Genetic Fusion of an {alpha}-Subunit Gene to the Follicle-Stimulating Hormone and Chorionic Gonadotropin-ß Subunit Genes: Production of a Bifunctional Protein

Masatoshi Kanda1, Albina Jablonka-Shariff, Asomi Sato2, Mary R. Pixley, Ebo Bos3, Takashi Hiro’oka, David Ben-Menahem and Irving Boime

Departments of Molecular Biology and Pharmacology and Obstetrics and Gynecology Washington University School of Medicine St. Louis, Missouri 63110


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common {alpha}-subunit assembled to a hormone-specific ß-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the {alpha}-subunit sequences required for association with either the LHß or FSHß subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different ß-assembled to a single {alpha}-subunit). To examine whether the {alpha}-subunit can interact with more than one ß-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged ß-subunits fused to a single {alpha}-subunit gene (FSHß-CGß-{alpha}). This approach permitted structure-function analyses of {alpha}/ß domain complexes without the possibility of subunit dissociation. We reported previously that the CGß or FSHß subunit gene can be genetically fused to the {alpha}-gene and the resulting single chains (CGß{alpha} and FSHß{alpha}, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHß-CGß-{alpha} is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the {alpha}-subunit can interact with each ß-subunit in the same complex and that an {alpha}-domain fused to a ß- domain can still interact with an additional ß-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the {alpha}-subunit can resonate, i.e. shuttle between {alpha} heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
One of the distinguishing features of the human CG, FSH, LH, and TSH family is their heterodimeric structure, consisting of a common {alpha}-subunit and a hormone-specific ß-subunit (1). The assembly of the glycoprotein hormone subunits, which occurs in the endoplasmic reticulum (ER) (2, 3, 4), is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormone-specific posttranslational modifications, and signal transduction. Although the {alpha}-subunit is common to the four hormones, it appears that the contact sites in the subunit for the gonadotropin ß-subunits are not the same (5, 6). This raises the possibility that an {alpha}-subunit can interact intracellularly with more than one ß-subunit at steady state. For example, both LH and FSH are synthesized in the same cell and, presumably, the {alpha}-subunit comingles with a mixed population of ß- subunits. It is unclear then what determines the fate of a newly synthesized {alpha}-subunit that establishes the physiological ratio of LH and FSH in the reproductive cycle; there could be a compartmentalization of the nascent ER pools, or alternatively, the {alpha}-subunit can coinsert with a specific ß subunit in the ER.

Because ternary complexes comprised of two ß-subunits and one {alpha}-subunit would likely be unstable–such molecules have not been detected in vivo–we and others devised a strategy to examine this problem using a single-chain gonadotropin model in which the ß- and {alpha}-subunits are genetically linked (7, 8, 9, 10). We showed that these variants were secreted and displayed biological activity comparable to the corresponding heterodimers (7, 9). A distinct advantage for using this single-chain model is that it permits structure-function analyses of ß/{alpha} domain complexes without the possibility of subunit dissociation.

To assess the ability of the {alpha}-subunit to interact with more than one ß- subunit, we constructed a gene encoding two tandemly arranged ß-subunits fused to a single {alpha}-subunit gene. We report here that a triple-domain single chain of the structure FSHß-CGß-{alpha} generates FSH and hCG heterodimeric-like epitopes. Moreover, this protein exhibits high-affinity receptor binding to both hCG/LH and FSH receptors. These results show that the {alpha}-subunit can interact with more than one ß-subunit within the same complex.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Intracellular Properties of Triple-Domain Chimera
To generate triple-domain chimeras, the previously reported single chains, CGß{alpha} (7) and FSHß{alpha} (9), were used as substrates. In constructing the CGß{alpha} single chain (double domain), the carboxy terminus of CGß was fused directly to the N terminus of the {alpha}-subunit without an added spacer sequence (Fig. 1AGo; Ref. 7). The CGß subunit (length 145 amino acids) is distinguished among the other ß-subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (CTP). This sequence is a suitable linker, since it is serine/proline rich and thus lacks significant secondary structure, and would provide sufficient distance between Cys-110 and the first disulfide bond in the linked {alpha}-subunit. The FSHß subunit (length 111 amino acids) contains a shorter C-terminal region that could result in more interference between the cysteine 20–104 loop and the adjacent {alpha}-subunit sequences in the single chain. Thus, the CTP sequence was used as a linker in constructing the FSHß{alpha} single chain (double domain) (Fig. 1BGo; Ref. 9). In the case of the triple-domain single chains, the CTP sequence is inserted between the FSHß and the adjacent subunit (Fig. 1Go, C and D).



