0013-7227/90/1261-0376102.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 1 Printed in U.S.A.

The Biological Role of the Carboxyl-Terminal Extension of Human Chorionic Gonadotroin /?-Subunit M. M. MATZUK*, A. J. W. HSUEH, P. LAPOLT, A. TSAFRIRI, J. L. KEENEt, AND I. BOIME Departments of Pharmacology and Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110; and the Department of Reproductive Medicine, University of California-San Diego (A.J. W.H., P.L., A.T.), La Jolla, California 92093

ABSTRACT. hCG is a member of a family of glycoprotein hormones which share a common a-subunit, but differ in their hormone-specific /3-subunits. The CG |8-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the 0linked oligosaccharides and the carboxyl-terminal extension of hCG/3 on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCGa and hCG/3 expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the /3-subunit O-linked oligosaccharide units. In addition, a mutant hCG/3 gene (CG/JAT) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCGa gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino

H

CG IS a member of the glycoprotein hormone family which also includes LH, FSH, and TSH. These heterodimeric hormones share a common a subunit, but differ in their /3-subunits, which confer biologic specificity (1). It is likely that the four different /3-subunits evolved from the same ancestral gene, since there is a high degree of amino acid homology between these proteins (1). This is readily apparent for the jS-subunits of hCG and LH which share 85% sequence identity in their first 114 amino acids (2, 3) and may be responsible for the similar receptor binding specificity of their dimers. However, hCGjS is distinct among the /3-subunits, since it contains a carboxyl-terminal extension with four serine O-linked oligosaccharides (4-6). This extension Received August 21,1989. Address requests for reprints to: Dr. Irving Boime, Department of Pharmacology, Washington University School of Medicine, Box 8103, 660 South Euclid Avenue, St. Louis, Missouri 63110. * Participant in the Medical Scientist Training Program supported by National Institute of General Medical Sciences Grant GM-07200. Present address: Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104 t Present address: MonSanto Company, 700 Chesterfield Village Parkway, St. Louis, Missouri 63198

acids 115-145 of hCG/3 (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG/S and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses. (Endocrinology 126: 376-383,1990)

probably resulted from a read-through event in the ancestral LH/hCG/3 gene, presumably due to a single base deletion at codon 114 (3). The result of this deletion is that hCG/3 contains a 31-amino acid hydrophilic terminus, whereas LH/3 has a 7-amino acid hydrophobic stretch (3). This hydrophilic terminus may explain the observed longer plasma half-life (7-10) and increased biological potency of hCG vs. LH (11-13), which could be due to the 4 O-linked oligosaccharides, the carboxylterminal extension, or both (11, 14). Since LH and hCG bind to the same gonadal LH/hCG receptors, it is presumed that the carboxyl-terminus of hCG/3 has little role in the interaction of hCG with its receptor. Several studies using antibodies to hCG have shown that the carboxyl-terminus is exposed when bound to the receptor, and antibodies to this region fail to block hCG receptor binding and biological responses (15-19). We chose to examine the role of the hCG/3 carboxylterminal extension and the O-linked oligosaccharides by using site-directed mutagenesis and an O-glycosylation mutant cell line. This approach has the advantage that the hCG produced lacks only the O-linked oligosaccharides or the carboxyl-terminal extension. Alternative 376

