). Our understanding of the precise molecular determinants of hormone binding is limited, because there are no structural data for any of the glycoprotein hormone receptors. Overexpression of the ECD of the receptor has been attempted in various expression systems. Prokaryotic expression does not yield properly folded ECD. Eukaryotic expression, on the other hand, results in mostly heterogeneous, intracellularly trapped protein, but the secreted ECD is completely folded. Accordingly, we have tethered the single-chain hormone, yoked hCG, to the N terminus of LHR-ECD (yoked hormone-extracellular domain). Yoked hCG is secreted at high levels; binds LHR with high affinity; and, when tethered to the N terminus of full-length LHR, it binds and constitutively activates the receptor. Using recombinant baculovirus, yoked hormone-extracellular domain is secreted from insect cells at levels greater than 1 µg/ml, nearly 20-fold higher than that previously reported in eukaryotic expression systems. The protein was purified and binds exogenous 125I-hCG with high affinity but, significantly, only after protease treatment to remove the tethered hormone. Thus, the fusion protein seems to form a functional hormone-receptor complex that is expressed at levels sufficient for its biophysical characterization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NORMAL REPRODUCTIVE FUNCTION in primates and equids is dependent on the actions of three gonadotropins, pituitary-derived LH and FSH and the placental-derived human (h)CG. Along with TSH, these molecules form the family of glycoprotein hormones that share a similar heterodimeric architecture of a common {alpha}-subunit and a hormone-specific ß-subunit. Their biological signals are transduced at the target tissue via their respective G-protein-coupled receptors (1). In the case of LH and hCG, the same receptor (LHR) is used. This is not surprising, given the high sequence similarity (85%) between these two hormones, with the major difference being ascribed to the 30-amino-acid residue C-terminal extension of hCG (C-terminal peptide, CTP) (1), whose four sites of O-glycosylation are important for the increased circulatory half-life of hCG (2). The crystal structure of hCG has been determined (3, 4), and it has provided a framework on which to interpret the numerous studies involving mutagenesis, peptides, and chimeras and has aided in our understanding of hormone binding and signal transfer. However, there are no structural data for any of the glycoprotein hormone receptors.

LHR binds hCG via its large, soluble extracellular domain (ECD) [341 amino acids in rat LHR (rLHR)]. This glycosylated domain is apparently responsible for most, if not all, of the contacts necessary to form a specific and high-affinity interaction with hCG (dissociation constant, Kd = 0.2 nM) (5). The ECD of LHR and the other glycoprotein hormone receptors (FSHR and TSHR) have an imperfect, but discernible, leucine-rich repeat (LRR) motif (6). The structure of the inhibitor of ribonuclease A, which consists entirely of LRRs, revealed the extended and nonglobular fold of the motif, whose tandem {alpha}/ß hairpin repeats form a cusp (7). This motif is used frequently in nature for protein-protein interactions and provides an extended binding surface for numerous contact sites with ligand (8). Accordingly, several laboratories have proposed homology models of the LHR-ECD, based on the ribonuclease A inhibitor structure (6, 9, 10). Though significant, the models cannot substitute for direct biophysical data of the receptor or receptor-hormone complex. Indeed, a crystal structure of the ECD would profoundly enhance our understanding of hormone binding and signal transfer; moreover, a structure of the ECD-ligand complex would solidify or reform current paradigms.

LHR presents major hurdles for its resolution at a molecular level. In being an integral membrane protein, it is difficult to express and purify in significant amounts. Therefore, several groups have attempted expression of the soluble LHR-ECD, as well as the ECDs for TSHR and FSHR (Table 1Go). Limited data of significant production of the ECDs for FSHR and TSHR have been reported. The highest expression level of ECD was reported for insect cell-expressed porcine (p)LHR-ECD at approximately 100 µg/ml, whereas the highest level of secreted ectodomain for the same receptor was 70 ng/ml in mammalian cells. In eukaryotic expression systems, secreted ectodomain displays nearly homogeneous binding activity and has a more complex set of glycans, suggesting a rigorous folding requirement from the cellular protein folding components in the endoplasmic reticulum and golgi (11). Moreover, there are no published reports of the generation of significant quantities of ligand-receptor complexes for any of the glycoprotein hormone receptors for biophysical characterization.


