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J. Biol. Chem., Vol. 276, Issue 32, 29871-29879, August 10, 2001

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The Position of the  and  Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes^*

David Ben-Menahem, Albina Jablonka-Shariff^§, Ricia K. Hyde, Mary R. Pixley, Shivaji Srivastava, Peter Berger^¶, and Irving Boime

From the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^¶ Institute for Biomedical Aging Research, Austrian Academy of Science, A-6020 Innsbruck, Austria

Received for publication, May 22, 2001, and in revised form, June 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common  subunit and a hormone-specific  subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the  and  subunits are linked (NH₂-CG-; CG orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of  was fused to the CG amino terminus (NH₂--CG; CG orientation). The secretion rate of CG from transfected Chinese hamster ovary cells was less than that seen for CG. The CG tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in CG, it was co-transfected with a monomeric  or CG gene. In each case, CG/ and CG/CG complexes were formed indicating that CG dimer-specific epitopes were established. The CG/ complex bound to receptor indicating that the  domain in the CG tether was still functional. In contrast, no significant receptor binding of CG/CG was observed indicating a major perturbation in the  domain. These results suggest that although dimeric-like determinants are present in both CG/ and CG/CG complexes, the receptor binding determinants in the  domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The glycoprotein hormones lutropin (LH),¹ follitropin (FSH), thyrotropin, and choriogonadotropin (CG) are heterodimers, which consist of a common  subunit and a unique  subunit that confer the receptor specificity of the ligand. The subunits combine non-covalently early in the secretory pathway, and formation of the heterodimer is crucial for binding to the gonadal and thyroid receptors. Although it is well established that both subunits contain residues critical for bioactivity, recent evidence indicates that the quaternary interactions between the two subunits are essential for intracellular behavior but not for in vitro bioactivity (1-3). This conclusion was based on the single chain gonadotropin model where the amino end of the common  subunit was genetically fused to the carboxyl end of CG subunit (designated CG; Fig. 1) (4). This analog was secreted efficiently and was active in in vitro and in vivo bioassays. The CG orientation was chosen to keep the carboxyl end of the  subunit unmodified because this region is critical for high affinity receptor binding (5-11). However, it was unclear whether the orientation of the tethered subunit domains affects the heterodimeric alignment between the two subunits, resulting in functional determinants for secretion and bioactivity, or if the two domains can swivel with respect to each other producing heterodimer-like contacts regardless of their position. To examine this issue, we constructed a single chain hCG in which the carboxyl terminus of the  subunit was genetically fused to the amino end of CG subunit (CG; Fig. 1). This design reverses the relative position of the linked subunit domains compared with the first generation of single chain analog described above.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   The orientation of the  and CG subunit domains in single chains. The CG subunit (open box) is genetically fused to the  subunit (stippled box). CG indicates amino-terminal location of the  subunit, whereas CG denotes that the carboxyl end position is occupied by the  subunit. In CGT the carboxyl-terminal peptide (CTP) subunit is absent. In the case of cCGT, the CTP was deleted from the carboxyl end of the CG subunit and inserted between the carboxyl end of the  and amino terminus of the CG subunits.

Several parameters were examined for determining the function of the reverse-oriented constructs as follows: 1) the secretion rate and recovery from the media, 2) the ability of the tethered / domains to combine and form heterodimeric-specific epitopes with co-transfected monomeric subunits, and 3) formation of receptor binding determinants. Here we show that the relative position of the  and CG tethered domains in the single chain is critical for bioactivity but not for secretion. In addition, CG/ and CG/CG complexes were observed when the single chain was co-expressed with individual subunits. Dimer-specific conformational epitopes were detected in both complexes, but only CG/ was bioactive. This implies that formation of heterodimeric interactions does not ensure bioactivity. The results support the hypothesis that epitopes for gonadotropin assembly and bioactivity are distinct and can be uncoupled from each other.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Single Chains-- Engineering of the single chain CG was described previously (4). To construct CG, the CG gene was inserted in frame at the carboxyl end of the  subunit gene using overlapping polymerase chain reaction. The following primers were used in the construction: 1) 5'-CTACAGGAAAACCCATT-3'; 2) 5'-GCGGCTCCTTGGAAGATTTGTGATAAT-3'; 3) 5'-ATTATCACAAATCTTCCAAGGAGCCGC-3'; 4) 5'-TGAGTCGACATGATAATTCAGTGATTGAAT-3'. From the  minigene, product

View larger version (7K): [in this window] [in a new window]   Product A.