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377

BIOLOGICAL ROLE OF hCG/3 CARBOXYL-TERMINAL EXTENSION methods, such as chemical or enzymatic treatments, to analyze this region have several disadvantages: 1) removal of the O-linked oligosaccharides cannot be accomplished without also altering the hCG AT-linked oligosaccharide units; 2) proteases or chemical treatments may be used to remove the peptide extension, but could produce a heterogeneous terminus, making it difficult to analyze the role of the hCG/3 carboxyl-terminal extension; and 3) chemical or enzymatic attempts to remove the hCG|3 O-linked oligosaccharides or carboxyl-terminus may affect other portions of the /3-subunit. Using gene transfer experiments in which the hCGa and hCG/3 genes were inserted into an O-linked glycosylation mutant, IdlD (20), we showed that these oligosaccharides are not involved in secretion or assembly of hCG in Chinese hamster ovary (CHO) cells (21). Here, we constructed a mutant hCG/8 gene in which a premature termination codon is substituted for amino acid 115. Cotransfection of this mutant gene (CG/3AT) with the hCGa gene enabled us to generate a mutant hCG derivative (truncated hCG) which lacked the unique hCG/3 carboxyl-terminal amino acids 115-145. The O-linked oligosaccharide-deficient hCG secreted from the IdlD cells and the truncated hCG mutant allowed us to directly address whether the O-linked oligosaccharides or the carboxyl-terminal extension play a role in vitro and in vivo.

Materials and Methods Materials

Enzymes used in the preparation of DNA vectors and constructs were purchased from Bethesda Research Laboratories (Gaithersburg, MD) and New England Biolabs (Beverly, MA). All other reagents for the recombinant DNA studies are as described by Matzuk and Boime (22). Mutagenesis, vector constructions, and DNA transfection The HmdIII-BamHI fragment [3250 basepairs (bp)], containing exons II and III of the CG/3 gene, was inserted into M13mpl9, and the single stranded viral recombinant DNA was isolated for mutagenesis. A 27-nucleotide oligomer, synthesized by the Washington University Sequencing Facility, was used to generate the mutant CG/3 AT with a change at codon 115 (Fig. 1). Mutagenesis and hybridization reactions were performed as described previously (22); the tetramethylammonium chloride wash temperature for the 27-mer was 68-69 C. The mutant gene was sequenced and rechecked by restriction enzyme analysis. The /fmdlll-BamHI fragment containing the mutations was subcloned into a vector containing exon I to reconstitute the entire hCG/3 gene with three exons (21). This subcloned gene was rechecked by hybridization to ensure that the mutation was still present and that there was no wild-type contamination. The Bglll-BamHl fragment containing the 3600-bp CG/3AT gene was inserted into the eukaryotic expres-

•Pro113 CCC

Argm CGC

phe115. TTC

•Pro 113

Arg 114

Ter

CCC

CGC

TAA

FlG. 1. Carboxyl-terminal extensions of wild-type and mutant CG/3 subunits. Amino acids 113-115 of wild-type CG/3 are shown along with the DNA triplets encoding these amino acids. The final two bases of the Phe codon (underlined) were converted to AA by site-directed mutagenesis (see Materials and Methods) to generate the termination codon TAA. The CG/?AT derivative terminates at Arg114 at the position where the LH/3 and CG/3 sequences diverge.

sion vector pM2 (21) down-stream of a strong promoter. This vector is called pM2CG/3AT. Construction of the wild-type a and /3 expression vectors, pM2CGa and pM2CG/3, respectively, has been described (21). The DNA sequence and the predicted protein sequence of wild-type CG/3 and CG/3AT are shown in Fig. 1. Clones of wild-type CHO cells or the O-glycosylation mutant CHO cells (IdlD) that secret hCG have been described (21). pM2CGi8AT and pM2CG« were cotransfected into CHO cells and selected as described previously (21, 22). Selection of a cell line expressing both a and CG/3AT (truncated hCG) was detected by immunoprecipitation of metabolically labeled cells as described previously (21). Cell culture Clones were maintained in medium I [Ham's F-12 medium supplemented with penicillin (100 U/ml), streptomycin (100 Hg/w\), and glutamine (2 mM)] containing 0.25 mg/ml of the neomycin analog G418 (Gibco, Grand Island, NY), and 5% (vol/vol) fetal calf serum (Sigma, St. Louis, MO) in a humidified 5% CO2 incubator. Cells secreting wild-type hCG and truncated hCG were plated and grown to confluency in medium I, except that a-Minimum Essential Medium was substituted for Ham's F-12, fetal calf serum was increased to 10% (vol/ vol), and G418 was deleted. After 48 h of incubation, the medium was centrifuged to remove cell debris and concentrated several-fold using an Amicon concentrator (CentripreplO, Amicon, Lexington, MA). The IdlD clone secreting hCG dimer was grown to confluency and preincubated in medium I supplemented with 1% dialyzed calf serum (Gibco) containing both galactose (10 /xM) and iV-acetylgalactosamine (100 /xM) or only galactose. After 12 h, the medium was removed, and fresh medium was added. After 48 h, medium was removed and centrifuged to remove cell debris. Cells incubated with both galactose and iV-acetylgalactosamine have the normal N- and O-linked oligosaccharide structures (hCG O-linked), whereas cells incubated with only galactose have no O-linked oligosaccharides (hCG O-linked), as shown previously (20, 21). Wildtype hCG, truncated hCG, and hCG with or without O-linked oligosaccharides from IdlD cells were quantitated by two different RIAs using the dimer-specific monoclonal antibodies B107