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  		Table 1. Expression summary for the ectodomains of the glycoprotein hormone receptors LHR, TSHR, and FSHR

 
To improve the production of this challenging glycoprotein, our laboratory has cloned a fusion protein consisting of a single-chain yoked hCG (YhCG), N-ß-{alpha}-C, linked to the N terminus of the rLHR-ECD (12). Bioactive YhCG is secreted at high levels and, when fused to the full-length receptor, forms a complex with the receptor (yoked hormone receptor; YHR) that promotes signal transduction and inhibits binding of exogenous 125I-hCG (12, 13). Significant binding of 125I-hCG can be recovered only after treatment with factor Xa protease that cleaves at the engineered site between the hormone and receptor and allows for the dissociation of YhCG. Therefore, if a yoked hormone-extracellular domain (YECD) folded similarly, as when connected to the full-length receptor, then it would represent a functional hormone-receptor complex. Accordingly, with future scale-up production of the protein in mind, baculovirus-promoted expression in insect cells was used for the characterization of YECD, as opposed to mammalian cells, which were used exclusively in previous studies on YHR (13).

Herein, we describe the cloning, expression, characterization, and purification of YECD. When YhCG is fused to the ECD, the protein is secreted at levels well over 1 µg/ml, i.e. 20-fold greater than secreted levels reported previously (Table 1Go), indicating a practical expression level for scale-up production and biophysical characterization. The protein can be purified to a high degree, in one step, using an affinity tag. Significantly, the purified protein can bind 125I-hCG with high affinity but only after dissociation of YhCG is facilitated by digestion with factor Xa protease, suggesting a functional hormone-receptor association. This approach of fusing ligand to its cognate receptor to increase expression and form a biologically significant ligand-receptor complex is, in principle, applicable to the other glycoprotein hormone receptors and to other receptor systems that involve protein-protein or peptide-protein interactions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning and generation of recombinant virus for epitope tagged YECD
The complementary DNA for a histidine-tagged (HT) YECD was constructed using the yoked hormone receptor complementary DNA (YHR), i.e. N-ß-{alpha}-LHR-C, as a template. PCR was used to amplify a product containing the entire 341 amino acid residues of the ECD of rLHR fused to YhCG via residues 116–145 of the CTP of the ß-subunit, with a six-histidine tag on the C-terminus. The product was then cloned into the transfer vector pVL1393 (PharMingen, San Diego, CA). To generate a second tagged product, the HT in the initial construct was replaced with a flag tag (N-DYKDDDDK-C) using PCR with the following primers: 5'ATGGAGATATTCCAGGGGCTGCTG-3', and 5'-TTACTTATCGTCATCGTCCTTGTAGTCCCTAAGGAAGGCATAGCC-3'. The product was directly cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and subcloned into pVL1393. Recombinant baculoviruses coding for YECD-HT and for YECD-flag were generated as previously described for YhCG (12). The virus was plaque purified and amplified, with viral titers of greater than 1 x 108 plaque-forming units/ml being routinely obtained.

YECD-HT and YECD-flag expression
Plaque-purified and amplified recombinant virus was used to infect Sf9 cells, both in monolayer and in suspension cultures. For initial expression screening, monolayer infections were performed. Approximately 2.5 x 106 cells were seeded on 60-mm dishes, and virus was added with a multiplicity of infection (MOI) of 1.0 and incubated at 27 C for 1 h in a reduced volume (1 ml), followed by addition of medium to 4 ml. Medium was then collected after 72 h and, following centrifugation, the amount of YECD was quantified using the ß-hCG RIA (ICN, Costa Mesa, CA), which recognizes free hCG-ß and the heterodimer. For expression optimization and purification, cells were adapted in suspension cultures per manufacturer’s instructions (Life Technologies, Inc., Grand Island, NY) and infected at a density of 8 x 105 cells/ml with an MOI of 0.1, 1.0, or 10.0. Medium was collected, centrifuged, and assayed using the ß-hCG RIA.