Primer 2 contains the first 4 codons of the CG subunit and the last 5 codons of the  subunit. In another reaction, primers 3 and 4 were used with CG as template. Primer 3 encodes the first 4 amino acids of the CG subunit (residues 1-4) and the last 5 amino acids of the  subunit (residues 88-92). Primer 4 contains some of the intron sequence between CG exons 2 and 3 and also includes a newly created SalI site. The fragment derived from this reaction is product

View larger version (7K): [in this window] [in a new window]   Product B.

In a second reaction, fragments A and B were used as overlapping templates with primers 1 and 4 to generate product

View larger version (6K): [in this window] [in a new window]   Product C.

To construct the cCGT single chain, a carboxyl-terminal peptide (CTP) was deleted from the CG subunit and inserted between the  and CGT domains. To facilitate construction of cCGT, the CTP-derived sequence linked to the carboxyl-terminal of the  subunit was truncated at amino acid residue 117 (instead of residue 114) and included 28 residues rather than 31 amino acids. This analog bearing the 28-amino acid linker exhibited the same intracellular and extracellular characteristics (data not shown) as reported previously for single chains containing a 31-amino acid linker (4).

DNA Transfection and Cell Culture-- All variants were inserted into the mammalian expression vector pM²HA (4) and were transfected into CHO cells by the calcium phosphate method. Stable clones were selected ~11 days after transfection by using the neomycin analog G418 (250 µg/ml). The clones were maintained in Ham's F-12 medium (supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 2 mM glutamine) containing 5% fetal bovine serum and G418 (125 µg/ml) at 37 °C in a humidified atmosphere of 5% CO₂, 95% air, as described previously (12).

Metabolic Labeling-- Pulse-chase experiments were performed as described previously (4, 13). Aliquots of cell lysate and medium were immunoprecipitated with polyclonal antisera directed against either the common  or the CG subunit, and the proteins were resolved on 12.5% SDS-polyacrylamide gels (4). The secretion half-time (t_1/2) and recovery efficiency of the single chains were estimated by determining the time (min) when half of the maximal secreted variant was detected in the medium; the recovery of the secreted variants is expressed as a fraction (%) of the total (intracellular + secreted) (4, 13).

Western Blot Analysis-- Media samples were resolved on 12.5% SDS-polyacrylamide gels without heating and under nonreduced conditions to prevent dissociation of the hCG heterodimer and were blotted onto nitrocellulose. The blots were probed with antisera or monoclonal antibodies (mAbs) as described in the figure legends. Purified hCG (CR 127) was kindly supplied by Dr. A. Parlow (National Hormone and Pituitary Program, Torrence, CA). Rabbit polyclonal antisera against the human  or CG subunits were raised in our laboratory. The hCG conformational sensitive mAbs that recognize primarily the heterodimer, but not the monomeric CG subunit, were provided by Dr. Steven Birken (Columbia University Medical School, New York). The blots were visualized with the Western Light Detection System (Tropix, Inc., Bedford, MA) following the manufacturer's protocol. Western blot analysis was performed 3-5 times using 3 independent collection media.

