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Endocrinology Vol. 143, No. 10 3773-3778
Copyright © 2002 by The Endocrine Society

ARTICLE

Thyrotropin, Follitropin, and Chorionic Gonadotropin Expressed as a Single Multifunctional Unit Reveal Remarkable Permissiveness in Receptor-Ligand Interactions

Vicenta Garcia-Campayo, T. Rajendra Kumar and Irving Boime

Department of Molecular Biology and Pharmacology, Washington University School of Medicine (V.G.-C., I.B.), St. Louis, Missouri 63110; and Departments of Pathology and Molecular and Cellular Biology, Baylor College of Medicine (T.R.K.), Houston, Texas 77030

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The glycoprotein hormones [chorionic gonadotropin (CG), FSH, LH, and TSH] are composed of a common {alpha}-subunit and a hormone-specific ß-subunit. Subunit assembly is vital to the in vivo function of these hormones. However, recent in vitro studies using double domain (ß-{alpha}) and triple domain (ß-ß-{alpha}) single chains have shown that gonadotropin receptor recognition can accommodate conformationally modified ligands. To investigate the extent of flexibility of ligand-receptor interactions, we constructed a single chain tetramer containing three different ß-subunits (TSHß, FSHß, and CGß) and a single {alpha}-subunit. This analog was inefficiently secreted from transfected Chinese hamster ovary cells, but surprisingly, the protein exhibited all activities comparable to the corresponding heterodimers. Because the {alpha}-subunit presumably cannot form the entire array of heterodimeric contacts with all ß-subunits simultaneously in the tetra-domain analog, the data show that the complete quaternary subunit-subunit interactions are essential for the efficient intracellular trafficking of the glycoprotein hormones, but not for receptor recognition. From an evolutionary perspective, the organization of such a multifunctional analog is consistent with the hypothesis that glycoprotein hormone genes were originally linked in tandem and subsequently evolved as independent genes. Our results also indicate that both gonadal and thyroid stimulatory functions can be combined in a unique analog.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FAMILY of glycoprotein hormones consists of TSH, FSH, and LH synthesized in the anterior pituitary, and chorionic gonadotropin (CG), which is produced by the placenta (1). They are heterodimers composed of a common {alpha}-subunit and a hormone-specific ß-subunit, which are noncovalently associated. Glycoprotein hormone subunit assembly is vital for binding to their cognate receptors and signaling on the thyroid gland and gonads (1). The crystal structures of human (h)CG and FSH heterodimers show that the {alpha}- and ß-subunits have remarkably similar conformations and are tightly associated with each other along much of their surfaces in a head to tail arrangement (2, 3, 4). It has been shown that covalently linking the {alpha}- and ß-subunits in a single chain results in biologically active analogs (5, 6, 7, 8). Studies using these variants imply that the extensive interaction between the {alpha}- and ß-subunits is not essential for the biological action of these hormones (9, 10, 11). In addition, triple domain, single chain variants comprised of two different ß-subunits covalently fused to a single {alpha}-subunit (12, 13) displayed dual hormone activities, suggesting that gonadotropin receptors have sufficient flexibility to recognize and bind these larger multidomain structures. We engineered a unique tetra-domain analog comprised sequentially of TSHß, FSHß, and CGß subunits that are covalently linked to a single {alpha}-subunit. Because the {alpha}-subunit presumably cannot heterodimerize with three different ß-subunits simultaneously, such an analog is an excellent candidate to test the hypothesis that hormone signaling is not dependent on the determinants generated by {alpha}/ß heterodimeric contacts. We show that this tetrameric analog is secreted from Chinese hamster ovary (CHO) cells and is recognized by specific antibodies directed against each of the hormones. Interestingly, this analog demonstrated all the three hormonal activities comparable to the corresponding individual heterodimers. These results prove that glycoprotein hormone-receptor interactions do not rely on the complete intact quaternary interactions. That three functionally different hormonal activities with distinct in vivo expression patterns can be efficiently combined in a unique tetrameric structure is evolutionarily intriguing. It has been proposed that both {alpha}- and hormone-specific ß-subunits evolved by duplication of a single ancestral gene (1, 14, 15). If, as shown for a number of protein families (16), internal duplication also occurred within this family, one polypeptide chain could comprise a tandem array of two or more linked functional domains. Our results suggest that the glycoprotein hormone family could have existed as a polycistronic unit, evolving into the individual hormonal entities.