Polyacrylamide gel analysis
Samples were resolved on 10% polyacrylamide gels under reducing conditions. Gels used for Western analysis were transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon P, Millipore, Bedford, MA). The membrane was probed for the CTP with the rabbit polyclonal antiserum raised against a peptide corresponding to amino acid residues 109–145 of the ß-subunit of hCG. The flag tag (Sigma, St. Louis, MO) and the HT (Invitrogen) were detected using commercially available monoclonal antibodies per manufacturer’s instructions, and all blots were visualized by the ECL method (Amersham Pharmacia Biotech, Piscataway, NJ). For total protein detection, gels were silver-stained (Bio-Rad Laboratories, Inc., Hercules, CA).

Purification of YECD-HT and YECD-flag
For purification of YECD-HT and YECD-flag, 1-liter Sf9 cell suspension cultures were clarified by centrifugation at 2000 x g for 20 min, and the supernatant was filtered (0.45 µM). For YECD-HT, the expression medium was concentrated 10-fold using an Amicon ultrafiltration cell fitted with a 30-kDa cutoff membrane (Millipore Corp., Bedford, MA). The concentrated medium was then dialyzed at 4 C overnight against start buffer (20 mM sodium phosphate, pH 7.9, 0.5 M NaCl, 10 mM imidazole). The dialyzed sample was incubated with preequilibrated (start buffer) superflow nickel-nitrilotriacetic acid (Ni2+-NTA) resin (QIAGEN, Valencia, CA) overnight at 4 C and then packed into a column for FPLC. After an initial wash with 10 column volumes of start buffer, a linear gradient of 10–200 mM imidazole was applied to the column. Elution of YECD-HT was followed using the ß-hCG assay. For purification of YECD-flag, clarified and filtered expression medium was adjusted to a pH of 7.5 and then loaded on a C10/10 column (Amersham Pharmacia Biotech) packed with M2-flag Ab resin (Sigma) that was preequilibrated with Tris-buffered saline (TBS)a (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl) at 0.5 ml/min (4 C). The column was then washed extensively with TBSa (500 ml per inch of gel) at 1 ml/min. The protein was eluted with 0.1 M glycine, pH 3.5, and the eluted sample was immediately collected in tubes containing 1 M Tris-HCl, pH 8.0.

Factor Xa digestion of YECD-HT and YECD-flag
Purified samples of YECD were concentrated and dialyzed against factor Xa buffer (20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 2 mM CaCl2) overnight at 4 C. The purified, concentrated, and dialyzed sample was quantified using the ß-hCG RIA. YECD was then digested with factor Xa enzyme (New England Biolabs, Inc., Beverly, MA) in a 1:25 enzyme to substrate mass ratio for 4–6 h at room temperature. Undigested samples were incubated at room temperature without the addition of enzyme.

Binding of 125I-hCG to YECD-HT and YECD-flag
Purified and protease-treated samples (50–100 ng) were blotted onto PVDF membrane (Millipore Corp.) using the Biodot Dot blotting manifold (Bio-Rad Laboratories, Inc., Beverly, MA). The membrane was then blocked in TBSb (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl) with 0.2% Tween 20, 3% BSA for 4 h at room temperature. Individual squares (6 x 6 mm) were cut from the loaded and blocked membrane and added to tubes containing blocking solution with increasing amounts of label (125I-hCG) in the presence or absence of excess unlabeled hCG. The membranes were shaken overnight at room temperature, then washed 4 times in TBSb containing 1% NP-40, followed by two washes in TBSb. The squares were then air-dried, counted for {gamma}-radiation, and exposed to film overnight at -70 C with Cronex intensifying screens (Eastman Kodak Co., Rochester, NY). Data were analyzed by nonlinear regression using the Prism software (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Using YHR as a template, PCR was used to generate the YECD construct. Targeting to the ER is directed by the ß-subunit’s signal sequence, and the CTP of hCG-ß was used as a linker between the C-terminus of the {alpha}-subunit and the ECD. The factor Xa recognition site was retained in YECD, and either a 6-histidine or flag tag was added to the C-terminus to generate the construct shown in Fig. 1AGo. High titer recombinant baculovirus was used to infect Sf9 cells, and expression was detected by RIA and Western blot analysis. The RIA recognizes the monomeric or heterodimeric form of the ß-subunit of hCG, and it was a useful assay for the rapid analysis of the medium for YECD expression. However, the assay assumes an equal affinity of the antiserum for the tracer antigen, the standards, and the unknown (YECD). Because YECD may represent a functional hormone-receptor complex, the affinity of the antiserum for this protein may be different, given suggestions of conformational differences in the hormone when bound to receptor (14, 15). Furthermore, epitopes important for binding of the antiserum to ß-hCG may be masked in YECD, and the glycosyl chains present on an insect-expressed YECD are likely to be quite different than those of the human-derived standards. Despite these limitations, positive recognition by the assay corresponded reproducibly with the presence of YECD on Westerns, and dilutions within a given assay gave consistent values for the complex. Western blot analysis of the expression medium was performed using the polyclonal rabbit CTP antibody and the mouse-derived monoclonals specific to the particular C-terminal tags, 6-histidine, and flag (Fig. 1BGo). Medium taken from cells infected with YECD-(HT or flag) showed a major band with an apparent molecular mass between 83–89 kDa. In the case of YECD-HT, minor bands at 63 kDa and 28 kDa were also present. The exact nature of these proteins has not been investigated, but they probably represent degradation products or minor cross-reacting species in the expression medium. In medium from cells infected with YECD-flag virus, the lower 28-kDa protein was not present.



