Derivation of Functional Antagonists Using N-Terminal Extracellular Domain of Gonadotropin and Thyrotropin Receptors

Yutaka Osuga, Masataka Kudo, Antti Kaipia, Brian Kobilka and Aaron J. W. Hsueh

Division of Reproductive Biology (Y.O., M.K., A.K., A.J.W.H.), Department of Gynecology and Obstetrics and Howard Hughes Medical Institute (B.K.), Stanford University Medical School, Stanford, California 94305


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Receptors for the glycoprotein hormones, LH/CG, FSH, and TSH, are a unique subclass of the seven-transmembrane, G protein-coupled proteins with a large N-terminal extracellular (ecto-) domain. Although ecto-domains of gonadotropin receptors confer ligand binding, expression of soluble binding proteins has been difficult. We fused the ecto-domains of LH or FSH receptors to the single-transmembrane domain of CD8 and found that hybrid proteins anchored on the cell surface retained high-affinity ligand binding. Inclusion of a junctional thrombin cleavage site in the hybrids allowed generation of soluble receptor fragments that interfered with gonadotropin binding to their receptors and blocked cAMP production stimulated by gonadotropins. Cross-linking analyses confirmed the formation of high molecular weight complexes between receptor ecto-domains and their specific ligands. A similar approach also generated a soluble TSH receptor fragment capable of blocking TSH-induced signal transduction. When administered to rats, the soluble FSH receptor fragment retarded testis growth and induced testis cell apoptosis. These findings demonstrate the feasibility of generating ligand-binding regions of glycoprotein hormone receptors to selectively neutralize actions of gonadotropins and TSH, thus allowing future design of novel contraceptives and management of different gonadal and thyroid dysfunction. The present study represents the first successful derivation of soluble, ligand-binding domains from glycoprotein hormone receptors as functional antagonists. Similar approaches could allow generation of ecto-domains of related receptors to neutralize actions of ligands or receptor antibodies and to facilitate structural-functional analysis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gonadotropins stimulate gonadal development following activation of specific target cell receptors. Gonadotropin receptors belong to the large family of G protein-coupled receptors characterized functionally by their interaction with guanine nucleotide-binding proteins and structurally by their seven hydrophobic, {alpha}-helical transmembrane domains. Members of this superfamily are functionally diverse and include receptors ranging from the cAMP receptor in slime mold to mammalian neurotransmitter and glycoprotein hormone receptors (1).

The glycoprotein hormone receptor subfamily, including receptors for LH/CG, FSH, TSH, diverges structurally from other G protein-coupled receptors in having a large N-terminal extracellular (ecto-) domain required for interaction with the large glycoprotein hormones (2, 3, 4). These ecto-domains of glycoprotein hormone receptors confer ligand specificity and are homologous to those of the leucine-rich repeat protein family, including the ribonuclease inhibitor (5). In addition to gonadotropin and TSH receptors, several other G protein-coupled receptors also have large ecto-domains. Included are those in the secretin/VIP/calcitonin/CRF/PTH receptor family (6, 7, 8), the metabotropic glutamate receptor (9), and parathyroid calcium ion-sensing receptors (10). For the PTH/PTH-related peptide receptor, the ecto-domain is also important for ligand binding based on studies using chimeric receptors (11).

Many receptors for hormones of the hematopoietin family have soluble isoforms derived either from alternative mRNA splicing (receptors for granulocyte colony-stimulating factor, interleukin-4, leukemia-inhibiting factor, etc.) or from proteolytic cleavage of the extracellular region of the receptor (receptors for interleukin-1, tumor necrosis factor, etc.) (reviewed in 12 . Furthermore, isolation of circulating GH-binding proteins indicated that they are truncated forms of tissue receptors with deletion of the transmembrane region (13). Among these truncated receptors, some of them act as functional antagonists. In contrast to soluble fragments derived from these receptors with a single transmembrane domain, generation of the ecto-domains of seven-transmembrane, G protein-coupled receptors as soluble binding proteins has been difficult. Although the large ecto-domains of gonadotropin receptors confer ligand binding, truncated gonadotropin receptors are trapped intracellularly (14, 15).