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Figure 1. Maps of Single-Chain Chimeras

The construction of double domain single chains corresponding to hCG (CGß{alpha}; panel A) and FSH (FSHß{alpha}, panel B) have been previously reported. They were used as substrates for constructing the chimeras shown in panels C and D. The designation CTP refers to the last 28 amino acids of the CGß subunit, which was used as linker in constructing the chimeras. The primers (1–4 as described in Materials and Methods) were used for overlap PCR to construct FSHß-CGß-{alpha}.

 
Metabolic Labeling
Synthesis of the single chains bearing two (CGß{alpha} and FSHß{alpha}) or three (FSHß-CGß-{alpha} and CGß-FSHß-{alpha}) subunit domains from stably transfected Chinese hamster ovary (CHO) cells was examined by metabolic labeling with [35S] cysteine, and the proteins were immunoprecipitated from cell lysate and medium with polyclonal {alpha} antiserum (Fig. 2Go). The apparent molecular masses of the FSHß{alpha} and CGß{alpha} single chains are 53 ± 0.4 kDa and 51 ± 0.1 kDa, respectively. It is clear that both triple-domain variants, FSHß-CGß-{alpha} and CGß-FSHß-{alpha}, are secreted (Fig. 2Go; lanes 5–8), and their apparent molecular masses are 88 ± 0.6 kDa and 88 ± 0.9 kDa, respectively, which correspond to the presence of an additional subunit domain and CTP cassette. The shift in mobility observed between the lysate/medium forms is due to the processing of Asn-linked oligosaccharides and the addition of carbohydrates to serine acceptor sites in the CTP (11). It is noteworthy that no smaller species corresponding to a heterodimeric-like protein is detected.



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Figure 2. Synthesis of Single-Chain Chimeras from Transfected CHO Cells

Cells were incubated for 6 h with [35S] cysteine. Polyclonal {alpha}-antiserum was used to precipitate cell lysate (L) and medium (M) fractions, and the reduced proteins were resolved on 12.5% SDS-PAGE. The migration of molecular mass markers is shown. The apparent molecular mass for FSHß-CGß-{alpha} (lane 8) and CGß-FSHß-{alpha} (lane 6) is ~88 kDa (n = 2 experiments). The extended band width of the FSHß{alpha} (lane 1) and CGß{alpha} (lane 4) media forms is due to distortion created by comigration of the unlabeled IgG recovered from the immunoprecipitation assay.

 
The secretion of the chimeras from stably transfected CHO cells was examined by pulse-chase metabolic labeling, and the proteins were resolved on reduced SDS-PAGE gel (Fig. 3Go). As previously observed, the CGß{alpha} (7) and FSHß{alpha} (9) single chains were secreted efficiently (t1/2 = 90 min and t1/2 = 100 min, respectively). The secretion of the mutants was considerable slower; the t1/2 of CGß-FSHß-{alpha} and FSHß-CGß-{alpha} are 205 min and 325 min, respectively. While CGß{alpha} and CGß-FSHß-{alpha} were quantitatively secreted (>80% recovery), only 30–50% of the FSHß-CGß-{alpha} chimera is recovered. These data show that the orientation of the two ß-subunits affects secretion and recovery, and for the FSHß-CGß-{alpha} chain, a significant fraction was degraded.