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  		Figure 1. Secretion of YECD by insect cells. A, Schematic representation of YECD. YhCG is tethered to the N-terminus of the ECD of the receptor via amino acid residues 116–145 comprising the CTP of ß-hCG. The factor Xa recognition sequence is present between the hormone and receptor, and the C-terminus of the construct contains either a six-histidine or flag tag. The ß-subunit’s signal sequence (ssß) is used for targeting the protein for secretion. B, Expression of YECD-HT and YECD-flag was detected by Western blot analysis using antibodies against the CTP and the two C-terminal tags. All lanes were loaded with 50 µg total protein. Odd-number lanes contain samples collected from the medium of cells infected with the indicated YECD virus. Even-number lanes contain medium from cells infected with a control virus expressing the XylE protein. The sizes of the molecular mass standards are indicated.

 
Expression of YECD was then optimized in suspension cultures of Sf9 cells. Using the RIA, media from suspension cultures of Sf9 cells infected with various MOIs were assayed over a time period of 4 days (Fig. 2Go, which shows results for YECD-flag only). At 72 h, the amount of protein produced by an infection of 0.1 MOI was equivalent to that induced by the 1.0 MOI infection, and the highest level was secreted after 96 h. At yields exceeding 1 µg/ml, this is nearly 20-fold greater than the highest reported levels of secreted LHR-ECD (Table 1Go).



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  		Figure 2. Expression of YECD-flag in insect cells. Sf9 cells were adapted to suspension cultures and infected at various MOIs. Medium was harvested from the cultures every 24 h for 4 days. The results are the mean ± SEM of two expression surveys assayed by the ß-hCG RIA.

 
Before functional analyses of YECD were performed, the protein from the serum-free medium was purified using the C-terminal tags on the different constructs for affinity chromatography. Overnight batch binding of YECD-HT to the Ni2+-charged NTA resin was employed because only low amounts of protein bound with a single pass through a packed column. Elution of YECD-HT from the Ni2+-NTA column revealed a significant amount of nonspecific binding to the column, despite relatively stringent starting conditions (Fig. 3AGo). More highly purified fractions, eluting toward the end of the gradient, were pooled and used in subsequent analyses (Fig. 3AGo, lanes 7and 8 and right panel). Despite good purification, only 50% of the YECD-HT bound to the column; and, to achieve higher purity, significant losses were encountered in collecting the most homogenous fractions from the gradient. This resulted in yields of less than 5% of the starting material. For YECD-flag, binding to the antibody-conjugated resin was dramatically more specific than the metal affinity chromatography method used with YECD-HT (Fig. 3BGo). Routinely, 60%–70% of the starting material bound to the column, and elution from the column resulted in nearly homogeneous protein. In this case, final yields were much improved over that obtained with YECD-HT. For both proteins, fractions were pooled and concentrated (Fig. 3Go, A and B, pooled and concentrated lanes).