Using an anchored fusion receptor approach followed by proteolytic cleavage, we have generated ligand-binding regions of gonadotropin and TSH receptors as soluble binding proteins to neutralize the action of specific glycoprotein hormones. This approach could allow structural analysis of the ligand binding region of glycoprotein hormone receptors as well as future design of contraceptives and novel treatment protocols for gonadal and thyroid dysfunction associated with aberrant secretion of glycoprotein hormones.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Anchoring of Ligand Binding Regions of Gonadotropin Receptors and Derivation of Soluble Receptor Fragments
Using PCR-based mutagenesis, we fused the ecto-domains of human FSH and LH receptors to the single-transmembrane domain of CD8 through a thrombin cleavage site and named them as FtCD8 and LtCD8, respectively (Fig. 1AGo). Human 293 cells transfected with FtCD8 or LtCD8 showed a high-affinity cell surface binding to their specific ligand with expression levels comparable to those of the wild type receptors (Fig. 1BGo). For anchored FtCD8, the binding affinity to radiolabeled FSH was 3-fold higher than that of the wild type receptor, whereas anchored LtCD8 showed a binding affinity comparable to that of the wild type receptor (Kd values: FtCD8, 0.31 nM; wild type FSH receptor, 1.03 nM; LtCD8, 0.61 nM; wild type LH receptor, 0.28 nM). The expression levels of anchored chimeric receptors are about 50% of that found for the wild type receptors. These results demonstrated that the ecto-domains of gonadotropin receptors can be expressed on the cell surface independent of the seven-transmembrane region and still retain their ligand-binding ability.



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Figure 1. Ecto-domains of FSH and LH Receptors Anchored on the Plasma Membrane Retain High-Affinity Binding to Specific Ligands: Derivation of Hormone-Specific Binding Proteins following Thrombin Cleavage

A, Diagram of wild type and anchored receptors, FtCD8 and LtCD8. The ligand-binding ecto-domains of human gonadotropin receptors were fused to the single transmembrane region of CD8 through a thrombin cleavage site. B, Binding kinetics of wild type FSH receptor and FtCD8, as well as wild type LH receptor and LtCD8. After transfection with cDNAs encoding chimeric or wild type receptors, 293 cells were incubated with labeled ligands (FSH or hCG) to determine equilibrium binding constants using Scatchard plot analysis. C, Cross-linking of labeled ligands to solubilized ecto-domains of anchored receptors following thrombin cleavage. Cells expressing wild type or anchored receptors were treated with 10 IU/ml {alpha}-thrombin before incubation of conditioned media with labeled ligands followed by cross-linking with disuccinimidyl suberate. Thrombin treatment was omitted for control groups whereas some groups were incubated with excess nonlabeled ligands to demonstrate binding specificity. Migration patterns of molecular mass markers and labeled ligands are shown. D, Competition of complex formation between solubilized ecto-domains of gonadotropin receptors and their specific labeled ligands by increasing doses of nonlabeled gonadotropins. wt FSHR, Wild type FSH receptor; wt LHR: wild type LH receptor.

 
To solubilize ligand-binding regions of these anchored receptors, we treated cells expressing FtCD8 or LtCD8 with {alpha}-thrombin to release gonadotropin receptor ecto-domains. Conditioned medium was incubated with labeled ligands followed by cross-linking with disuccinimidyl suberate. As shown in Fig. 1CGo, labeled FSH migrated as a 45 kDa band after gel electrophoresis, whereas the conditioned media from FtCD8-expressing cells pretreated with thrombin showed a higher molecular mass band (90 kDa), indicating the formation of complexes between labeled FSH and the ecto-domain of FSH receptor. Complex formation was inhibited with the inclusion of excess nonlabeled FSH and was not found in cells expressing wild type receptors or without thrombin pretreatment. Likewise, thrombin pretreatment allowed the formation of a 105 kDa complex between labeled human (h) CG and the ecto-domain of LH receptor (Fig. 1CGo). The deduced large size of the ecto-domains of both gonadotropin receptors is consistent with our studies using N-glycanase treatment, suggesting that these molecules are fully glycosylated (data not shown).