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Figure 3. Secretion Kinetics of Single-Chain Chimeras

CHO cells expressing the chimeras were pulse-labeled and chased for the indicated times (h). Cell lysates and media were imunoprecipitated with {alpha}-antiserum, and reduced proteins were resolved on 12.5% SDS-PAGE.

 
Western Blot Analysis
To characterize the subunit domain interactions in the mutants, Western blots were performed under nonreduced conditions using CG- and FSH dimer-specific monoclonal antibodies (mAbs) (Fig. 4Go). In addition to the triple-domain single chains, we probed the double-domain single chains, CGß{alpha} and FSHß{alpha}, as controls. As shown previously (7, 9), the ß- and {alpha}-domains in CGß{alpha} (molecular mass = 51 ± 0.5 kDa) and FSHß{alpha} (molecular mass = 53 ± 0.5 kDa) retained the majority of the heterodimeric interactions which was reflected by the signals detected by dimer-specific mAbs B109 (CG) (panel A, lane 1) and 117 (FSH) (panel B, lane 2). As expected, no signals were detected for FSHß{alpha} and CGß{alpha} in the CG and FSH immunoblots, respectively. FSHß-CGß-{alpha} was immunoreactive with both B109 (panel A, lane 3; molecular mass = 83 ± 1.9 kDa) and 117 (panel B, lane 3; molecular mass = 83 ± 1.3 kDa) antibodies. However, while no detectable signal was seen when CGß-FSHß-{alpha} was probed with mAb B109 (panel A, lane 4), this mutant was reactive to mAb 117 (panel B, lane 4; Mr = 83 ± 1.8 kDa). Together, these data show that both the CGß and FSHß subunits form heterodimeric-like interactions with the {alpha}-subunit in the FSHß-CGß-{alpha} mutant but only the FSHß component is dimer configured in the CGß-FSHß-{alpha} variant. Similar to the results obtained with the reduced SDS-PAGE (Fig. 2Go), no significant accumulation of bands corresponding to a heterodimer-related protein was seen. Slower migrating species were detected on the blots (Fig. 4Go; Mr = 134 ± 6 kDa), and although the nature of these proteins is not clear, such aggregates are observed during the purification of native hCG (12, 13).



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Figure 4. Western Blot Analysis of Secreted Mutants under Nonreducing Conditions

Samples were electrophoresed on 12.5% polyacrylamide gels in the presence of 0.1% SDS. Proteins were detected with CG dimer-specific mAb B109 (panel A) and FSH dimer-specific mAb 117 (panel B). The migration of molecular weight markers is shown on the left side of both panels. The apparent molecular mass for FSHß-CGß-{alpha} (lane 3) and CGß-FSHß-{alpha} (lane 4) is ~83 kDa. The migration of the double-domain single chains (controls) CGß{alpha} (molecular mass, ~51 kDa) and FSHß{alpha} (molecular mass, ~53 kDa) is shown in lanes 1 and 2, respectively. This experiment was repeated five times.

 
Receptor Binding and Signal Transduction
The quantitation of the triple-domain structures is a critical issue since there are no comparable standards to calibrate these new mutants. To address this point, we determined the amount of FSH and hCG in mutants using RIA and sandwich-type enyzme-linked immunosorbent assay (ELISA) methods. Using a polyclonal-based RIA the amount of FSH and hCG in the FSHß-CGß-{alpha} mutant was 25.3 ± 1.8 µg/ml and 19.4 ± 0.7 µg/ml, respectively. For the CGß-FSHß-{alpha} chimera, the FSH and hCG content was 22.5 ± 1.9 µg/ml and 15.1 ± 1.2 µg/ml, respectively. The ELISA contains subunit-specific monoclonal antibodies; the FSH and hCG centers in the FSHß-CGß-{alpha} calculated by this assay were 31 µg/ml and 55 µg/ml, respectively. Although there is some difference in the immunoreactivity of the FSH/hCG components as determined by these methods (likely due to the use of different antibodies and standards), the results obtained by two independent immunological assays are within reasonable agreement. Moreover, the extent of binding of an equivalent amount of the triple-domain chimera and the heterodimer, as determined by ELISA, was measured by the BIACORE (BIACORE Products, Life Sciences, Uppsala, Sweden). No significant difference in the extent or kinetics of binding was observed when the reactivity of the triple domain was compared with that of the corresponding heterodimers (data not shown). This corroborates the reliability of the immunoassay determinations and confirms the expected 1:1 molar ratio of the ß-subunits arranged in the chimera. Taken together, these data verify that the immunoreactivity of the chimera is similar to the corresponding heterodimers.

