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Endocrinology Vol. 142, No. 12 5203-5211
Copyright © 2001 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Independent Activities of FSH and LH Structurally Confined in a Single Polypeptide: Selective Modification of the Relative Potencies of the Hormones

Vicenta Garcia-Campayo and Irving Boime

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Irving Boime, Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110. E-mail: iboime{at}pcg.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human glycoprotein hormones CG, LH, FSH, and TSH are heterodimers composed of a common {alpha} subunit noncovalently associated with a hormone-specific ß subunit. Recently, it was reported that a covalently fused triple-domain gonadotropin analog containing FSHß, CGß, and {alpha} subunits was dually active because it bound to both FSH and human CG (hCG)/LH receptors. However, it is not known whether both activities can be uncoupled from each other or whether they change in tandem when modifications are made in the molecule. To address this point, we constructed a triple-domain analog containing FSHß, LHß, and {alpha} subunits, and variants of this analog differing in the carboxyl-terminal region of LHß. All of the analogs exhibited bifunctional action, i.e. they bound to both LH/hCG and human FSH receptors. FSH binding and signal transduction were similar for all variants and differed less than 2-fold from that of the heterodimer. In contrast, the triple-domain variants manifested distinct individual differences in LH activity. Binding affinity of the longest variant was 30-fold lower than that of the heterodimer. Shortening the length of the LHß carboxyl-terminal region resulted in decreasing affinities between 210- and more than 480-fold. The potency of adenylate cyclase activation for LH/hCG also decreased as the carboxyl length of LHß subunit decreased. Thus, while minimally affecting the FSH activity, truncating the carboxyl end of the LHß subunit in the triple-domain analogs alters the alignment of the LHß-{alpha} domains, presumably at the junction between the subunits, and perturbs epitopes required for receptor binding. These data imply that the relative potencies of the two gonadotropin components of a triple-domain structure are independent from each other and can be selectively modified. Because there is a strong rationale for FSH/LH combinations for clinical protocols and patients exhibit variations in metabolic responses in the ratio of FSH/LH, the ability to vary the individual activities represents a potential addition to the therapeutic repertoire for treating infertility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN GLYCOPROTEIN hormones CG, LH, FSH, and TSH are heterodimers composed of a common {alpha} subunit and a hormone-specific ß subunit that are noncovalently associated. The assembly of the subunits, which occurs primarily in the endoplasmic reticulum (1), is essential for receptor binding and signal transduction in the gonads. A number of reports have now shown that covalently linking the ß and {alpha} subunits in a single chain results in biologically active analogs (2, 3, 4, 5, 6, 7). Furthermore, studies using the single-chain model have suggested that a tight association between the subunits is not required for receptor recognition and signal transduction (5, 8, 9, 10). This implies that the cognate glycoprotein hormone receptor is highly flexible and capable of accommodating varying configurations of the ligand. This hypothesis is supported by recent data showing that an unusually large analog consisting of two different ß subunits (FSHß and CGß) covalently linked to a single {alpha} subunit interacted with both receptors [FSH-R and LH/human CG (hCG)-R] and stimulated adenylate cyclase (11). It is not clear, however, how such a large and structurally modified complex interacts with either receptor, but it likely involves linear epitopes from the common {alpha} and hormone-specific ß subunit rather than unique quaternary interactions of the subunits. Here, we address whether the two hormonal activities displayed by a dually functional analog change in tandem when altering the amino acid sequence of one ß subunit domain or whether the individual activities can be uncoupled. It was observed previously that the dual activity of the above chimera was dependent on the orientation of the ß subunits, i.e. whether CGß or FSHß occupied the amino-terminal position. Thus, we could not exclude the possibility that steric alterations created by the insertion or deletion of clusters of amino acids would abolish all activity of the triple-domain analog. We selected FSH and LH as the bioactive centers and constructed a triple-domain analog consisting of FSHß, LHß, and {alpha} subunits. In addition to providing information regarding ligand-receptor contact sites, these dually active analogs represent a therapeutic regimen. For example, conditions such as hypogonadotropic hypogonadism have been considerably improved by infertility treatments consisting of typical FSH therapy supplemented with exogenous LH (12, 13, 14). In these treatments, the availability of a single compound containing both hormonal activities would be highly advantageous. Furthermore, given the distinct patient responsiveness to LH and to FSH dosing, individualizing the therapeutic FSH/LH ratio represents a potentially valuable tool for further improving infertility protocols in those patients (12, 15). To vary the relative FSH and LH activities, we modified the unique hydrophobic carboxyl-terminal end of LHß subunit, which has shown to be critical for the intracellular behavior of the dimer (16). We constructed FSHß-CTP-LHß-{alpha} variants differing in the length of the carboxyl-terminal region of LHß subunit (Fig. 1Go). In these variants, CTP represents the 28-amino-acid-long hydrophilic carboxyl-terminal peptide of CGß and serves as a linker (2, 5). All of the analogs were secreted by transfected Chinese hamster ovary (CHO) cells and exhibited comparable wild-type FSH receptor binding affinity and signal transduction. In contrast, the LH activity was variable and correlated with the length of the carboxyl terminus of the LHß subunit. The results demonstrate that the relative potencies of the two gonadotropin activities comprised in a triple-domain structure are uncoupled and not necessarily tandemly linked when modifications are introduced into a single domain.