To demonstrate that the solubilized receptor fragments still retained high-affinity binding to their specific ligand, competition analyses were performed. As shown in Fig. 1DGo, addition of increasing amounts of nonlabeled ligand competed for complex formation in a dose-dependent manner, and the kDa values for soluble FSH and LH receptor ecto-domains were estimated to be 200 pM and 2 nM, respectively. These results demonstrated that high-affinity ligand-binding domains of gonadotropin receptors could be generated using the anchored receptor approach. The soluble receptor fragments were named FBP (FSH-binding protein) and LBP (LH/hCG-binding protein), respectively.

Receptor Fragments Blocked Gonadotropin Receptor Ligand Signaling
We used solubilized ligand-binding regions of FSH and LH receptors to block gonadotropin actions in vitro. In a radioligand binding assay, 293 cells expressing wild type FSH receptors were incubated with labeled FSH in the presence or absence of increasing concentrations of FBP or LBP. As shown in Fig. 2AGo, addition of FBP prevented binding of labeled FSH to the wild type receptors in a dose-dependent manner, reaching a level comparable to nonspecific binding. In contrast, inclusion of LBP was ineffective. Furthermore, inclusion of the same preparations of receptor fragments in the hCG binding assay using wild type LH receptors led to a dose-dependent inhibition of binding by LBP but not by FBP (Fig. 2BGo). Thus, FBP and LBP specifically prevented the binding of their respective ligands. We further evaluated the ability of these binding proteins to interfere with signal transduction induced by gonadotropins. 293 cells expressing wild type FSH or LH receptors were incubated with increasing concentrations of FSH or hCG for 3 h at 37 C to stimulate cAMP production. As shown in Fig. 2CGo, addition of FBP completely blocked cAMP production induced by FSH in FSH receptor-expressing cells whereas LBP was ineffective. Conversely, addition of LBP inhibited cAMP stimulation by hCG in LH receptor-expressing cells whereas FBP was ineffective (Fig. 2DGo). Thus, these binding proteins are capable of selective blockage of gonadotropin-induced signal transduction in vitro.



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Figure 2. Competition for Receptor Binding and Neutralization of Gonadotropin Actions in Vitro by Soluble Ecto-domains of FSH and LH Receptors

A, Competition of labeled FSH binding to FSH receptors by soluble ecto-domain of FSH receptor (FBP) but not LH receptor (LBP). Cells expressing human FSH receptors were incubated with labeled FSH with or without increasing amounts of FBP or LBP. B, Competition of labeled hCG binding to LH receptors by LBP but not FBP. Cells expressing LH receptors were incubated with labeled hCG with or without LBP or FBP. C, Antagonism of FSH stimulation of cAMP production by FBP but not LBP. Cells expressing FSH receptors were incubated with increasing amounts of FSH in the presence or absence of FBP or LBP (107 cells equivalent per well) for 3 h at 37 C before cAMP determination by RIA. Concentration of binding proteins was determined based on their ability to inhibit the binding of respective labeled ligands to wild type receptors. D, Antagonism of hCG stimulation of cAMP production by LBP but not FBP. Cells expressing human LH receptors were incubated with hCG with or without LBP or FBP.