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BIOLOGICAL ROLE OF hCG/3 CARBOXYL-TERMINAL EXTENSION

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Endo • 1990 Vol 126 • No 1

and B109 (18). CR119 hCG was used as the standard, and the RIA was repeated three times. As further proof that absence of the carboxyl-terminal extension does not greatly influence the quantitation of truncated hCG compared to wild-type hCG, Western blots were used in these experiments. Samples were subjected to polyacrylamide gel electrophoresis on native gels, and radiolabeled monoclonal antibody B105, which binds LH/3 (no COOH-terminal extension) and CG/3 at comparable affinities, was used to quantitate the amount of dimer present in the samples. Comparison of the Western blots and the RIAs are discussed in Results.

Kontes ground-glass tissue grinder (Kontes Co., Vineland, NJ) in ice-cold Dulbecco's PBS containing 0.1% BSA. The homogenate was centrifuged at 20,000 X g for 30 min at 4 C, and the crude membrane pellet was resuspended in the buffer. Aliquots of this crude membrane preparation were incubated at room temperature for 18-20 h with [125I]hCG (20,000 cpm) with or without unlabeled hCG (100 IU Pregnyl; Organon Pharmaceuticals, West Orange, NJ) to determine nonspecific and total binding, respectively. Various concentrations of wild-type and truncated hCG were added, and their effects on the binding of [125I]hCG to rat testicular membrane were determined.