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  		Figure 1. Construction of triple-domain FSHß-CTP-LHß-{alpha} mutant and its LHß variants (see Materials and Methods). FSHß-CTP-LHß{Delta}T-{alpha} lacks the carboxyl-terminal heptapeptide of LHß, and FSHß-CTP-LHß{Delta}T-CTP-{alpha} contains the hCGß subunit CTP segment replacing the heptapeptide.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Restriction enzymes were purchased from Promega Corp. (Madison, WI), New England Biolabs, Inc. (Beverly, MA), and Life Technologies, Inc. (Grand Island, NY). Oligonucleotides used for PCR amplification and sequencing were prepared by Washington University Sequencing Facility (St. Louis, MO). [35S]Cysteine was purchased from NEN Life Science Products (Boston, MA). Media and reagents for cell culture were prepared by Washington University Center for Basic Research (St. Louis, MO), except F-12 Ham’s, which was purchased from Sigma (St. Louis, MO). Monoclonal antibody FSH117 was obtained from Dr. P. Berger (Institute for Biomedical Aging Research, Innsbruck, Austria), monoclonal antibody LH40 was obtained from Organon (Oss, The Netherlands), and the {alpha} subunit-specific antiserum was prepared in this laboratory. Purified hCG (CR127) and recombinant FSH (rFSH) were obtained from Dr. A. Parlow (NIH Pituitary Hormone Program, Baltimore, MD). Plasmid pBS-KS was purchased from Stratagene (La Jolla, CA), and plasmid pM2-HA was constructed as described previously (2, 17). Pansorbin was purchased from Calbiochem (San Diego, CA). RIA kits for LH, FSH, and hCG were purchased from Diagnostic Products (Los Angeles, CA), and the Adenyl Cyclase Activation Flash Plate kit was obtained from NEN Life Science Products. Ultrafree centrifugal filter devices were purchased from Millipore Corp. (Bedford, MA).

Engineering triple-domain chimeras
Triple-domain single chains comprising the {alpha}, the FSHß, and different variants of the LHß subunits (Fig. 1Go) were constructed as follows:

Step 1. We generated a PCR fragment (PCR I) comprising FSHß exon 3, the complete CTP sequence, and the first five residues of LHß exon 2 by using the FSHß-CTP-{alpha} single chain (5) as a template and primers 1 (containing an EcoRI site in the intron of the FSH ß gene) and 2 (encoding the first five residues of LHß exon 2 and the last five residues of CTP). A second PCR (PCR II) was performed using the LHß{alpha} single chain (6) as DNA template and primers 3 (encoding the last five residues of CTP and the first five residues of LHß exon 2) and 4 (containing a newly created SalI site in the intron of the LHß gene), resulting in a fragment comprising the last five residues of the CTP, the complete sequence of LHß exon 2, and the newly created SalI site. Primers 1 and 4 were used for an overlapping PCR (PCR III) in which PCR I and PCR II fragments were ligated into a single product containing FSHß exon 3 and the complete sequences of CTP and LHß exon 2 flanked by EcoRI and SalI sites at the 5' and 3' ends, respectively.