Each chimera was tested independently for binding to the FSH and LH/hCG receptors using transfected CHO cells expressing either the human LH/hCG or human FSH receptors (Fig. 5Go and Table 1Go). The FSHß-CGß-{alpha} mutant displayed high-affinity binding to the LH/hCG receptors, although the binding affinity was reduced 4- and 8-fold relative to the hCG heterodimer and CGß{alpha}, respectively. In the case of the FSH receptor binding, there was a 4- to 5-fold decrease in binding by this triple domain chimera compared with the FSH heterodimer and FSHß{alpha} (Fig. 5Go, A and B, and Table 1Go). In contrast to these results, CGß-FSHß-{alpha} exhibited little binding to either the hLH/hCG or hFSH receptor (Fig. 5Go and Table 1Go).



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Figure 5. Receptor Binding of Single-Chain Chimeras to the hLH/hCG and FSH Receptors

Stably transfected CHO cells expressing the receptors were incubated for 16–18 h at room temperature in the presence of varying concentrations of single-chain mutants. Panels A and B correspond to hCG and FSH binding, respectively. The corresponding heterodimeric forms were used as standards. The concentration of each hCG and FSH center in the chimeras was determined by RIA and was used for calculating the inputs of LH/hCG and FSH to the binding assays, respectively. Data are mean of ± SEM of four experiments.

 

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Table 1. The LH/hCG and FSH Receptor Binding and cAMP Stimulation by the Triple Domain Single Chains Using CHO Cells Expressing Either LH/hCG or FSH Receptors

 
The signal transduction activity of the chimeras was assessed by their ability to stimulate the cAMP production in transfected CHO cell lines expressing human LH/hCG or FSH receptors. While the corresponding signal transduction activity by the chimeras (Fig. 6Go, A and B, and Table 1Go) paralleled the receptor binding affinity, there was some apparent uncoupling in binding and cAMP activation.



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Figure 6. Signal Transduction of Single-Chain Chimeras

Stimulation of cAMP accumulation by wild-type heterodimers and single-chain variants was assayed in transfected CHO cells expressing either the LH/hCG or hFSH receptors. The concentration of hCG and FSH in the chimeras was determined as described in the legend of Fig. 5Go. The analogs were incubated for 16–18 h at room temperature, and the total amount of cAMP (extra- and intracellular) was quantitated. Panels A and B correspond to hCG and FSH assays, respectively. Data are the mean ± SEM of three experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Here we show that the single chains comprised of three genetically fused gonadotropin subunit domains are secreted from cells and are biologically active. It is striking that while FSHß-CGß-{alpha} had a greater binding affinity than CGß-FSHß-{alpha} to both the FSH and hCG receptors, its secretion rate and recovery was less. These data further support the hypothesis that for the glycoprotein hormone family, the subunit-specific epitopes responsible for intracellular behavior are uncoupled from those required for bioactivity. Regarding the intracellular differences, since the {alpha}-subunit is at the carboxyl end in both chimeras, this apparently precludes a direct affect of this domain in the secretory pathway. Previously we showed that the uncombined FSHß subunit was secreted poorly whereas the free CGß subunit was secreted quantitatively (14). Because the FSHß subunit occupies the amino-terminal end of the FSHß-CGß-{alpha} chimera, it may behave similarly to the free FSHß subunit where exposure of epitopes to one or more chaperones impedes secretion. By contrast, the noncombined CGß subunit was secreted efficiently consistent with the secretion of the mutant bearing CG at the amino terminal. These data imply that the amino-terminal region of the ß-subunits influences the intracellular behavior of the chimeras.