In vitro hormone assays

Animals

The receptor-binding activity of recombinant wild-type and hCG derivatives was determined with a radioligand receptor assay using intact MA-10 cells (23) and [125I]hCG (CR119). On day 0, 400,000 MA-10 cells were plated into 6 X 35-mm clusters in Waymouth MB752/1 (pH 7.4) supplemented with 15% horse serum (Gibco) and gentamicin (40 jig/ml). Cells were fed on day 2, and on day 3, cells were placed at 4 C for at least 1 h, washed twice with 2 ml 4 C assay medium [Waymouth MB752/ 1 containing 1 mg/ml BSA (Sigma) and gentamicin (40 ng/ ml)], and incubated 15 h or more in 1 ml assay medium containing [125I]hCG (100,000 cpm/ml) and varying concentrations of wild-type hCG and hCG derivatives. Cells were then washed twice with cold assay medium, and the [125I]hCG bound to the cells was determined. All binding data are reported as a percentage of the control bound (5-10%; 5,000-10,000 cpm); nonspecific binding was determined in the presence of 1 iig/m\ unlabeled CR119 and accounted for about 4% of the total binding (~200 cpm). Assays of the in vitro bioactivity of wild-type hCG and hCG derivatives were also determined using MA-10 cells (see also Refs. 24-26). For steroidogenesis assays, MA-10 cells were prepared as described above, except that on day 3, cells were washed twice with assay medium at 37 C and incubated in this medium containing varying concentrations of wild-type hCG and derivatives. After 4 h, progesterone in the medium produced by the MA-10 cells was determined by RIA using a kit supplied by ICN Biomedical, Diagnostics Division (Carson, CA). For the intracellular cAMP assays, cells were prepared in 35-mm dishes and incubated with wild-type hCG and derivatives similar to the progesterone assays, except that 0.5 mM isobutylmethylxanthine was added. After 45 min, cells were placed on ice and washed twice with cold HEPES-buffered salt solution containing 1 mg/ml BSA, 40 Mg/ml gentamicin, and 1 mM theophylline, and then 0.5 ml cold 0.5 N HC104 containing 1 mM theophylline was added before placing the dish at —80 C. Cells were thawed at 22 C and scraped, and the plates were washed twice with 0.2 ml HC104 solution to retrieve remaining cells. Cell suspensions were placed at 70 C for 15 min, centrifuged to remove cell debris, and neutralized by the addition of 0.34 ml 2 M KHCO3. After an additional centrifugation, the supernatants were assayed for cAMP using an RIA kit supplied by DuPont-New England Nuclear (Wilmington, DE). For the rat LH/hCG receptor binding assays, hCG (CR121) was iodinated using the Iodogen method (27). Decapsulated testes from adult Sprague-Dawley rats were homogenized in a

Immature female rats were hypophysectomized and implanted with a silastic capsule containing about 10 mg diethylstilbesterol on day 22 by a commerical breeder (Johnson Laboratories, Chicago, IL). The rats were shipped to our laboratory at 25 days of age and housed in standard vivarium facilities, with laboratory chow and 0.9% saline available ad libitum. Room temperature (24 C) and lighting (lights on, 0600-1800 h daily) were controlled throughout the studies. At 26 days of age, the rats received a priming dose of 15 IU PMSG (Calbiochem, La Jolla, CA), sc, at 0900 h. In vivo hormone assays Fifty-two hours after PMSG priming, the female rats received either intrabursal or iv injections. For experiments involving intrabursal injections, rats were anesthetized with halothane, and the left ovary was exposed via an incision through the left flank. The bursa surrounding the ovary was injected via a 30-gauge needle through the ovarian fat pad with varying amounts of concentrated conditioned medium containing either wild-type or truncated hCG or concentrated control medium. The ovary was returned to the ip cavity, and the incision was closed with wound clips. The following morning (20-22 h later), ovulation (number of oocytes) was checked by removing and opening both right and left oviducts to examine their contents for the number of ovulated oocytes. For experiments involving iv injections, varying amounts of wild-type or truncated hCG or control medium were injected into the exposed jugular vein of anesthetized rats, and ovulation was determined as described above. Ovine LH, injected ip or iv, was used as a control for these experiments.

Results Generation of wild-type and mutant hCG To examine if the O-linked oligosaccharides attached to the hCG/3 carboxyl-terminal extension interacted with the LH/hCG receptor and were involved in signal transduction, we used hCG secreted from IdlD cells (20) which we had previously transfected with hCGa and hCG/3 vectors and were shown to produce intact dimer hCG (21). The IdlD cells have a reversible defect in O-linked glycosylation, and by eliminating JV-acetylgalactosamine from the culture medium, hCG derivatives can be pro-