Step 2. Primers 5 (containing a newly created SalI site in the intron of the LHß gene) and 6 (universal primer for pM2HA) and the previously reported LHß{alpha} single chain were used to generate a PCR product (PCR IV) containing LHß exon 3 and the complete {alpha} sequence flanked by SalI sites at both 5' and 3' ends. Parallel reactions containing primers 5 and 6 and LHß{Delta}T{alpha} or LHß{Delta}T-CTP-{alpha} single-chain variants as templates generated a PCR fragment comprising LHß exon 3 devoid of its carboxyl-terminal heptapeptide and the complete {alpha} sequence, and this truncated LHß containing the CTP and complete {alpha} sequence, respectively. In all cases, the PCR products were flanked by SalI sites at both 5' and 3' ends.


The overlapping PCR III product was ligated into the EcoRI/SalI sites of FSHß{alpha} [BlueScript KS(+)], which was then digested with BamHI/SalI. The released fragment containing the complete FSHß sequence, CTP, and LHß exon 2 was ligated into pM2HA vector. This construct was digested with SalI and, in separate reactions, ligated to each of the PCR IV products, which contained the different LHß exon 3 variants and the entire {alpha} sequence. The final products, FSHß-CTP-LHß-{alpha}, FSHß-CTP-LHß({Delta}T)-{alpha}, and FSHß-CTP-LHß-({Delta}T)-CTP-{alpha}, were sequenced to ensure that no errors occurred during PCR. Thermal cycling conditions for all DNA amplifications consisted of 20 cycles of a 3-step reaction: 30 sec denaturation at 94 C, 30 sec annealing at 55 C, and 1 min extension at 72 C.

Transfection, cell culture, and metabolic labeling
Plasmids were transfected into CHO cells as previously described (18, 19). Clones were selected using the neomycin analog G418 and maintained in Ham’s F-12 medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), and FBS (5%) in a humidified 5% CO2 incubator at 37 C. For continuous labeling experiments, cells were plated into 12-well dishes and labeled overnight with 25 µCi/ml [35S]cysteine in cysteine-free F-12 medium. Lysates and media fractions were precleared with 7.5 µl normal rabbit serum (1 h) and 75 µl Pansorbin (15 min). Supernatants were immunoprecipitated with an excess of {alpha} subunit-specific antiserum (2 h), followed by 50 µl Pansorbin (15 min). All of the incubations were performed at room temperature in a shaker. Complexes were dissociated and separated from Pansorbin by boiling for 5 min in 1x sample loading buffer (50 mM Tris-HCl, pH 6.8, 5% ß-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and the proteins were resolved by 12.5% SDS-PAGE. Gels were soaked for 15 min in 1 M sodium salicylate, dried, and autoradiographed.

Western blot analysis
Single-chain gonadotropin mutants in concentrated conditioned media were quantitated by both FSH- and LH-dimer-specific RIAs according to the manufacturer’s instructions. Equal amounts of analogs were resolved on 12.5% SDS-PAGE in the absence of heat and reducing agent and transferred onto nitrocellulose. Proteins were probed with FSH dimer-specific (FSH117) or LH/hCG (LH40) monoclonal antibodies and detected with Tropix chemiluminescent system (Tropix, Bedford, MA).