 
Solubilized TSH Receptor Fragment Interfered with TSH Induction of cAMP Production
We used the same anchored receptor approach to generate the soluble ecto-domain of TSH receptor and named it TBP. Although the exact binding affinity of TBP to TSH could not be adequately evaluated due to high nonspecific binding of the tracer (4), ligand cross-linking analysis indicated that soluble TBP released from cells transfected with a plasmid encoding anchored ecto-domain of human TSH receptor (TtCD8) formed high molecular mass (105 kDa) complexes with labeled TSH (Fig. 3AGo). Complex formation was blocked following the addition of excess unlabeled TSH. Furthermore, TSH stimulation of cAMP production by cells expressing wild type TSH receptors was substantially blocked by cotreatment with TBP but not with LBP coaddition (Fig. 3BGo), whereas the same TBP preparation was ineffective in interfering with the hCG stimulation of cAMP in LH receptor-expressing cells (Fig. 3CGo).



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Figure 3. Ecto-domain of Human TSH Receptor Is a Functional Antagonist

To generate TSH-binding proteins, 293 cells expressing TtCD8 were treated with thrombin as described for anchored gonadotropin receptors. A, Cross-linking analysis. Conditioned media from cells expressing TtCD8 were incubated with labeled bovine TSH (10,000 cpm/tube) followed by cross-linking with disuccinimidyl suberate. SDS-PAGE analysis indicated the presence of a high molecular mass complex of 97 kDa. In contrast, incubation with excess nonlabeled TSH (1 µg/tube) completely blocked complex formation. Migration patterns of molecular mass markers are shown. B, Neutralization of TSH stimulation of cAMP by TBP. Cells expressing wild type TSH receptors were incubated with increasing amounts of TSH in the presence or absence of TBP or LBP (107 cells equivalent per well) for 3 h at 37 C before determination of total cAMP production using RIA. C, Antagonism of hCG stimulation of cAMP production by LBP but not by TBP. Cells expressing human LH receptors were incubated with hCG with or without LBP or TBP.

 
FSH Receptor Fragment Suppressed Testis Function in Rats
To test whether FBP could be used as a functional antagonist in vivo, we scaled up the production of FBP using a baculovirus expression system. Experimental data indicated that FBP produced by FtCD8-expressing insect cells retained its ability to inhibit the binding of labeled FSH to wild type FSH receptors in vitro. We administered conditioned media from insect cells containing FBP into immature male rats. Treatment with FBP every 6 h for 2 days attenuated testis growth by 33% (untreated, 192 ± 28 mg; FBP-treated, 128 ± 30 mg; n = 14). Analysis of testis DNA fragmentation using a 3'-end-labeling method followed by gel electrophoresis further indicated major increases in testis cell apoptosis in the FBP-treated group, as evidenced by the appearance of internucleosomal DNA fragmentation (Fig. 4AGo). In rats treated with a GnRH antagonist to suppress both LH and FSH secretion, comparable decreases in testis weight (140 ± 18 mg) and increases in apoptotic DNA fragmentation were found. In contrast, no retardation of testis growth (201 ± 26 mg) or alteration of testis cell apoptosis was seen in rats treated with conditioned media of insect cells infected with the wild type baculovirus. In situ apoptosis analysis further indicated that DNA fragmentation was restricted to primary spermatocytes in rats treated with FBP or the GnRH antagonist but not found in untreated rats (Fig. 4BGo, arrowhead). These results suggested that FBP is capable of neutralizing the action of endogenous FSH, which is essential for testis germ cell survival (16, 17).



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Figure 4. Induction of Testis Cell Apoptosis following FBP Administration in Vivo