Radioreceptor Assay-- Conditioned media were concentrated using either a Centricon concentrator (Amicon Inc., Beverly, MA) or an ultra-free concentrator (Millipore Corp., Bedford, MA). Subsequently, the samples were washed in phosphate-buffered saline and quantitated using a double polyclonal based radioimmunoassay kit (Diagnostic Products Corp., Los Angeles, CA), containing antiserum that recognizes the CG subunit. Receptor binding and cAMP production were determined using a transfected CHO cell line, expressing the human LH/CG receptor (14). Total binding was 15%, and nonspecific binding (in the presence of 5 µg of hCG) was 1.5% of total counts. The cAMP accumulation was determined using the Adenyl Cyclase Activation Flash Plate kit (PerkinElmer Life Sciences) as per manufacturer's instructions. Briefly, 5 × 10⁴ stably transfected CHO cells were incubated for 2 h at room temperature with ligands; ¹²⁵I-cAMP was then added, and the cells were incubated for 17 h at room temperature. The flash plate was then read in a Packard Top Counter. Each experiment was performed at least 3 times, and the data are presented as the mean ± S.E. of 3 independent culture collections.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Biosynthesis of hCG Tether Variants-- To compare the secretion of CG and CG from stably transfected CHO cells, pulse-chase experiments were performed. Cells were labeled with ³⁵S-Promix, chased with unlabeled amino acids, and aliquots of lysate and medium immunoprecipitated with polyclonal  antiserum (Fig. 2). Although recovery of both variants from the media was similar, CG was secreted slower (t_1/2 = 155 min) than CG (t_1/2 = 90 min).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Secretion kinetics of CG and CG. CHO cells expressing the chimeras were pulsed-labeled and chased for the indicated times (hr). Cell lysates and media were immunoprecipitated with  subunit antiserum, and the reduced and heated proteins were resolved on 12.5% SDS-polyacrylamide gel electrophoresis. The recovery (%) and secretion half-times (t1/2) were calculated as described under "Experimental Procedures."

To examine if the two subunit domains in CG could form intrachain heterodimeric-like interactions, media samples were electrophoresed under nonreduced conditions without heating, and the blots were probed with polyclonal  antiserum and a panel of CG dimer-specific mAbs (Fig. 3). The  antiserum recognizes both native CG heterodimer and the non-combined  subunit (Fig. 3, panel A, lane 1) and the two single chain variants (lanes 2 and 3) that migrate with an apparent molecular mass of 50 kDa. Larger forms were also observed, and although their composition is unclear, aggregates have been observed in the synthesis of a variety of single chain mutants (1-3) and during hCG purification (15, 16). Aggregation is more extensive for CG, implying that its structure differs from that of CG. When the blots were probed with a CG dimer-specific mAb (panel B), the native dimer (lane 1) and CG were seen (lane 2). No signal corresponding to the 50 kDa of CG protein was observed (lane 3). Similar results were obtained with six additional dimer-specific mAbs (not shown). These data suggest that compared with CG, the two tethered domains in CG cannot form intrachain heterodimeric determinants recognizable by these conformational sensitive antibodies. If absence of CG immunoreactivity to mAbs is due to inhibition of the native dimeric interactions between the subunit domains, CG should be in a more open configuration than CG. To test this prediction, both variants were probed with mAb 68 that primarily reacts with the monomeric form of the CG subunit rather than the heterodimer (panel C). As expected, the non-combined CG subunit control secreted from transfected cells was immunoreactive (lane 4), and the heterodimer was not detected (lane 1). Whereas CG was poorly recognized by the CG-specific mAb (lane 2), CG was much more immunoreactive (lane 3). These data imply that the conformation of one or both subunit domains in CG is altered, which inhibits an intrachain heterodimeric-like interaction.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Western blot analyses of CG and CG. The samples were electrophoresed under non-reduced conditions and probed with the following:  antiserum (panel A), mAb (designated 40) specific for the hCG heterodimer (Di mAb; panel B), and an mAb (designated 68) specific for the monomeric CG subunit (panel C). Di refers to the native heterodimer. The migration of the free  and  subunits and the non-aggregated single chain (tether) are indicated by the arrows. This experiment was repeated 5 times with 3 independent collection media.