   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 for Ham’s F-12, which was purchased from Sigma (St. Louis, MO). Monoclonal antibody FSH117 was obtained from Dr. P. Berger (Institute for Biomedical Aging Research, Innsbruck, Austria), and monoclonal CG (B109) was provided by Dr. Steven Birken (Columbia University Medical School, New York, NY). The {alpha}-subunit-specific antiserum was prepared in this laboratory. Purified hCG, pituitary hTSH, and TSH antiserum were obtained from Dr. A. Parlow (NIH Pituitary Hormone Program, Baltimore, MD). Recombinant human FSH was provided by Organon (Oss, The Netherlands). Plasmid pM2-HA was constructed as described previously (5, 17). Pansorbin was purchased from Calbiochem (San Diego, CA). RIA kits for LH, FSH, hCG, and TSH were purchased from Diagnostic Products (Los Angeles, CA). The Adenyl Cyclase Activation Flash Plate kit, [125I]hCG, and [125I]FSH were obtained from NEN Life Science Products. [125I]TSH was obtained from Braham Diagnostics (Atlanta, GA). Ultrafree centrifugal filter devices were purchased from Millipore Corp. (Bedford, MA). CHO cells expressing the hTSH receptor were provided by Dr. Basil Rapoport (Cedars-Sinai Research Institute and University of California School of Medicine, Los Angeles, CA).

Engineering tetra-domain glycoprotein analog
The single chain comprising the TSHß, FSHß, CGß, and {alpha}-subunits (Fig. 1Go) was constructed as follows: pM2-HA-TSHß-CTP-{alpha} (18) was used as a template for oligos 1 and 2 to generate a PCR fragment comprising the complete TSHß sequence, the CTP, and the first five residues of exon 2 of FSHß. A second PCR was performed using the FSHß-CTP-CGß-{alpha} triple domain analog (12) as template and primers 3 and 4. This yielded a fragment comprising the last five residues of the CTP, the complete exons 2 and 3 of FSHß, CTP, and exon 2 of CGß. Primers 1 and 4 were used for an overlapping PCR in which the above PCR products were ligated into a single product containing the complete sequences of TSHß and FSHß joined by a CTP sequence, and exon 2 of CGß linked to FSHß through a CTP unit. This PCR product, which was flanked by BamHI and SalI sites, was subcloned into the BamHI/SalI sites of pM2-HA (5, 17). This construct was finally digested with SalI and ligated to a SalI/SalI fragment containing CGß exon 3 and the complete {alpha} sequence released from pM2HA-CGß-{alpha} (5) single chain. The final TSHß-CTP-FSHß-CTP-CGß-{alpha} product was 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. The following primers were used: primer 1, universal primer for pM2HA; primer 2, 5'-CAG CTC ACA GCT ATT TTG TGG GAG GAT CGG-3'; primer 3, 5'-CCG ATC CTC CCA CAA AAT AGC TGT GAG CTG-3'; and primer 4, 5'-CCT ACC AGA GAG GTC GAC CAC CCT TCC-3'.



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  		Figure 1. Schematic representation of the tetra-domain single chain glycoprotein analog. CTP, Carboxyl-terminal peptide of hCGß.

 
Transfection, cell culture, and metabolic labeling
Plasmids were transfected into CHO cells, and clones were selected and maintained using neomycin analog G418 as previously described (19). 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 medium fractions were immunoprecipitated with {alpha}-subunit-specific antiserum. The reduced proteins were resolved by 8% SDS-PAGE (19).

RIA and Western blot analyses
Estimates of the concentrations of the three components of the single chain analog in concentrated conditioned media were performed by TSH, FSH, and CG dimer-specific RIAs according to the manufacturer’s instructions. Each of the three RIAs used is hormone specific and shows low cross-reactivity (<0.1%) to the other glycoprotein hormones. The tetramer single chain exhibited 7.0 IU CG and 1.6 mIU TSH for each international unit of FSH immunoreactivity detected. The responses obtained from serially diluted samples of analog (log-logit graphs) parallel those of the respective heterodimers (the slopes of heterodimer vs. that of the tetramer were 1.00, 0.96, and 1.04 for CG, FSH, and TSH, respectively). Proteins were probed with dimer-specific FSH and hCG monoclonal antibodies, and with {alpha} and TSHß antisera and were detected with Tropix chemiluminescent system (Tropix, Bedford, MA).