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  		Figure 3. Purification of YECD. A, SDS-PAGE of fractions eluted from Ni2+-charged NTA resin loaded with medium from cells infected with YECD-HT virus. FT, 50 µg total protein was loaded from the flow through fraction; lanes 1–8, 20 µl aliquot from 1-ml fractions along the imidazole gradient. The right panel contains pooled and concentrated (30-kDa cutoff) fractions from lanes 7 and 8. B, M2-Ab-conjugated resin purification of YECD-flag from medium from cells infected with YECD-flag virus. Lanes 1–8 of the SDS-PAGE gel represent fractions collected during elution of YECD-flag from the resin with 0.1 M glycine, pH 3.5; equivolume samples (20 µl) were loaded. The right panel is a pool of all fractions from the elution that was concentrated (30-kDa cutoff). The gels were silver-stained.

 
The purified and concentrated samples were treated with factor Xa protease, which cleaves after the sequence, IEGR, present between the hormone and receptor (Fig. 4AGo). Silver-stained gels of the digested protein revealed an efficient and specific cleavage (YECD-flag only shown, Fig. 4BGo). The hormone and the receptor components migrate with the same apparent molecular mass of 44 kDa (Fig. 4BGo) and were distinguished using Western blot analyses (Fig. 4CGo). The CTP antibody is specific to the linker regions of the hormone, thus identifying this component of the protein. Upon digestion, the hormone migrates with an apparent mobility of 42 kDa (Fig. 4CGo, lane 2). While the molecular mass is higher than predicted by the amino acid sequence, the observed value is consistent with the presence of eight putative sites for O-glycosylation and four sites for N-glycosylation. Indeed, the diffuse band is characteristic of extensive glycosylation. The flag antibody recognizes the tag at the C-terminus of the receptor; and, after treatment with protease, the ECD migrated with an apparent molecular mass of 47 kDa (Fig. 4CGo, lane 1). Again the observed mass of the protein is greater than the predicted size, but the receptor contains four putative sites for N-glycosylation that could account for this difference. Analysis with both antibodies revealed that the cleavage specificity of factor Xa, under the conditions used, was excellent and that most of the YECD-flag was cleaved by the enzyme. The difference in the amount of undigested YECD-flag seen with two antibodies can be explained by the presence of two CTPs in the YhCG portion of the complex, thus enhancing the sensitivity of detection when probing with this antibody.



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  		Figure 4. Factor Xa digestion of YECD-flag. A, Schematic representation of digestion of YECD with factor Xa. Digestion by the protease results in cleavage of the tether between the yoked hormone and ECD. The yoked hormone retains the two CTPs of the construct. B, Silver-stained gel, comparing the mobility changes of YECD-flag digested with factor Xa (+) and undigested YECD-flag (-). Approximately 10 ng YECD-flag was loaded. C, Western blot analysis of the digestion of YECD-flag using the anti-CTP and antiflag antibodies. All lanes contain 10 ng of digested or undigested sample. Lanes 1 and 3 were probed using the antiflag antibody, and lanes 2 and 4 were probed using the anti-CTP antibody. (+), Factor Xa treated; (-), untreated.

 
To assess the functionality of YECD, binding of 125I-hCG to the purified protein was tested. Initially the binding assays were performed by receptor binding to polyethylenimine-charged (GF/B) filters (Whatman, Clifton, NJ) (16) or by precipitation with polyethylene glycol (17). Despite obtaining positive results for binding using these methods, the background or nonspecific counts (counts obtained in the presence of an excess of unlabeled hCG) were inconsistent and high. Therefore, a method commonly used for ligand blotting was modified (18). The purified protein was dot-blotted on PVDF membranes and then incubated in binding solution containing the appropriate amounts of labeled and unlabeled hCG. These blots were incubated in the presence of relatively high concentrations of 125I-hCG (0.5–1 nM) overnight at room temperature, conditions chosen to favor the dissociation of the digested, yoked hormone from the receptor and allow for association of 125I-hCG.