It is curious that in CGß-FSHß-{alpha} chimera, only the FSHß domain formed heterodimeric-like epitopes; no signals characteristic of a CG dimer were detected with mAb B109. These data suggest that the FSHß subunit is less flexible than the CGß subunit since the latter permitted the amino-terminal FSHß domain to interact with the {alpha}-subunit in the FSHß-CGß-{alpha} chimera. In the case of CGß-FSHß-{alpha}, only the adjacent FSHß subunit domain, but not the CGß domain, interacted with the {alpha}-subunit to establish a dimeric-like association.

Given the increased size of the triple-domain structure, it was surprising that the complexes were biologically active. Although determinants from both {alpha}- and ß-subunits are critical for bioactivity, our previous studies suggested that ligands with different conformations can also bind to the receptor (15, 16). In addition, others have reported that ligand conformation can be altered during binding (17, 18). The high-affinity binding of the triple-domain complex supports this hypothesis. That the receptor productively interacts with three subunit domain gonadotropins suggests that the receptor has sufficient flexibility to accommodate a much larger ligand.

Although the trimeric entity exhibits both hCG and FSH dimeric-like epitopes, we cannot exclude the possibility that the higher molecular mass forms also contributed to the observed bioactivity. However, we consider this unlikely since in the case of the double-domain single chain, there was no correlation with biological activity and the extent of aggregation in a series of tethered gonadotropin mutants (15, 16).

A critical question arising from these observations is whether the FSHß-CGß-{alpha} chimera possesses dual activity, i.e. does a single form of this protein exhibit both FSH and CG activity? If such a molecule is dually active, it implies that an {alpha}-subunit can serve two ß-subunits simultaneously and achieve a heterodimeric-like configuration for each hormonal unit, as exemplified by FSHß-CGß-{alpha}. Consistent with this suggestion is the evidence for different contact sites in the {alpha}-subunit for the FSHß and CGß subunits (5, 6). It is likely that such dual interactions would not be stable but rather transitory heterodimeric complexes pro tem. This would be analogous to a resonance interaction in which the {alpha}-subunit phases between both ß-subunits. However, we cannot exclude the existence of at least two distinct stable species with heterodimeric-like features in the chimeric population, CGß/{alpha} or FSHß/{alpha}, i.e. no dually active species. That both the hCG and FSH bio- and immunoreactivities in the FSHß-CGß-{alpha} chimera are comparable implies that a stochastic generation of equal distribution of the two stable, dimer-like conformers would have to occur. On the other hand, it is unnecessary to assume a priori that the native heterodimeric configuration is formed in this chimera. As discussed above, single-chain mutants were biologically active despite changes in the quaternary structure (15, 16, 19). Thus, if the subunits were properly folded but not configured strictly as native heterodimers, the protein was biologically active. If the {alpha}-subunit interacts simultaneously with two ß-subunits, this would imply that a multimeric gonadotropin could be generated in the secretory pathway. This point is especially relevant to FSH/LH because both are synthesized in the pituitary gonadotropes and presumably the two ß-subunits and {alpha}-subunit comingle in the same ER pools. Thus, complexes of {alpha}, FSHß, and LHß could exist during the early stages of synthesis and accumulation in the ER before the segregation of the subunits to form native hormone.

The ability to design dually active single-chain analogs (17) may be useful as therapeutics in addition to structure-function issues. For example, there could be more effective control of the half-life and duration of activity of gonadotropins. If LH and FSH were given as single entities, there is the potential for unexpected difference in the in vivo response. This point is important given the variations in metabolic clearance by the patient population.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of Triple-Domain Tethers
The general orientation of the subunit domains in the tethers is: NH2-ß-ß-{alpha}-COOH. We wanted the C-terminal end of the {alpha}-subunit to be free since this region is critical for the receptor binding (20, 21, 22, 23). To generate mutants, the previously reported single chains, CGß{alpha} (7) and FSHß{alpha} (9), were used as substrates (Fig. 1Go, A and B).