Bioassay
The hCG and FSH receptor binding activities of the variants were determined by radioligand receptor assays using CHO cells stably transfected with hLH/CG or hFSH receptors, respectively, as previously described (12). Nonspecific binding was measured in the presence of 50 IU (3 µg) hCG or 10.5 IU (2 µg) of 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. TSH binding assays were performed similar to the conditions previously reported by Filetti et al. (20). Briefly, 12-well plates containing CHO cells stably transfected with TSH receptors (4 x 105 cells/well), varying concentrations of unlabeled analogs, and [125I]TSH (15,000 cpm/well) were incubated for 16–18 h at 4 C. The cells were then washed three times with cold modified Krebs-Ringer buffer (21), and the radioactivity was measured after solubilization in 1 M NaOH. The average specific binding (TSH) was 15% of the total counts added, and nonspecific binding was 6%. Displacement curves are presented as the percentage of maximal binding at each concentration of unlabeled hormone.

The cAMP levels were assayed in the stimulated CHO cells containing hLH/CG, hFSH, or TSH receptors as previously described (12). Varying concentrations of unlabeled hormones were incubated with the corresponding receptor cells (5 x 104 cells/well) in the Flash Plates (NEN Life Science Products) for 2 h at room temperature. After adding [125I]cAMP and a further 16- to 18-h incubation at room temperature, the plates were read in a {gamma}-counter, and the cAMP content was expressed in picomoles per milliliter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the tetra-domain analog in CHO cells
The expression and secretion of the TSHß-CTP-FSHß-CTP-CGß-{alpha} variant from stably transfected CHO cells were examined by metabolic labeling with [35S]cysteine (Fig. 2Go). The analog was secreted from CHO cells and exhibited an apparent molecular mass of 110–120 kDa (Fig. 2Go, lane 2). Although much of the mutant was retained intracellularly (Fig. 2Go, lane 1), these data show that the structural features necessary for secretion are conserved in the tetra-domain analog and imply that the fusion protein is stable. The apparent increase in mass of the secreted form (Fig. 2Go, lane 2) compared with the corresponding lysate form (Fig. 2Go, lane 1) reflects both terminal processing of the Asn-linked carbohydrate units and addition of O-linked chains to Ser residues in the CTP units just before secretion (22).



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  		Figure 2. Expression of the tetra-domain single chain analog in CHO cells. Transfected CHO cells were incubated at 37 C for 18 h in the presence of [35S]cysteine. Intracellular (L) and secreted (M) forms were immunoprecipitated with {alpha} antiserum and separated on 8% SDS-PAGE gels.

 
To confirm that each of the gonadotropin ß-subunits was present in the mature secreted protein, we performed Western blot analysis under nonreducing conditions (Fig. 3Go). Blots were probed with TSHß polyclonal antiserum (Fig. 3AGo), monoclonal antibodies specific for FSH (Fig. 3BGo) and CG (Fig. 3CGo), and {alpha} polyclonal antiserum (Fig. 3DGo). The analog was recognized by all of these antibodies, demonstrating the integrity of the recombinant fusion protein and also that epitopes specific for each of the gonadotropin subunits were preserved. A high molecular mass band was also observed in the blots. This band was most apparent in the blot probed with the CG-specific antibody, which also recognized lower molecular mass species.



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  		Figure 3. Western blot analysis of secreted tetra-domain single chain analog. Equal amounts of analog (100 IU FSH) and equal amounts of each heterodimer (50 ng) were resolved on nonreducing 8% SDS-PAGE gels and transferred to nitrocellulose membranes. The samples were probed with TSHß antiserum (A), FSH-dimer specific monoclonal antibody (B), hCG-dimer specific antibody (C), and {alpha} antiserum (D). The migration of molecular mass markers is shown. The control heterodimers are designated di; T, tetra-domain single chain analog.