Indeed, using capture with PVDF and these binding conditions, background binding was dramatically reduced, and the specific binding of 125I-hCG to YECD could be readily assessed (Fig. 5AGo). In the presence of 1 nM 125I-hCG, YECD bound specifically to the labeled hormone. The specific radioactivity bound to the membrane was increased considerably when the YECD was digested with factor Xa (Fig. 5Go, A and B). In the presence of lower concentrations of label (<0.5 nM), there was no detectable significant specific binding of 125I-hCG to membranes blotted with undigested YECD (Fig. 5CGo). However, YECD that had been digested with factor Xa bound 125I-hCG efficiently. The protein complex was analyzed further by saturation binding experiments. The digested YECD bound with an affinity (Kd = 1–2 nM) somewhat lower or comparable with full-length LHR and previously measured affinities of the ECD (Fig. 6Go) (5, 17, 19, 20, 21). Although, the calculated maximal binding (Bmax) was surprisingly low (1–2 pg 125I-hCG/ng YECD), the data support the idea that the cleaved yoked hormone can still interact productively with the ECD and reduce the total binding of labeled hormone.



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  		Figure 5. Binding of 125I-hCG to YECD-HT. A, PVDF membranes were blotted with different amounts of factor Xa-digested and undigested YECD and incubated with solutions containing either 1 nM 125I-hCG alone or 1 nM 125I-hCG plus 5 µg/ml hCG. The membranes were washed and exposed to film overnight at -70 C. B, Squares from these membranes were cut out and counted for {gamma}-radiation. The nonspecific counts (from membranes incubated in the presence of 5 µg/ml hCG) were subtracted from the total. C, Squares incubated with 0.5 nM 125I-hCG alone or 0.5 nM 125I-hCG plus 5 µg/ml hCG were washed, dried, and counted. The nonspecific counts were subtracted from the total. The data shown are from a representative experiment. These experiments have been repeated at least five times, with independent YECD preparations.

 


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  		Figure 6. A representative saturation binding experiment with factor Xa-digested YECD-HT (A) or YECD-flag (B). Digested YECD protein (50 ng) was blotted to PVDF membrane cut into squares and incubated with increasing amounts of 125I-hCG in the presence or absence of excess unlabeled hCG (5 µg/ml). The squares were washed, dried, and counted for {gamma}-radiation. The data shown represent the average ± range of duplicate determinations, and the experiments were repeated at least twice with different protein preparations. Conc., Concentration.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The binding of LH and hCG to the ECD of their common receptor is essential for male sexual differentiation and normal reproduction. Despite the importance of this event, our understanding of it is based on indirect observations and models. With no biophysical data available for any of the glycoprotein hormone receptors, many laboratories are attempting to produce the ECDs of the receptors at levels sufficient for structural studies (Table 1Go). Herein we show that a fusion of single-chain ligand (YhCG) and receptor (ECD), via the innocuous CTP as a linker, results in the secretion of immunoreactive protein (YECD) at levels nearly 20-fold greater than previously reported (22). By using a flag tag vs. the more standard 6-histidine tag, affinity purification of YECD with the monoclonal Ab-conjugated resin resulted in significantly higher purity and final yields, compared with Ni2+ affinity chromatography.

The purified YECD (both -HT and -flag) binds 125I- hCG with high affinity but only after the treatment with factor Xa. This important observation suggests that hormone and receptor ECD are able to fold successfully as independent domains while in a tethered context and, moreover, that they are able to associate in a productive manner to form a functional hormone-receptor complex. This interpretation is supported by previous studies in which expression of YhCG fused to the full-length receptor (YHR) resulted in a constitutively active complex (13). Binding of exogenously added hormone is blocked by the tethered hormone but can be recovered by treatment of YHR with factor Xa. Further support was provided when the pLHR-ECD and transmembrane domains and subunits of hCG were coexpressed as separate polypeptide chains in mammalian cells (23). The proteins were able to coalesce into an active receptor, indicating the ability of the proteins to fold independently while in an overexpressed environment and presumably in close proximity. Intracellular association of hCG with the ECD was not determined.

The lowest MOI used (0.1) produced the most secreted protein. This is in accordance with another report involving an intracellular protease, where very low MOIs produced considerably more protein at late harvest times (24). At low MOIs, there is an increased cell survival time, which may result in more robust secretion at later time points.