A major difference between the two single chain variants is the presence of the native CTP in the CG subunit (17) between the subunit domains in CG. As discussed previously, the CTP sequence lacks extensive secondary structure and serves as a natural linker (18). The CG analog was constructed without a linker to maintain consistency with the subunit structure of CG. To assess if the intracellular effects of CG are related to the absence of a linker sequence, a different chimera was constructed where the CTP was deleted from the carboxyl end of the CG subunit and inserted between the  and CG subunits (cCGT) (Fig. 1). When examined by pulse-chase kinetics (Fig. 4, panel A), the secretion rate was 3-fold greater (t_1/2 = 50 min), and the recovery was higher (80%) than CG (t_1/2 = 155 min; recovery = 70%). To examine the ability of cCGT to generate heterodimer-like determinants, blots containing this variant were screened with the mAbs described above (Fig. 4, panels B and C). In contrast to the results seen with CG, cCGT is recognized by dimer-specific mAb (panel B, lanes 3 and 4), although its immunoreactivity to CG or  monomer-specific mAbs is reduced considerably (panel C, lanes 1-4). Thus, the presence of the linker sequence in cCGT increases the heterodimeric alignment of the  and  domains.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Intracellular behavior of cCGT. The chimera cCGT was subjected to pulse-chase kinetics (panel A) as described in Fig. 2. Panels B and C are Western blots containing non-reduced proteins. Blot in panel B was probed with  antiserum (lanes 1 and 2), and mAb 40 against the hCG heterodimer (Di mAb; lanes 3 and 4). Blot in panel C was probed with mAbs specific for either the monomeric CG (mAb 68; lanes 1 and 2) or the monomeric  subunit (mAb 80; lanes 3 and 4). This experiment was repeated 3-5 times with 3 independent collection media.

Biological Activity of the Variants-- The receptor binding affinity of the variants was determined using CHO cells stably expressing the human LH/CG receptor. CG displayed high affinity binding, displacing the iodinated tracer similar to that of native hCG (Fig. 5A; Table I). In contrast, no binding was observed with CG. However, cCGT does bind to receptor, although its affinity is reduced about 100- and 70-fold when compared with CG and the heterodimer, respectively. Signal transduction of the chimeras was assessed by quantitating adenylate cyclase activation (Fig. 5B; Table I). Native hCG, CG, and cCGT caused a concentration-dependent increase in cAMP accumulation. Although, the stimulation of cCGT was 25-30-fold less than the controls, the induced levels of cAMP were greater than expected based on the receptor binding data (see "Discussion"). The results show that for the variant in the configuration (cCGT), the linker can rescue a significant portion of the receptor binding activity, but the lack of a free carboxyl end in the  subunit nevertheless reduces the binding affinity. These observations are similar to data seen with the heterodimer containing an  subunit with a CTP unit at its carboxyl end (10). In that study it was shown that the receptor binding affinity of this analog was reduced over 100-fold.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Bioactivity of hCG single chain chimeras. Varying concentrations of single chains were incubated for 16-18 h at room temperature with stably transfected CHO cells expressing the LH/CG receptor (panel A). Signal transduction of the ligands was determined under the same conditions by measuring cAMP (panel B). Data are mean ± S.E. of 4-6 experiments.

Intrachain Subunit Domains and Assembly Competence-- It is evident that compared with CG, CG is biologically inactive, indicating that reversing the positions of the subunits dramatically alters the overall structure of the single chain. A key question is whether or not the reduction in receptor binding is related to modifications of one or both of the / domains. To address this point, we co-transfected CG with either the  or CG subunit gene. We reasoned that if the intrachain domains were substantially misfolded, this would be reflected in their inability to form heterodimeric-like contacts with either of the co-transfected monomeric subunits. For example, co-transfecting the single chain gene with the monomeric CG subunit gene will assess the assembly determinants in the  domain of the single chain. Because CG and the free subunits do not bind to the LH/CG receptor (Fig. 5A) (11, 19-21) and are not recognized by dimer-specific mAbs, any observed biologic activity and formation of heterodimeric-like epitopes would be presumptive for the presence of a CG/monomeric subunit functional complex.

We first examined the integrity of the tethered CG domain by co-expressing the monomeric  subunit with CG (Fig. 6). The secreted proteins were analyzed in Western blots using polyclonal  antiserum (panel A) and a CG dimer-specific mAb 40 (panel B). The  antiserum recognized heterodimer and uncombined  subunit (panel A, lane 3) and CG (lane 2 and 3). In addition, a 70-kDa band corresponding to CG/ was detected in cells co-expressing both the single chain and the monomeric subunit (lane 3). The interaction between the incoming  subunit and the tethered CG subunit domain formed heterodimeric-like epitopes because the complex was recognized by a dimer-specific mAb (panel B, lane 3).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Interaction of the CG single chain with the monomeric  subunit. Cells expressing CG co-transfected with the monomeric  subunit (CG+) were probed with  antiserum (panel A) or an hCG dimer-specific mAb 40 (Di mAb; panel B). The electrophoretic migrations of CG/ and of the non-aggregated single chain CG (Di(CG)) are indicated by the arrows (seen in lane 3 of panels A and B). The presence of uncombined  subunit is seen in lane 3 of panel A. This experiment was repeated 3-5 times with 3 independent collection media.