Bioassay
Receptor-binding activity of the variants was determined by radioligand receptor assay using CHO cells stably transfected with either human FSH (hFSH) or human LH (hLH)/CG receptors as previously described (11). Briefly, varying concentrations of unlabeled analogs were incubated with cells (4 x 105 cells per tube) and either 125I-hCG or 125I-FSH (100,000 cpm/tube) for 16–18 h at room temperature. The cells were washed twice with PBS/0.1% BSA, and the radioactivity was determined in a gamma counter. Nonspecific binding was measured in the presence of 3 µg (50 IU) hCG or 2 µg (10.5 IU) FSH, and this value (0.5%) was subtracted to yield specific binding. The average specific binding (hCG or FSH) was 8% of the total counts added. Displacement curves are presented as the percentage of maximal binding at each concentration of unlabeled hormone; for each sample, the 50% inhibitory concentration (IC50) value corresponds to 50% binding. The cAMP production was assessed in the above CHO cells as previously described (11). Varying concentrations of unlabeled hormones were incubated with the corresponding receptor cells (5 x 104 cells per well) in flat plates for 2 h at room temperature. After the addition of 125I-cAMP and a further 16–18 h incubation at room temperature, the plates were read in a gamma counter, and the cAMP content was expressed in picomoles per milliliter. For each ligand, the 50% effective concentration (EC50) value corresponds to the half-maximal adenylate cyclase stimulation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of triple-domain gonadotropins in CHO cells
Synthesis of the triple-domain analogs was examined by metabolic labeling of transfected cells with [35S]cysteine (Fig. 2Go). Intracellular and secreted forms were immunoprecipitated with polyclonal {alpha} antiserum and analyzed by SDS-PAGE. All variants were secreted with comparable efficiencies, i.e. for each mutant, the ratio of secreted vs. intracellularly retained forms was similar. The apparent molecular mass of the secreted forms were 66 kDa for FSHß-CTP-LHß-{alpha} (lane 2) and FSHß-CTP-LHß({Delta}T)-{alpha} (lane 4), and 80 kDa for the FSHß-CTP-LHß({Delta}T)-CTP-{alpha} variant (lane 6) containing the additional CTP. The increase in mass of the secreted forms (lanes 2, 4, and 6) compared with the corresponding lysate forms (lanes 1, 3, and 5) reflects both terminal processing of the Asn-linked carbohydrate units and addition of O-linked chains to Ser residues of the CTP before secretion (20). Although no significant heterogeneity was observed by the secreted forms of the triple-domain variants, intracellularly retained forms were clearly heterogeneous, which was more evident in the variants lacking the carboxyl-terminal heptapeptide of LHß [FSHß-CTP-LHß({Delta}T)-CTP-{alpha}, lane 5; and FSHß-CTP-LHß({Delta}T)-{alpha}, lane 3]. Absence of this sequence increased intracellular heterogeneity presumably due to differences in the processing of the oligosaccharides. These data show that gonadotropin triple-domain LH- containing mutants are synthesized and efficiently secreted by CHO cells.



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  		Figure 2. Expression of triple-domain gonadotropin analogs in transfected CHO cells. Stable clones were labeled with 25 µCi/ml [35S]cysteine for 16 h. Lysate (L) and media (M) were immunoprecipitated with polyclonal {alpha}-antiserum, and the proteins were separated on 12.5% SDS-PAGE gels. The migration of molecular weight markers is shown.

 
The secreted forms of the analogs were characterized on Western blots under nonreducing conditions (Fig. 3Go). The triple-domain variant containing the CGß subunit (lane 4) was also analyzed. Equal amounts of protein, determined by RIA, were separated on SDS gels in the absence of heat denaturation and ß-mercaptoethanol. The proteins were probed with either a FSH dimer-specific monoclonal antibody (FSH117) (Fig. 3AGo) or a LH monoclonal antibody that binds both free LHß subunit and LH dimer (Fig. 3BGo) (this monoclonal antibody also reacts with hCG). The FSH dimer-specific monoclonal antibody reacted with all variants (lanes 1–4) and with control rFSH dimer (lane 5). As expected, the electrophoretic migration of mutants containing two CTP sequences, FSHß-CTP-LHß({Delta}T)-CTP-{alpha} (lane 3) and FSHß-CTP-CGß-{alpha} (lane 4), was slower than those containing only one CTP sequence, FSHß-CTP-LHß({Delta}T)-{alpha} (lane 2) and FSHß-CTP-LHß-{alpha} (lane 1). These results imply that the FSHß subunit forms productive heterodimeric contacts with {alpha} subunit in the triple-domain fusions and that these interactions are not significantly influenced by the other ß domain (CGß or LHß) present in the polypeptide.