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BIOLOGICAL ROLE OF hCG/3 CARBOXYL-TERMINAL EXTENSION duced which lack the four carboxyl-terminal O-linked oligosaccharides (see Refs. 20 and 21). hCG devoid of 0linked oligosaccharides was compared to native hCG secreted from wild-type CHO cells for the ability to bind to the LH/hCG receptor and stimulate adenylate cyclase and steroidogenesis. Since both LH and hCG bind to the same receptor and yet have different carboxyl-terminal extensions, we also tested whether the entire hCG/3 carboxyl-terminus was involved in receptor interactions. We generated a mutant hCG/3 gene (CG/3AT) which contained a 2-bp change at codon 115 and, therefore, contained a termination codon (TAA) instead of the TTC triplet that normally codes for phenylalanine (Fig. 1). A vector containing this CG/3AT gene was cotransfected into CHO cells with a hCGa vector, and a stable cell line that produced large quantities of hCG lacking the carboxyl-terminus of hCGjS (truncated hCG) was selected. Experiments using cells secreting CG/3AT only or both a and CG/3AT show that the secretion and assembly of this carboxyl-terminal mutant were not greatly perturbed compared to those of wild-type hCG/3 (28, 29), similar to studies analyzing the hCG/3 O-linked oligosaccharides using the transfected O-glycosylation mutant cells (21). In vitro biological activity To analyze the receptor binding of mutants lacking the O-linked oligosaccharides or carboxyl-terminus of CG/3, we used a mouse Leydig tumor cell line, MA-10 (23). Wild-type (produced from both wild-type CHO cells and idlD cells), O-linked oligosaccharide-deflcient, and truncated hCG forms were quantitated using two different dimer-specific monoclonal antibodies. Based on their antigenicity, varying concentrations of hCG lacking the O-linked oligosaccharides and truncated hCG were compared to wild-type hCG produced from CHO cells for their ability to displace [125I]hCG from the LH/hCG receptor on MA-10 cells (Fig. 2). Comparison of the 0linked oligosaccharide-deficient hCG to hCG secreted from CHO cells (Fig. 2) or IdlD cells reveals that absence of the O-linked oligosaccharides has little effect on the ability of hCG to bind to the receptor. Similarly, there is less than a 50% difference in the affinity of the truncated hCG for the LH/hCG receptor compared to either Olinked oligosaccharide-deficient hCG or wild-type hCG (Fig. 2). However, since the amounts of truncated and wild-type hCG were quantitated using RIAs, these results could be skewed if the immunoreactivity was altered by deletion of the carboxyl-terminus. Further evidence that the quantitation reflects the actual amount of hormone present are the following. 1) Two different monoclonal antibodies (B107 and B109) which recognize dimer only were used in the quantitation and gave nearly identical

379

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^ hCG (-) O-Linked •

0.01

0.1 hCG (pmol/ml)

Truncated hCG

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FIG. 2. Binding of wild-type (WT) and mutant hCG to mouse LH/ hCG receptor. The displacement of [125I]hCG with increasing concentrations of hCG lacking either the O-linked oligosaccharides (hCG 0linked) or the carboxyl-terminal extension (truncated hCG) are compared to wild-type hCG produced in CHO cells. Cultured MA-10 Leydig tumor cells (23) were used as the source of the LH/hCG receptor.

values. 2) Analysis of [35S]cysteine-labeled hormones secreted from CHO cells was consistent with the results of the RIAs (29). 3) Samples containing truncated and wild-type hCG were analyzed on native gels and subjected to Western blotting using a monoclonal antibody which recognizes /3-subunits lacking the carboxyl-terminal extension (LH/3) as well as CG/3 (data not shown). These experiments suggested that the amount of truncated hCG present is 40% more than that measured by RIA, which would indicate that the receptor binding of truncated hCG would more closely approximate that of wild-type hCG (Fig. 2). Thus, the carboxyl-terminal extension and the O-linked oligosaccharides do not seem to play a significant role in receptor binding of hCG. Previous studies of the AT-linked oligosaccharides on hCG have shown that although receptor binding is unaffected by absence of the JV-linked oligosaccharides, signal transduction is greatly decreased (30-34). Thus, we compared the O-linked oligosaccharide-deficient hCG and the truncated hCG mutant to wild-type hCG in their ability to stimulate adenylate cyclase and steroidogenesis. Intracellular cAMP produced by MA-10 cells (2426) as a function of the concentration of mutant and wild-type hCG was examined (Fig. 3). The hCG derivative lacking the carboxyl-terminal extension (truncated hCG) and wild-type hCG stimulate the production of comparable amounts of cAMP, consistent with their similiar affinity for the LH/hCG receptor. Likewise, absence of the O-linked oligosaccharides causes only a small decrease in the cAMP response. Since the production of progesterone in MA-10 cells by hCG is coupled to cAMP stimulation (23-26), these results suggest that absence of the carboxyl-terminal extension and O-linked oligosaccharides should also have little effect on steroi-