Two triple-domain tethers were constructed: 1) CGß-FSHß-{alpha} (Fig. 1CGo) and 2) FSHß-CGß-{alpha} (Fig. 1DGo). CGß-FSHß-{alpha} was engineered in a series of cut-and-paste experiments. To construct FSHß-CGß-{alpha}, PCR was used with the following primers (5' -> 3'):

1. CAA GCA GTA TTC AAT TTC TGT CTC; contains a newly created EcoRI site in the intron of FSHß gene.

2. CGG CTC CTT GGA TTG TGG GAG GAT CGG; this sequence encodes amino acids 141–145 and 1–4 of the CGß subunit.

3. CCG ATC CTC CCA CAA TCC AAG GAG CCG; corresponds to amino acids 141–145 and 1–4 of the CGß subunit.

4. CTG ACC AGA GAG GTC GAC CAC CCT TCC; contains a SalI site in the CGß intron.

Primers 1 and 2 generated a fragment containing the EcoRI site of the FSHß gene, exon 3 of the FSHß subunit, amino acids 118–145 of the CGß subunit (CTP), and amino acids 1–4 of the CGß subunit. A fragment corresponding to amino acids 141–145, 1–4 of the CGß subunit, and the SalI site in CGß intron 2 was synthesized using primers 3 and 4. Primers 1 and 4 were used to ligate the above fragments by overlapping PCR, and this product contained the EcoRI/SalI site in the FSHß intron, CTP, CGß exon 2, and the SalI site in the intron between exons 2 and 3 of the CGß subunit. After EcoRI/SalI digestion, this product was ligated into the EcoRI/SalI site of FSHß{alpha} [BlueScript KS(+)] and after subcloning, the vector was digested with BamHI/SalI, and the resulting fragment was subcloned into pM2HA (7). This construct was then digested with SalI. In a separate reaction pM2HA bearing the CGß{alpha} single chain (7) was digested with SalI. The fragment containing CGß exon 3 and the entire coding sequence of the {alpha}-subunit was inserted into the SalI site containing the FSHß-CTP-CGß-exon 2 sequence. The final product, FSHß-CTP-CGß-{alpha}, was sequenced to verify that no errors were introduced during the construction.

Transfection and Cell Culture
The tethered variants were transfected into CHO cells using the calcium phosphate method as previously described (7). Stable clones were selected approximately 11 days later using the neomycin analog G418 (250 µg/ml). The clones were maintained in Ham’s F-12 medium [supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine] containing 5% FBS and G418 (125 µg/ml) at 37 C in a humidified atmosphere of 5% CO2/95% air.

Metabolic Labeling
To examine the synthesis of single-chain chimeras, CHO cells expressing mutants were labeled for 6 h in Ham’s F-12 medium containing dialyzed BSA, [35S]cysteine (Amersham Pharmacia Biotech, Arlington Heights, IL), or a mixture of [35S]cysteine and methionine (Promix; Amersham Pharmacia Biotech) (15, 16). Aliquots of cell lysate and medium were immunoprecipitated with polyclonal antiserum directed against the common {alpha}-subunit, which was raised in the laboratory. The reduced proteins were resolved on 10% or 12.5% SDS-PAGE. To determine the secretion kinetics of chimeras, cells were pulse labeled and chased up to 24 h. Lysates and media were immunoprecipitated with {alpha}-antiserum, followed by SDS-PAGE. The secretion t1/2 of chimeras was calculated as the time (min) when half of the maximal secreted hormone was detected in the media.