The integrity of the intrachain  domain was examined with cells co-expressing CG and the monomeric CG subunit (Fig. 7). The dimer-specific mAb 53 recognizes CG (lane 1) but not CG (lane 2). However, the CG/CG complex is immunoreactive (lane 3, asterisk). Six other conformational sensitive mAbs also detected both the CG/CG and the CG/ complexes (data not shown). Thus, the intrachain  subunit can form a heterodimer-like interaction with a co-expressed monomeric CG subunit. Both CG/ and CG/CG complexes and the heterodimer dissociate after heating (3 min, 95 °C) in the absence of -mercaptoethanol (data not shown). This result indicates that the association of the monomeric subunit with the tethered domain is non-covalent, comparable to subunit interactions seen in the native heterodimer. These experiments show that both the  and the CG subunit domains in CG retain the ability to assemble with a monomeric subunit despite their inability to exhibit an intrachain heterodimeric configuration.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Complex formation between CG and co-transfected monomeric CG or CGT subunits. Media derived from cells co-expressing the CG single chain and monomeric CG subunit (CG+CG; lane 3) or monomeric CG devoid of the CTP (CG+CGT; lane 4) were blotted under non-reduced conditions and probed with mAb 53, which is specific for the hCG heterodimer. The asterisks in lanes 3 and 4 denote the CG/CG and CG/CGT complexes, respectively. This experiment was repeated 3-5 times with 3 independent collection media.

Determinants for Bioactivity in CG-- The CG/ complex binds to the human LH/CG receptor although with reduced affinity compared with the heterodimer (Fig. 8A; Table I). The observed dose-dependent binding could only result from synthesis of a CG/ complex since neither CG nor the monomeric  subunit alone exhibits significant receptor binding. In contrast, CG/CG did not displace the tracer (Fig. 8A) despite the formation of immunoreactive dimer-specific epitopes (see Fig. 7, lane 3). Because both CG and the monomeric  subunit contain the CTP at their carboxyl termini, which are glycosylated and sialylated, the absence of CG/CG receptor binding could be related to charge interference of two CTPs. To examine this point CG was co-transfected with a CG monomeric variant lacking the CTP (CGT). Although CG/CGT complex exhibited heterodimeric-like interactions (see Fig. 7, lane 4), it nevertheless did not bind to the receptor (Fig. 8A; Table I). The data imply that lack of bioactivity in the CG/CG complex is not due to the additional CTP. The cAMP levels induced by the complexes paralleled receptor binding (Fig. 8B; Table I). Thus, although the monomeric subunits in both CG/ and CG/CG form heterodimeric contacts, only the CG/ complex is bioactive. These results indicate that determinants for receptor binding/signal transduction are preserved in the intrachain  subunit of CG but not in the  domain.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Bioactivity of CG single chain complexed with either the monomeric  (CG/), CG (CG/CG), or CGT (CG/CGT) subunits. The experiment was performed as described in the legend to Fig. 5. Data are mean ± S.E. of 4-6 experiments.

The data described above demonstrated that the presence of the CTP as a linker in cCGT partially restores activity of the CG. We examined if this linker would reconstitute binding determinants in the tethered subunit domains and enhance the binding affinity when co-transfected with the monomeric , CG, or CGT subunits (Fig. 9). (As expected, cCGT/ was immunoreactive with dimer-specific mAbs (data not shown).) The affinity of the cCGT/CG complex was the same as cCGT (panel A). Although there was a significant increase in binding of cCGT/ compared with cCGT, it was similar to CG/. In addition, insertion of the linker sequence does not restore the bioactivity to the levels seen for the heterodimer or CG. These data imply that not only the lack of a free carboxyl end in the  subunit but also modifying the amino end in the CG subunit reduces receptor binding. Similar to the CG complexes, the analog-induced cAMP levels paralleled receptor binding (panel B). The results of the immuno- and bioactivities of the analogs are summarized in Table II.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Bioactivity of the cCGT variant synthesized in cells co-expressing either the monomeric  (cCGT/) or CG (cCGT/CG) subunits. Data are mean ± S.E. of 4-6 experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previously, we and others (4, 21-24) observed that single chain variants of the glycoprotein hormone family exhibit biological activity comparable to the corresponding heterodimers. In such chimeras, e.g. CG, the  subunit occupies the amino-terminal end of the molecule linked to the  subunit. This orientation was chosen to preserve the free carboxyl end in the  subunit due to its importance for maximal receptor binding efficiency (5-11).