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BIOLOGICAL ROLE OF hCG/? CARBOXYL-TERMINAL EXTENSION

380

600 r 500 •

hCG-WT Truncated hCG hCG(-)O-linked

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0.01 0.1 1 hCG (pmol/ml)

FlG. 3. Intracellular cAMP accumulation stimulated by wild-type (WT) and mutant hCG. The concentrations of cAMP (±range) that accumulates in the MA-10 cells upon addition of increasing concentrations of wild-type hCG, truncated hCG, and hCG lacking O-linked oligosaccharides are shown. 600 500

• A

400 300

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bursae of immature hypophysectomized female rats pretreated with PMSG (15 IU) 52 h earlier. The intrabursal route was used to insure high local concentrations of these proteins. Injection of ovine LH was used as a positive control, and injection of medium lacking hormone was used as a negative control. As shown in Fig. 5, ip or intrabursal injection of medium alone did not cause any animals to ovulate. However, ip injection of 5 and 10 ng ovine LH caused all 8 animals to ovulate, with approximately 30 ovulated oocytes from both the left and right ovaries. Intrabursal injections of 70 ng wild-type hCG resulted in 1 of 4 animals ovulating, whereas 210- and 700-ng doses caused all four animals to ovulate with approximately the same number of oocytes released, similar to ovine LH treatment. However, injection of truncated hCG failed to induce ovulation at 70- and 210-ng doses, whereas 700 ng caused all 4 animals to ovulate about 20 oocytes. In all ovulated groups, the number of oocytes recovered was similar in both ovaries. Thus, even though these hormones have similar abilities to bind to the LH/ hCG receptor and stimulate cAMP and steroid production in vitro, truncated hCG is approximately 3-fold less potent in ovulation induction in vivo. Furthermore, these hormones induced ovulations bilaterally even though the intrabursal injections were only into the left ovarian bursae, suggesting that these hormones can circulate to the contralateral side. To further confirm these findings, wild-type hCG and truncated hCG were also injected iv, and results were compared to those in rats treated with ovine LH and

FlG. 4. Stimulation of steroidogenesis by wild-type (WT) and mutant hCG. Progesterone elaborated into the medium by the MA-10 cells upon addition of varying concentrations of wild-type hCG, truncated hCG, or hCG lacking O-linked oligosaccharides is shown. Maximum progesterone (percentage of control ± SE; n = 5) produced by the derivatives when 0.67 pmol/ml wild-type or mutant hCG derivatives is added is as follows: wild-type hCG, 100%; hCG O-linked oligosaccharides, 93.2 ± 4.0%; and truncated hCG, 94.4 ± 1.7%.

dogenesis. The data in Fig. 4 confirm that absence of the O-linked oligosaccharides and the carboxyl-terminal extension from hCG/3 resulted in no significant changes of the steroidogenesis dose-response curves and maximum steroid produced compared to those of wild-type hCG. Thus, absence of the O-linked oligosaccharides and carboxyl-terminal extension from hCG/3 does not significantly alter the ability of hCG to either bind to the LH/ hCG receptor or stimulate cAMP and steroid production in vitro. In vivo biological activity To test the in vivo activity of wild-type and truncated hCG, we injected these glycoproteins into the left ovarian

Endo • 1990 Vol 126 • No 1

50 T 0/6 4/4 4/4

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0/4 0/4

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ES Right ovary Bi Left ovary

40-|30-

20-

|

10 +

o .