For expression of FtCD8 in insect cells, recombinant baculovirus was prepared using the Bac-to-Bac baculovirus expression system, and SF9 cells were infected with the recombinant viruses. After a 72-h culture at 27 C in media containing 5% serum and thrombin, cells were centrifuged and conditioned media concentrated before filtration with 0.2-µm filters. Immature male rats (21 days of age) were injected subcutaneously (every 6 h) with conditioned media of SF9 cells (108 cells equivalent per injection) containing soluble FBP. Two days later, testis weight was determined and testis cell apoptosis quantitated using a 3'end-labeling method. Some animals were treated with a potent GnRH antagonist (ANT; 50 µg/rat) to suppress pituitary release of LH and FSH whereas others were treated with conditioned media of SF9 cells infected with wild type baculovirus. In situ analysis of specific cell types undergoing apoptosis was performed using the Apotag kit. A, Pattern of apoptotic DNA fragmentation (two samples per group). B, In situ analysis of DNA fragmentation (bar length, 25 µm). Apoptotic primary spermatocytes are indicated by arrowheads.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present studies demonstrate that an anchored fusion receptor approach can be used to generate soluble ligand-binding regions of glycoprotein hormone receptors of the seven-transmembrane protein family. The soluble receptor fragments are capable of binding specific ligands and blocking signal transduction induced by FSH, LH/hCG, and TSH, thus serving as potent functional antagonists. Reproductive dysfunction due to aberrant gonadotropin secretion (such as polycystic ovarian syndrome and central precocious puberty) has been treated with potent GnRH analogs to suppress serum levels of both LH and FSH. The availability of FBP and LBP capable of specific neutralization of circulating gonadotropins could allow formulation of novel treatment protocols for patients with these diseases and could be used for treating patients with ovarian hyperstimulation syndrome by neutralizing the endogenous gonadotropins (18). In addition, FBP and LBP may be useful in the treatment of gonadal steroid-dependent prostate and breast cancers. Similar to the gonadotropin-binding proteins, the soluble TSH-binding protein can be used to block excess stimulation of thyroid function by endogenous TSH in patients with TSH-secreting tumors.

The present receptor antagonists bind specific hormones and are unlikely to show agonistic activity in direct contrast to hormone antagonists that interact with target organ receptors and usually exhibit agonistic activity at high concentrations. Furthermore, receptor antagonists do not have to be delivered to the target tissues and, due to their similarity to wild type receptors, probably have low antigenicity. Although immunization against hCG and other gonadotropins has been proposed as a potential immunocontraceptive device, the immune approach suffers from irreversibility and large individual variability. If the delivery system is optimized, the present gonadotropin-binding proteins could serve as reversible and reliable contraceptives in both sexes. Of interest, chimeric molecules containing the Fc region of IgG and the extracellular ligand-binding region of single-transmembrane receptors for tumor necrosis factor, hepatic growth factor, and related hormones, have been shown to be useful receptor antagonists (19, 20, 21). Indeed, splicing variants of GH receptors containing only the extracellular region are natural binding proteins found in circulation (13).

Stimulatory and inhibitory antibodies against the ecto-domain of TSH receptor cause thyroid dysfunction (22, 23), whereas premature ovarian failure has also been associated with circulating antibodies against FSH receptors (24, 25). The present anchored receptor approach might allow future generation of receptor fragments for the treatment of different autoimmune disorders. Patients with Graves’ disease could be treated with mutant forms of TBP defective in TSH binding but still capable of neutralizing the stimulatory effect of the stimulating IgG because the binding domains for TSH and Graves’ IgG on the ecto-domain of the TSH receptor are not identical (26).

Many other seven-transmembrane, G protein-coupled receptors also have large ecto-domains, including receptors for the PTH family (6, 7, 8), metabotropic glutamate receptor (9), and parathyroid calcium ion-sensing receptor (10). The ecto-domains of PTH (11) and CRF receptors are important for ligand binding, but the exact role of this region for other receptors is unclear. It is likely that the ecto-domains of many of these receptors can be generated using the present approach to neutralize the action of endogenous ligands and allow modulation of vascular (receptors for calcitonin gene-related peptide and vasoactive intestinal peptide), metabolic (receptors for glucagon and glucagon-like peptide 1), and other conditions. Recent data suggested that the entry of human immunodeficiency virus into target cells (27, 28) requires several seven-transmembrane chemokine receptors that, similar to the related C5a receptor, also contain ecto-domains believed to be involved in ligand recognition (29). Thus, the present approach could allow characterization of the ecto-domains for diverse G protein-coupled receptors and potential derivation of soluble binding proteins to neutralize the actions of specific ligands, antibodies, or viral proteins.