The ECD of LHR, as well as those for FSHR and TSHR, is responsible for most of the interactions necessary for high-affinity ligand binding (5). It has been expressed heterologously in Escherichia coli (E. coli), mammalian cell lines and insect cells. The bacterially expressed protein was trapped in a misfolded state in inclusion bodies (25). Refolded ECD was an active tetramer, but the reported expression levels did not indicate a recovery after refolding, a process which can often be laborious and time consuming. Baculovirus-promoted expression in insect cells has been successful in producing significant quantities of ECD, but most of this protein was trapped intracellularly and was incompletely processed, causing an inherent heterogeneity (21). It has been observed that secreted LHR-ECD is more homogeneous in binding activity, and this is probably a result of the cell’s protein-folding machinery in the secretion pathway, which aids in the more complete folding and processing of the protein (11). By tethering the YhCG to the ECD, we have observed dramatic increases in secretion of the ECD, to levels near 1.5 µg/ml, indicating a practical expression level for biophysical approaches to structure determination. To a lesser extent, this phenomenon was observed with the FSH receptor, in which coexpression of the ECD with individual subunits or with the hormone increased secretion of the FSHR-ECD, although expression values were not reported (26).

The increased secretion of YECD, compared with the ECD, may be attributed to several factors. First, in vivo during the first trimester of pregnancy and in vitro in various expression systems, hCG is expressed at high levels, and this property may contribute to the improved secretion of challenging proteins that are trapped intracellularly, like the LHR-ECD. Second, LRR proteins are hydrophobic and flexible. Though these properties enable such structures to form diverse interactions with other proteins (27), they may hinder their efficient folding and secretion in overexpression systems. With the ligand tethered, the flexibility of the ECD may be reduced, and its inherent hydrophobicity may be further buried at the interface of the hormone and receptor. This intramolecular interaction may facilitate folding and processing of the ECD by stabilization of the protein intracellularly and reduce the requirement for host chaperone machinery, which can be limiting to protein production in baculovirus-promoted expression in insect cells (28). Third, the yoked hormone may stabilize the receptor. YECD can retain binding activity for up to 3 months after preparation, when stored at 4 C. Secretion, in insect cells, of the very low density lipoprotein receptor ECD was not observed until coexpressed with the receptor associated protein, which is known to associate with high affinity (Kd = 0.7 nM) to the lipoprotein receptor (29). This reinforces the concept of associating proteins increasing the stability and expression of their partners and enhancing progression through the secretory pathway.

This novel approach of fusing ligand to receptor will be useful in generating yoked hormone-ECD complexes for the other glycoprotein hormones and may improve expression levels of these receptors as well. The availability of such fusion proteins will facilitate the determination of the structures of these hormone-receptor complexes and provide an appreciation for the precise determinants for hormone selectivity in this family of related hormones and receptors. In expressing the ECDs in tandem with their cognate ligands, the need for cocrystallization of two large glycoproteins is bypassed, which may facilitate the process of deciphering their structures. Also, in the case of hCG (3, 4) or FSH (30), having the ligand in association with the receptor may permit the use of molecular replacement for phasing of the diffraction data, thus avoiding the need for heavy atom isomorphous replacement. Furthermore, this would eliminate the need for Se-Met incorporation, which is inefficient in insect cells (31).

In summary, for the first time, a ligand has been fused to the ECD of a G-protein-coupled receptor to promote the expression and secretion of the protein and to form a functional complex. Secretion of the protein in insect cells was improved to levels near 1.5 µg/ml, and better purification was achieved using antibody affinity, rather than Ni2+-affinity chromatography. This approach could be extended to the other glycoprotein hormone receptors and other receptor systems that involve protein-protein or protein-peptide interactions, to improve their production and/or to provide a source of hormone-receptor complexes for structural studies.


   Acknowledgments
 
We thank Dr. Vernon Stevens for providing the CTP antiserum and Dr. Jan Potempa for his helpful suggestions regarding the binding assay on PVDF. We would also like to thank Dr. Chengbin Wu, who assisted with the initial set-up of the project and suggested the flag tag for purification.


   Footnotes
 
1 This work was supported by NIH Research Grant DK-33973. Back

Received September 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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