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  		Figure 3. Western blot analysis of secreted triple-domain gonadotropin analogs. Single-chain variants in the conditioned media were quantitated by RIA. Equal amounts of analogs were resolved on nonreducing 12.5% SDS-PAGE gels and transferred to nitrocellulose membranes. The variants were probed with FSH dimer-specific no. 117 (A) and specific no. 40 (B) monoclonal antibodies. The migration of molecular weight markers is shown.

 
Similar to the above, monoclonal antibody LH40 also reacted with the entire set of LH variants (Fig. 3BGo, lanes 1–3). However, we cannot conclude whether the LHß subunit is forming heterodimeric interactions with {alpha} subunit because, as shown in lane 5, this antibody reacts with both the free and heterodimeric forms of the LHß subunit. As previously reported for other gonadotropin mutants, high molecular weight species were detected in the blots (8, 9, 10, 11, 21, 22, 23). It is unclear whether these high molecular weight species significantly contribute to the overall biological activity of a specific mutant. Previous studies using a series of single-chain gonadotropin variants found no correlation between the proportion of aggregates formed by a particular mutant and the in vitro biological activity (8, 9). Because detection of LH dimer required overexposure of the blots, high molecular weight forms were more apparent in the Western blots probed with the LH-specific antibody. However, when the mFSH117 blots were overexposed, the high molecular weight forms were also observed (data not shown).

Biological activity of triple-domain single chains
The in vitro activity of the mutants was assessed by using CHO cells expressing either LH/hCG or hFSH receptors. The FSH-binding affinities of the different triple-domain variants were similar and comparable to that of the wild-type rFSH heterodimer (Fig. 4AGo and Table 1Go). Thus, we observed less than 2.4-fold reduction in the IC50 values of the different analogs compared with the wild-type rFSH. In contrast, the analogs displayed significant variation in their LH/hCG binding affinities (Fig. 4BGo). The triple-domain variant containing CGß subunit exhibited the highest binding (IC50, 520), which was five times lower than that of the hCG heterodimer (IC50, 104). Gradually decreasing affinities were displayed by the variants containing the LHß domain, and the extent of this decrease was correlated with the length of the LHß carboxyl-terminal region (Table 1Go). Thus, the binding affinity of FSHß-CTP-LHß({Delta}T)-CTP-{alpha} (IC50, 3000) was 30-fold lower than that of the heterodimer. Binding was reduced more than 200-fold for FSHß-CTP-LHß-{alpha} (IC50, 22,000) and more than 480-fold for FSHß-CTP-LHß({Delta}T)-{alpha}. These data show that manipulating the carboxyl-terminal extension of LHß subunit in the triple-domain structures dramatically affects the LH/CG-binding affinities of the different analogs while maintaining near-constant FSH activities. Because these variants are quantitated by RIA in conditioned medium, the question arises as to whether or not the variable LH activity is due to alterations in the quantitation by the structural modifications. If so, one might expect significant variations in the ratios of LH to FSH activities. However, RIA analyses indicated that 1 IU of FSH was equivalent to 1.1–1.2 IU of LH in those analogs containing a single CTP linker [FSHß-CTP-LHß-{alpha} and FSHß-CTP-LHß({Delta}T)-{alpha}] and corresponded to 2.4 IU of LH and 2.5 IU of CG in the case of the mutants comprising two CTP units, FSHß-CTP-LHß({Delta}T)-CTP-{alpha} and FSHß-CTP-CGß-{alpha}, respectively. In addition, because the FSH bioactivity remained relatively constant in all of the variants, quantitative changes of the analogs in the immunoassays were unlikely to account for the differences in the observed LH activity.



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  		Figure 4. Receptor binding of triple-domain variants to the hFSH (A) and LH/hCG (B) receptors. Stably transfected CHO cells expressing either LH/hCG or hFSH receptors were incubated with 125I-hCG or 125I-FSH, respectively, in the presence of varying concentrations of analogs as determined by RIA. Displacement curves show the percentage of maximal binding of the isotope at each concentration of unlabeled sample. Data are mean ± SEM of three experiments each performed in duplicate. According to the instructions supplied by Diagnostic Products, 1 mIU/ml of hCG is equivalent to 0.07 ng/ml, and 1 mIU/ml of FSH corresponds to 0.18 ng/ml. No conversion factor was provided for LH.