To examine if the relative position of the two subunit domains is critical for generating a functional single chain gonadotropin, we engineered a tethered hCG in which the position of the two domains was reversed (CG) relative to the CG orientation. In both and configurations, the variants were secreted efficiently which shows that the relative position of the linked subunits was not critical for secretion. However, in contrast to CG, the intra-chain subunits in CG exhibited no detectable reactivity to hCG dimer-specific mAbs, although each domain was capable of forming a heterodimeric-like complex with its complementary monomeric subunit. This suggests that the structural constraints generated at the - linkage do not favor a heterodimeric alignment for the two domains. We also observed that CG did not bind to the receptor compared with CG, which implies that accessibility of receptor binding determinants is dependent on the position of the subunits in the single chain. A major structural difference between CG and CG is the presence of a CTP sequence between the two tethered domains in CG. As reported previously (18), we considered this sequence a natural linker since it is serine/proline-rich and thus lacks significant secondary structure. In the case of CG, the CTP is at the carboxyl end of the single chain, and thus it does not serve as a linker. When the CTP was deleted from the carboxyl end of the CG subunit and inserted between the  and  subunits, the resulting cCGT was rapidly and efficiently secreted and exhibited strong immunoreactivity to dimer- but not monomer-specific mAbs. The cCGT variant was bioactive, but the binding affinity was reduced compared with CG. These data suggest that in the absence of linker the alignment of the / domains is perturbed especially at the carboxyl end of the  subunit.

Our results are consistent with a recent report that a single chain hCG constructed in the configuration and a CTP between the subunits is biologically active (25). In that work the binding affinity of the chimera was reduced 25-30-fold, whereas signal transduction was decreased only 10-fold. Similarly, we find that whereas the binding affinity of cCGT is reduced 70-100-fold, adenylate cyclase activation is reduced about 20-fold. Thus, there is an apparent uncoupling of binding/signal transduction when the subunit domains in the single chain are in the / orientation. This hypothesis is supported by data that show if the hCG heterodimer contains an  subunit with a deleted 59-87 disulfide bond, this analog stimulated cAMP synthesis to a greater extent than expected based on its low binding affinity (12). Because cysteine residue 87 is at the carboxyl end of the subunit, the data imply that the conformational changes in this region expose determinants that are more efficient in stimulating downstream intracellular signaling reactions compared with the wild type heterodimer. That these -carboxyl-terminal mutants exhibited signal transduction is not in agreement with a previous study (26) that mutating or deleting carboxyl-terminal amino acids 88-92 of the  subunit abolished adenylate cyclase activation for the hCG heterodimer. One explanation to account for these variances is the use of a different bioassay for assessing signal transduction in that work (26). Recently, Gupta and Dighe (27) constructed a chimera composed of the  subunit attached to the amino terminus of the  subunit through a single glycine residue linker. The receptor binding of this analog was reduced only 10-fold. Thus, it is apparent from the above studies that the binding affinity of CG is reduced, but there are quantitative differences in the reported binding affinities. The disparity between our CG data and that of Gupta and Sighe (27) is due to the presence of the glycine linker in their construct, which may have increased the binding affinity. This is analogous to the results seen with cCGT.