 
Biologic activity of tetra-domain mutant
Receptor binding.
The in vitro biological activity of the analog was assessed by using stably transfected CHO cells expressing TSH, FSH, or LH/hCG receptors. The cells were incubated with varying amounts of unlabeled mutant and 125I-labeled TSH, FSH, or hCG. The tetra-domain variant productively interacted with each of the three types of receptors (Fig. 4Go). The FSH (Fig. 4BGo) and hCG (Fig. 4CGo) binding affinities were about 2-fold less than those of the corresponding FSH and hCG heterodimers, respectively (Table 1Go). In the case of TSH (Fig. 4AGo), the binding affinity was 4.5-fold higher than that of the heterodimer (Table 1Go).



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  		Figure 4. Receptor binding of the tetra-domain single chain analog to hTSH (A), hFSH (B), and hCG (C) receptors. Stably transfected CHO cells expressing one of the above receptors were incubated with the corresponding iodinated hormone in the presence of varying concentrations of analog as determined by RIA. Displacement curves show the percentage of maximal binding of the isotope at each concentration of unlabeled sample. Data are the mean ± SEM of three experiments, each performed in duplicate. di, Heterodimer.

 

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  		Table 1. Biological activities of tetra-domain gonadotropin mutant

 
Signal transduction.
The ability of the tetra-domain analog to stimulate adenylate cyclase was also determined using CHO cells expressing TSH, FSH, or hCG/LH receptors. Consistent with the binding data, the analog stimulated cAMP production in the three different receptor binding assays (Fig. 5Go). The potency of adenylate cyclase activation by the analog in the FSH and hCG receptor systems was 2-fold higher and 2-fold lower, respectively, than those of the corresponding heterodimers (Fig. 5Go, B and C, and Table 1Go). In the case of TSH (Fig. 5AGo), the production of cAMP, although assayed only up to 10 mIU/ml analog, clearly paralleled that of the TSH dimer. We were unable to obtain full TSH dose-response curves because concentrating the conditioned medium further resulted in strong interference of the assay.



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  		Figure 5. Signal transduction of tetra-domain single chain glycoprotein analog. Adenylate cyclase activation was assayed using CHO cells expressing hTSH (A), hFSH (B), or hCG (C) receptors. cAMP production was determined by Adenyl Cyclase Activation FlashPlate assay. Data are the mean ± SEM of three experiments, each performed in duplicate. di, Heterodimer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies using single chain gonadotropin mutants suggested that gonadotropin hormone receptor interactions are conformationally flexible and do not strictly depend on the integrity of the {alpha}- and ß-subunit interactions in the heterodimer (9, 10). To further investigate the extent of this flexibility, we constructed a tetra-domain single chain glycoprotein analog consisting of three different ß-subunits covalently linked to a single {alpha}-subunit. Previous work with triple domain constructs composed of FSHß, CGß, and {alpha}-subunits indicated that optimal configuration for dual activity required the presence of CGß adjacent to the {alpha}-subunit (12). Based on these observations, we placed TSHß at the amino terminus of the tetra-domain protein. Each of the hormonal centers in this complex analog exhibited receptor binding affinity comparable to those of the corresponding heterodimers. It is unlikely that the folding of this chimera is capable of configuring the {alpha} to form heterodimer-like interactions with each ß-subunit simultaneously in the same molecule. Consistent with this idea is the observation that the major species detected in Western blot analysis corresponds to the total mass of the four subunits. However, because weak intermolecular associations may be disrupted under the nonreducing conditions used here (even though the heterodimers are stable under these conditions), it is possible that higher molecular mass species, e.g. aggregates, contribute to the functionality of the tetramer single chain.

The lack of intact ß-subunit heterodimeric interactions with the {alpha}-subunit is also supported by the observation that secretion of the analog was reduced significantly compared with that of the corresponding heterodimers. As previously shown, the formation of the correct heterodimeric alignments is critical for maximal secretion, as less than 20% of the uncombined monomeric LHß, FSHß, and TSHß is secreted (19, 23, 24, 25). The results using the tetra-domain model further support the hypothesis that heterodimer formation is critical for secretion and gonadotropin-specific carbohydrate processing, but not for receptor recognition and signal transduction. Thus, the multiple activities manifest by the tetra-domain analog are consistent with the hypothesis that initial receptor recognition depends on a linear array of key epitopes from both {alpha}- and ß-subunits rather than the generation of complex quaternary signals between one ß- and the {alpha}-subunit. However, we cannot exclude the possibility that some contact points can be coordinately generated between all of the domains. Furthermore, because the receptor binding and biopotencies are comparable, a similar array of key determinants is presumably formed by the {alpha}- and individual ß-subunits in both the tetra-domain analog and the corresponding heterodimers. Thus, the minimal amino acid residue contact points between the {alpha}- and each of the ß-subunits required for each of the glycoprotein hormone receptor binding specificities could be determined by a comparative epitope and peptide mapping analyses of the tetra-domain analog and the individual heterodimers.