The present study demonstrates successful derivation of soluble, ligand-binding domains of receptors in the large G protein-coupled receptor family to serve as functional antagonists. Expression of the ecto-domains of glycoprotein hormone receptors and related proteins would also allow analysis of interactions between the ligand-binding region and the transmembrane, signal transduction domain (30). In addition, the generation of soluble receptor fragments is useful for elucidating the three-dimensional structure of ligand-binding regions for gonadotropin, TSH, and other seven-transmembrane receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of Chimeric Receptors
Chimeric receptor cDNAs were generated using overlapping PCR and confirmed by dideoxy sequencing. For chimeric receptor FtCD8, the ecto-domain of human FSH receptor (amino acids 1 to 358; 31 was fused to the transmembrane and cytoplasmic region of CD8 (amino acid 162 to C terminus; 32 through a stretch of the thrombin receptor sequence using the plasmid SK-ATE-CD8 containing the thrombin cleavage site (amino acids 36 to 66 of thrombin receptor) (33, 34). The resulting junction between FSH receptor and thrombin receptor encoded the sequence (. ...NPCED/ATLDP. ...), whereas the junction between thrombin receptor and CD8 encoded the sequence (. ...NESGL/IYIWA. ...). For LtCD8, the ecto-domain of human LH receptor (amino acids 1 to 355; 35 was also fused to CD8 through a stretch of the thrombin receptor sequence. The resulting junction between LH receptor and thrombin receptor encoded (. ...NPCED/ATLDP. ...), whereas the junction between thrombin receptor and CD8 remained the same as that in FtCD8. For TtCD8, the ecto-domain of human TSH receptor (amino acids 1 to 410; 4 was fused to CD8 through a stretch of the thrombin receptor sequence and the resulting junction encoded (. ...NPCED/ATLDP. ...). For expression of wild type and chimeric receptors in human embryonic kidney 293 cells, receptor cDNAs were subcloned into pcDNA3 (Invitrogen, San Diego, CA).

Cell Culture and Transient Expression of Wild Type and Chimeric Receptors
Cell culture and transient expression were performed as previously described (36). Briefly, 293 cells were cultured in the DMEM/F12 medium supplemented with 10% serum and antibiotics. After replacement of the medium by DMEM supplemented with 10% serum and antibiotics, 293 cells were transfected using 10 µg expression vector containing wild type and chimeric receptor cDNAs by the calcium phosphate precipitation method (37).

RRA and Cross-Linking Analysis
Human CG (CR129) and human (h) FSH (NIDDK-I-1) were obtained from the National Hormone and Pituitary Agency (Baltimore, MD) and iodinated using the lactoperoxidase method as previously described (36). The specific activities of the tracers were approximately 100,000 cpm/ng for [125I]-FSH and approximately 50,000 cpm/ng for [125I]hCG. Cells transiently transfected with wild type receptors were incubated with specific radioligand in 300 µl PBS containing 0.1% BSA at 23 C for 18 h. Nonspecific binding was determined by adding excess unlabeled hormones (100 IU hCG/tube or 4 IU FSH/tube). Following estimation of optimal binding conditions, RRAs for cells transfected with FtCD8 and LtCD8 were performed at 23 C for 5 h and 2 h, respectively. After incubation, cells were washed twice followed by centrifugation and determination of radioactivity using a {gamma}-counter. To determine equilibrium binding constants (Kd), cells were incubated with labeled ligands (FSH or hCG) with increasing amounts of unlabeled hCG or FSH. Data from displacement analysis were analyzed using Scatchard plots (38).