Western Blot
Media samples were resolved on 12.5% SDS-PAGE under nonreduced conditions without heating. Blotting was performed on a nitrocellulose membrane and visualized with the chemiluminescence detection system (Tropix Inc., Bedford, MA) according to the manufacturer’s protocol. The blots were probed with the hCG conformational sensitive mAb B109 (provided by Dr. Steven Birken, Columbia University Medical School, New York) that recognizes primarily the heterodimer but not the monomeric ß-subunit (24) and an hFSH-specific monoclonal antibody (mAb 117), which has a 100-fold greater affinity for the FSH heterodimer than to the noncombined FSHß subunit (25).

Receptor Binding and Signal Transduction
Collection media were concentrated using either a Centric Prep concentrator (Amicon Inc., Beverly, MA) or an Ultra-free centrifuge filter device (Millipore Corp., Bedford, MA). The triple-domain single chains were quantitated using two different methods. In the first method the amount of FSH and hCG was determined with a RIA kit (Diagnostic Products, Los Angeles, CA), which uses either hFSHß or hCGß polyclonal antisera. These assays score the ß-subunit whether or not it is configured in a heterodimeric-like association with the {alpha}-subunit. The second approach employs a sandwich-type ELISA method and includes two sets of monoclonal antibodies. The FSH assay contained an FSHß mAb as the capture reagent and an {alpha} mAb for detection. The hCG assay included a capture {alpha} mAb and an hCGß-specific mAb for detection. All mAbs reacted with both intact heterodimer and noncombined subunits in solution. Thus, the assay minimizes the conformational influences on detection. To further verify the concentrations of the FSH and hCG centers in the chimera and heterodimers determined by the ELISA, we characterized the immunoassays with a BIACORE system (26, 27). We used the same amount of the triple-domain single chain and wild-type heterodimers with BIACORE chips each containing an FSHß and CGß subunit-specific mAbs. These mAbs recognize different epitopes from those used in the ELISA.

Receptor binding was determined using stably transfected CHO cell lines expressing either the hLH/hCG or FSH receptors. The cells were incubated (4 x 105 cells per tube) with ligands and [125I]hCG or [125I]FSH (100,000 cpm/tube), respectively, for 16–18 h at room temperature. The binding reaction was terminated by washing the cells twice with PBS containing 0.1% BSA, and the radioactivity was quantified in a {gamma}-counter. Each experiment was performed three times with duplicate tubes.

The total (extra-and intracellular) amount of cAMP was determined using the Adenyl Cyclase Activation Flash Plate kit (NEN Life Science Products, Boston, MA) as per the manufacturer’s instructions. CHO cells (5 x 104 cells per well) expressing either the LH/hCG or FSH receptors were incubated with ligand for 2 h at room temperature, [125I]cAMP was added, and the cells were incubated for an additional 16–18 h at room temperature. The Flash Plates were read in a Packard Top {gamma}-Counter (Packard Instruments, Downers Grove, IL). Each experiment was performed three to four times with duplicate wells. The cAMP content was expressed in picomoles/ml.

Purified hCG (CR127) and recombinant FSH were obtained from the NIH and N.V. Organon (Oss, The Netherlands), respectively. The binding affinity (IC50) and the half-maximal adenylate cyclase stimulation (EC50) were calculated for each concentration-response curve (hCG and FSH) for each ligand.


    ACKNOWLEDGMENTS
 
We are grateful to Floortje Nagel and Anna-Marie De Haan at N.V. Organon for their assistance in the BIACORE experiment. We thank Ms. Mary Wingate for her excellent assistance in preparing the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Irving Boime, Department of Pharmacology, Washington University Medical School, 660 South Euclid, St. Louis, MO 63110.

Albina Jablonka-Shariff was supported by National Research Service Award Fellowship HD-08301. This work was supported by N.V. Organon.

1 Current Address: Department of Obstetrics and Gynecology, Rokko Island Hospital, 658-0032 Kobe, Japan. Back

2 Current address: Nishi Kobe Medical Center, 650-0017 Kobe, Japan. Back

3 N.V. Organon, 5340 BH Oss, The Netherlands. Back

Received for publication March 22, 1999. Revision received June 21, 1999. Accepted for publication July 20, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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