Does generating a single protein bearing three functional activities reflect a vestigial evolutionary step in the glycoprotein hormone gene family? Based on sequence alignments, Li and Ford (26) performed a phylogenetic analysis of the glycoprotein hormone subunits, which supports the hypothesis that members of this gene family evolved from one ancestral gene. Biochemical support for this idea was obtained from crystallographic studies of hCG and FSH. These analyses revealed that the ß- and {alpha}-subunits have remarkably similar structures, e.g. they each have two hairpin loops on one side of the plane and a single larger loop on the opposite side. It was demonstrated that intersubunit homodimeric chimeras created by swapping portions of {alpha} and CGß, such as the {alpha}2 for the ß2 loop, can form functional analogs (27). Despite these observations, no peptide sequence directly related to the glycoprotein hormone ß-subunit ancestral sequence has been identified, and thus, the origin and order of emergence of the different lineages are unclear (28). The activities of the tetra-domain analog are consistent with the hypothesis that the glycoprotein hormone genes evolved from a fusion gene encoding a multifunctional hormone intermediate. Such a unified structure, characterized by similar gene expression profile and stoichiometric production of the component protein(s), might have been advantageous in primitive organisms containing less complex biosynthetic pathways and physiological systems. In this respect, it is noteworthy that an unusual coordinate expression of FSH and LH at the time of ovulation has been observed in a few salmonid species (29). It is also curious that the TSH receptor is expressed in the gonads of at least two other fish species (30, 31). Such observations imply physiological behaviors of an ancient structure in which the different glycoprotein hormones are closely related, possibly by forming a multidomain complex. Additional regulatory requirements, i.e. a more autonomous control of the diverse and complex endocrine systems (reproductive and thyroid) controlled by the glycoprotein hormones by more evolved organisms, would have been a driving force for the independent evolution of these family of genes.

It is plausible that the unique, unusually large extracellular component of the glycoprotein hormone receptors could be a vestigial structure. This predicts that the extracellular domain, which plays a major role in determining the ligand-binding ability of the receptor (32, 33, 34), accommodated a large multidomain single polypeptide. Recently, a number of orphan receptors (LGRs) homologous to the known mammalian gonadotropin and TSH receptors have been found in mammals (35, 36, 37, 38) and also in invertebrates (39, 40, 41, 42). Interestingly, the extracellular domains of all of the mammalian and one of the Drosophila orphan LGRs (DLGR2) are significantly longer than those of the known receptors and contain between 13 and 18 LRRs compared with the 9 existing in the known receptors. Moreover, in contrast with the restricted tissue expression of the known FSH, LH/hCG, and TSH receptors, the mammalian orphan receptors are expressed in several tissues (35, 37). Taken together, these data are consistent with the idea that these receptors could accommodate unusually large ligands with diverse physiological functions, such as the tetra-domain analog described here.

Finally, our data show that both gonadal- and thyroid-stimulating functions can be combined in a multifunctional single chain protein. Because altered thyroid function is generally associated with mammalian reproductive disorders, including impaired fertility (43, 44, 45, 46), this analog may have potential for studying the mechanisms of interaction between thyrotropic and gonadotropic activities in this pathology.


   Acknowledgments
 
We thank Drs. A. Jablonka-Shariff, M. Muyan, and D. Ben-Menahem for critical reading of this manuscript. We also thank Ms. D. Redmond and Ms. L. Lobos for their assistance in preparing this manuscript.


   Footnotes
 
This work was supported by Institutional Trainee Grant NINDS 5-T32-NS-07129.

Abbreviations: CG, Chorionic gonadotropin, CHO, Chinese hamster ovary; h, human.

Received March 25, 2002.

Accepted for publication June 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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