To solubilize the ecto-domain of gonadotropin receptors, 293 cells expressing anchored chimeric receptors were treated with {alpha}-thrombin (10 IU/ml for 1 h) before incubation of the conditioned media (10 x concentrated using Centricon 30, Amicon, Bedford, MA) with saturating amounts of radiolabeled ligands. Media from FtCD8-transfected cells were incubated with labeled FSH (150,000 cpm/tube) at 23 C for 5 h whereas media from LtCD8-transfected cells were incubated with labeled hCG (100,000 cpm/tube) at 4 C for 16–18 h. For studies on solubilized TSH receptor fragment, media from TtCD8-transfected cells were incubated with 125I-labeled bovine TSH (Kronus, San Clemente, CA) for 5 h at 23 C. Complexes formed between labeled hormones and solubilized receptor fragments were cross-linked using disuccinimidyl suberate (2 mM) for 1 h, and the reaction was terminated by the addition of 3.6 mM Tris-HCl, pH 7.4. After the addition of Laemmeli buffer without reducing reagents, cross-linked complexes were visualized following fractionation using PAGE (10%) and autoradiography. In some experiments, increasing amounts of nonlabeled hCG or FSH were included, and radioactivities corresponding to high molecular mass complexes were determined to derive a competition curve for the estimation of Kd values (38).

Cyclic AMP Production
To analyze the ability of solubilized gonadotropin receptor fragments to interfere with signal transduction induced by FSH or hCG, 293 cells expressing wild type FSH or LH receptors were incubated in DMEM/F12-0.1% BSA with increasing amounts of specific ligands in the presence or absence of conditioned media from cells transfected with FtCD8 or LtCD8 (107 cells equivalent per well). After treatment for 3 h at 37 C, total cAMP production was determined by RIA (39). The concentration of solubilized receptor fragments from each preparation was estimated based on its ability to inhibit the binding of respective labeled ligands to wild type receptors. Similar approaches were used to evaluate the ability of solubilized TSH receptor fragments to interfere with cAMP production induced by human recombinant TSH (Genzyme, Cambridge, MA) in 293 cells transiently expressing human TSH receptors.

In Vivo Studies
To generate large amounts of FSH receptor fragments, we expressed FtCD8 in insect cells. FtCD8 cDNA was subcloned into pFast Bac vector, and recombinant baculovirus was prepared using the Bac-to-Bac baculovirus expression system (GIBCO-BRL, Gaithersburg, MD) according to the protocol of the manufacturer. SF9 cells (3 x 106/ml) in the SF900II medium containing 5% serum were infected with the recombinant viruses. After a 72-h culture at 27 C in media containing 5% serum and thrombin (10 IU/ml), cell debris was centrifuged and removed. The conditioned media were concentrated 30 times using DIAFLO ultrafiltration membrane XM50 (Amicon) and filtered with 0.2 µm filters (UNIFLO, Schleicher & Schuell, Keene, NH). Immature Sprague-Dawley male rats (21 days of age) were injected subcutaneously (every 6 h) with conditioned media of SF9 cells (108 cells equivalent per injection) containing solubilized FSH receptor fragments. Based on the ability to displace binding of labeled FSH to wild type FSH receptors, the amount of FBP in the conditioned media was equivalent to 2 µg FSH/injection. Two days later, testis weight was determined and testis cell apoptosis quantitated using a 3'-end-labeling method (16). Some animals were treated with a potent GnRH antagonist (Org30850, 50 µg/rat) to suppress pituitary release of both LH and FSH (40) whereas others were treated with conditioned media of SF9 cells infected with the wild type baculovirus. SF9 cells infected with the wild type baculovirus were treated with thrombin followed by ultrafiltration using identical conditions as for the preparation of FBP. In situ analysis of specific cell types undergoing apoptosis was performed using Apotag kit (Oncor, Gaithersburg, MD) as previously described (17). Animal care was in accordance with institutional guidelines.


    ACKNOWLEDGMENTS
 
We thank Dr. Shaun Coughlin (UC San Francisco) for providing the thrombin receptor-CD8 construct, Dr. G. Vassart (Belgium) for TSH receptor cDNA, Dr. H. Kloosterboer for the GnRH antagonist, and Emerald Perlas for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, 300 Pasteur Drive, Stanford, California 94305-5317.

This study was supported by NIH Grant HD-23273.

Received for publication May 19, 1997. Revision received July 8, 1997. Accepted for publication July 11, 1997.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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