Previously, we compared the secretion and biological activity of the CG lacking the CTP sequence and FSH single chains constructed devoid of a linker (28). We observed that for these variants the secretion rate was substantially reduced, but receptor binding/signal transduction was unaffected. Although absence of the linker sequence in the CG tether lowered the secretion rate, no biological activity was detected. One explanation that could account for these differences is the interference between the adjacent disulfide bonds at the junction between the subunits since the 26-110 disulfide bond in the CG subunit (conserved position 20-104 in the FSH subunit) is critical for secretion (13). Based on chemical modification and mutagenesis studies, it appears that the CG carboxyl and  subunit amino-terminal regions do not contain key receptor contact sites (10, 29). Presumably, this accounts for high affinity receptor binding of CG devoid of CTP and FSH without linker despite their altered secretion kinetics. In the CG single chain, however, there could be a significant perturbation of the last disulfide bridge, i.e. Cys-59-87 in the  subunit and the adjacent amino-terminally located disulfide bond in the  subunit. It is well documented that the 87-92-amino acid sequence in the  subunit is crucial for high affinity receptor binding by the heterodimers (5-11). That the Cys-59-87 bond does not impair secretion or assembly of the wild type heterodimer (12) might explain why the monomeric CG subunit can form a heterodimer-like complex with CG, yet CG/CG is biologically inactive. In the case of the CG/ complex, the carboxyl terminus of the bound monomeric  subunit is free, and thus CG/ binds to the LH/CG receptor and activates adenyl cyclase.

Despite the absence of the linker sequence in CG, it is evident that much of the native  subunit structure is intact since it has the capacity to form heterodimeric contacts with the co-transfected CG subunit (Table II). The absence of heterodimer-like interactions in CG per se implies that the two domains cannot swivel with respect to each other. The data support the hypothesis that flexibility at the carboxyl end of the  subunit is a critical component for receptor recognition by the single chain variants and by native heterodimers but not for the intracellular behavior of the subunit.

The data presented in Fig. 9 suggest that altering the amino end of the CG subunit affects the binding affinity. This is consistent with a previous study of Xia et al. (30), which examined the role of the lysine residue at position 2 in the CG subunit. They suggested that the conformation of the amino end might be associated with receptor binding. In another investigation (31), a synthetic peptide corresponding to residues 1-16 of the CG subunit was shown to inhibit binding of ¹²⁵I-hCG to porcine Leydig cells.

Our results also show that the presence of heterodimeric determinants does not necessarily result in a biologically active molecule (Table II), which is in agreement with the report of a naturally occurring mutation in the LH gene (32). A single amino acid substitution in the LH subunit (glutamine to arginine at residue 54) was associated with hypogonadism. It was shown that the mutated subunit formed a heterodimer that did not bind to the receptor in vitro. The uncoupling of a quaternary event and target binding are also supported by recent mutation studies of the Escherichia coli lactose repressor protein (LacI), where it was demonstrated that although assembly and folding of the two functional domains were not significantly affected, their affinity for the DNA target sequence was reduced dramatically (33). These results are consistent with our earlier studies that gonadotropin variants composed of more than 2 subunit domains bind and activate the receptor (21). That such variants with bulky constituents relative to the native heterodimer are bioactive implies flexibility in the ligand-receptor interaction. The data show that the relative position of the - and CG-tethered domains in single chain CG is critical for bioactivity but not for secretion. Moreover, since no amino acid mutations were created in the CG molecule, the data imply that receptor binding determinants in this variant are not accessible to the receptor due to an altered conformation or by interference created from the other intrachain  subunit. Thus, quaternary interactions are essential for the intracellular trafficking of the heterodimers but not for receptor recognition and signal activation. In the latter case, the role of the heterodimeric structure is to ensure that the appropriate epitopes in each subunit are brought in contact with the receptor triggering the biological response.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Vicenta Garcia-Campayo for advice during this study. We thank Dr. Raj Kumar for critical comments and Linda Creacy and Kristy Chamberlain for their assistance in preparing the manuscript.

	FOOTNOTES

^* This work was supported in part by a grant from the Organon Co. (Oss, The Netherlands).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Clinical Pharmacology, Ben-Gurion University, P. O. Box 653, Be'er Sheva, Israel 84105.

^§ Recipient of an NRSA award from the National Institutes of Health.

To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2556; Fax: 314-361-3560; E-mail: iboime@pcg.wustl.edu.

Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.M104687200

	ABBREVIATIONS

The abbreviations used are: LH, lutropin; FSH, follitropin; CG, human choriogonadotropin; CTP, carboxyl-terminal peptide of the CG subunit; CHO, Chinese hamster ovary; mAbs, monoclonal antibodies.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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