 

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  		Table 1. Biological activity of triple-domain gonadotropin analogs

 
To analyze the signal transduction activity of the triple-domain single chains, their ability to stimulate adenylate cyclase was determined using CHO cells expressing either LH/hCG or hFSH receptors (Fig. 5Go and Table 1Go). Consistent with the binding data, the potency of adenylate cyclase activation for FSH component in the different triple-domain structures was comparable to that seen with rFSH heterodimer (Fig. 5AGo and Table 1Go). EC50 values differed less than 2-fold from that of the heterodimer. The LH/hCG activation of adenylate cyclase generally paralleled the corresponding receptor binding affinities and was dependent on the carboxyl-terminal end of LHß subunit. The potency decreased as the carboxyl length of LHß subunit decreased (Fig. 5BGo and Table 1Go). Interestingly, similar (<2-fold different) coupling ratios were observed for all of the analogs except FSHß-CTP-LHß-{alpha}, which exhibited a ratio 11-fold higher than that of hCG. Regarding the FSH activity, the coupling ratios differed between 2- and 4-fold from the heterodimer. These data indicate that the {alpha} subunit that was covalently fused to FSHß and LHß subunits can establish functional interactions with both LH/hCG and FSH receptors. Moreover, variations in the carboxyl-terminal sequence of LHß appear to modulate LH/hCG receptor binding affinities and signal transduction of triple domains, but these modifications do not alter significantly the activity of the FSHß component.



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  		Figure 5. Signal transduction of triple-domain gonadotropin analogs. Adenylate cyclase activation was assayed using the cells described in Fig. 4Go, expressing the hFSH receptor (A) or LH/hCG receptor (B). cAMP production was determined by Adenylyl Cyclase Activation Flash Plate assay. Data are mean ± SEM of three experiments, each performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously showed that triple-domain single chains comprised of FSHß, CGß, and {alpha} subunits were secreted from CHO cells and displayed both FSH and CG activities (11). Here, we wanted to evaluate whether two structurally linked hormone units remain functionally independent, i.e. do they respond to specific modifications as a single entity or do they preserve their individual structure-function relationships? To address this issue, we targeted the unique hydrophobic carboxyl-terminal heptapeptide of LHß subunit as a determinant for structural and functional modifications. Our data show that the triple-domain FSH/LH gonadotropin analogs productively interacted with the two types of receptors. Furthermore, within the triple-domain single chain, the LH activity can be varied while maintaining a constant FSH activity. Thus, low levels of LH activity were detected in the case of the analog devoid of the hydrophobic carboxyl- terminal heptapeptide of LHß subunit. In addition, greater activity was achieved when the heptapeptide was replaced by the CTP sequence. The receptor binding affinities were relatively proportional to signal transduction (coupling ratios were <2-fold compared with the heterodimer) for all of the analogs except the variant containing the full-length LHß subunit (FSHß-CTP-LHß-{alpha}), which shows significant uncoupling. This result suggests that, compared with the FSHß-CTP-LHß({Delta}T)-CTP-{alpha} analog, determinants for signaling reactions in the FSHß-CTP-LHß-{alpha} configuration are less affected by the mutations than those related to receptor binding. These variations in LH/hCG binding affinities and signal transduction were independent of the FSH activity. Both IC50 and EC50 values for FSH binding of all the analogs remained within 2-fold of that of the heterodimer, with coupling ratios below 4. The data demonstrate that in those multidomain analogs, the biological activities can be modified independently by manipulating specific determinants of one subunit, and that both actions do not change tandemly.

The fact that altering the carboxyl-terminal sequence of LHß subunit changed the ratio of FSH/LH activities implies that the carboxyl-terminal sequence of the LHß subunit and/or the presence of CTP linker are at least indirect determinants for the LH receptor binding affinity and potency of the triple-domain mutants. Additionally, the amino- terminal part of LHß subunit could also be implicated. Previous work using double-domain LH single chains showed that although critical for secretion and stability, changes in the carboxyl-terminal end of the LHß subunit had no significant influence on receptor binding activity of LH-single chains (6). The amino end of the LHß subunit in those chimeras was free. Because the same LHß mutants in the triple-domain variants altered the binding affinity, this suggests that fusing the amino-terminal part of LHß subunit via the CTP to the FSHß subunit perturbed critical interactions for binding. Matzuk et al. (16) showed that the intracellular behavior of LH dimer is determined by cooperative interactions between the seven hydrophobic carboxyl-terminal amino acids and residues at the NH2-terminal region part of the LHß subunit. In addition, studies on the role of the lysine residue at position 2 in the CGß subunit (this corresponds to arginine in the LHß subunit) suggested that the conformation of the amino end might be associated with receptor binding (24). Furthermore, experiments using a synthetic peptide corresponding to residues 1–16 of the hCGß subunit inhibited binding of 125I-hCG to porcine Leydig cells (25).

A critical question concerns the ligand-receptor interactions. In vivo, the hCG heterodimer undergoes a structural change after it binds the LH receptor on intact cells that involves the interaction between the two subunits (26). This change occurs during the initial interaction of hCG with the extracellular domain (exodomain or ectodomain) (27), i.e. before the subsequent contact of the hCG/exodomain complex with the endodomain and generation of a hormone signal. Recent studies show that FSH induces a conformational change in the soluble exodomain of the FSH receptor after hormone binding (28). Thus, a structural change is clearly an intrinsic feature of the in vivo glycoprotein hormone-receptor interaction and signal transduction. That such large and presumably conformationally modified triple-domain analogs retain their capability to interact with two distinct types of receptors suggests that the ligand and/or the receptor are flexible. The unusually large extracellular domain of the glycoprotein hormone-receptors (29), which is essential for ligand binding (30, 31, 32, 33, 34, 35, 36), could be a determinant for such permissiveness. This domain, which is composed of eight imperfect Leu/Ile-rich repeats, each consisting of a ß strand and an {alpha} helix (29), represents a very flexible entity that could adapt to structurally and dimensionally modified ligands such as the triple-domain analogs. Supporting the idea of conformational flexibility, Wang et al. have shown that hybrid hCG/FSH analogs in which 11 amino acids in the CGß subunit were substituted with the corresponding residues of the FSHß subunit adopt different conformations upon binding with the LH and FSH receptors (37).

Dually functional gonadotropins offer a potential tool for improving fertility protocols. A number of studies have reported that some LH activity is necessary for supporting FSH-induced follicular development (12, 38, 39, 40, 41, 42). As independent entities, differences in the in vivo response to LH and FSH are plausible, given their distinctive biochemical features and the variations in metabolic clearance by the patient population. However, as a single entity, there could be a more effective control of the half-life and duration of LH and FSH activity, in addition to the beneficial effect of both activities being administered in a single injection. Furthermore, selectively altering the ratio of FSH/LH activity within the same molecule has important clinical implications. Hypogonadotropic hypogonadism patients undergoing FSH therapy require exogenous LH for an adequate follicular response. However, it has been shown that for a fixed dose of FSH, there are variations in the dose of LH required by individual patients to promote optimal follicular development (12). Similarly, varying the FSH activity during the stimulation phase could also be clinically beneficial. Thus, the availability of a wide set of differentially active bifunctional gonadotropin analogs would provide a very efficient system to closely calibrate ovulation induction protocols accurately adapted to individual patients’ requirements.


   Acknowledgments
 
We thank Dr. Albina Jablonka-Shariff, Dr. Rajendra Kumar, and Ben Gerber for critical reading of this manuscript. V.G.C. is the recipient of Institutional Trainee Grant NINDS (5-T32-NS07129).


   Footnotes
 
Abbreviations: CHO, Chinese hamster ovary; EC50, 50% effective concentration; hCG, human CG; hFSH, human FSH; hLH, human LH; IC50, 50% inhibitory concentration; rFSH, recombinant FSH.

Received June 11, 2001.

Accepted for publication August 13, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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