0 5 10 Mg o-LH >P

0

210 700 ng wildtype hCG ib 70

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210 700 ng truncated hCG ib

FlG. 5. Number of ovulated oocytes (mean ± SEM) recovered from the oviducts of PMSG-primed female rats after ip injection of ovine LH or intrabursal (ib) injections of wild-type (WT) hCG or truncated hCG. Intrabursal injections were into left ovaries. The number of rats that ovulated per total number of animals used is listed at the top over each column. • , Oocytes recovered from left ovaries; ;^, oocytes recovered from right ovaries. Data represent pooled results from two independent experiments, each with two or three rats per group.

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BIOLOGICAL ROLE OF hCG/3 CARBOXYL-TERMINAL EXTENSION control medium (Fig. 6). Similar to the above results, injection of control medium failed to stimulate ovulation, whereas ovine LH stimulated the release of high numbers of oocytes. Intravenous injection of 70 ng wild-type hCG increased the number of rats that ovulated and the number of ovulated oocytes compared to those when the intrabursal route was used (Fig. 5). Similarly, 210 ng truncated hCG administered iv stimulated more animals to ovulate with higher numbers of oocytes released compared to the intrabursal route, where no animals ovulated. Further, consistent with the intrabursal injection of wild-type and truncated hCG, comparison of the levels of hormone injected iv, which was needed to stimulate ovulation, revealed that truncated hCG resulted in significantly fewer ovulated oocytes at the 70- and 210-ng doses (P < 0.05) and confirmed that the truncated hCG is approximately 3-fold less potent than wild-type hCG. Although differences in the quantitation of the hormone present could be responsible for these differences, this seems unlikely, as discussed above. Furthermore, since the Western blotting experiments showed that there was 40% more truncated hCG present than the RIA indicated, this implies that the potency of wild-type hCG is more than 3-fold greater than that of truncated hCG in vivo. Although hCG binds similarly to rat and mouse LH/hCG receptors, we also wanted to confirm that the in vivo differences seen were not due to differences in the receptor binding of the truncated derivative to the rat receptor. Truncated and wild-type hCG interact similarly with the rat receptor (Fig. 7), as seen above for the mouse LH/hCG receptor (Fig. 2). This is a further confirmation that the amount of truncated hCG injected approximates the amount of wild-type hCG injected and 0/10

3/4

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70 210 1400 ng wildtype hCG iv

21 70 210 1400 ng truncated hCG iv

FlG. 6. Number of ovulated oocytes (mean ± SEM) recovered from the oviducts of PMSG-primed female rats after iv injection of ovine LH, wild-type hCG, or truncated hCG. The number of rats that ovulated per total number injected is listed at the top over each column. Data represent pooled results from three independent experiments, each with two to four rats per group.

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£100 o o .o 80 T3 60 c O CO

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hCG-WT Truncated hCG

40 20 0

3 10 hCG (ng/tube)

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FIG. 7. Receptor binding of wild-type (WT) and truncated hCG to rat testicular membranes. The displacement of [125I]hCG observed in the presence of increasing concentrations of wild-type hCG or truncated hCG is shown. The specific binding is expressed as a percentage of the binding observed in the absence of hCG. Each point represents the mean ± SEM of three independent experiments.

further emphasizes that there are not other proteins in the hormone preparations that are preventing the truncated hCG from interacting with the receptor in vivo.

Discussion Our studies have examined the role of the hCG/3 Olinked oligosaccharides and the carboxy-terminal extension in hCG receptor binding, steroidogenesis in vitro, and ovulation induction in vivo using recombinant hormones generated by site-directed mutagenesis and gene transfer methods. Previous investigations (15-19) using antibodies to the hCG/3 carboxyl-terminal extension have suggested that this region is still exposed to the surface after hCG binds to the LH/hCG receptor. One group (17) also suggested that since the carboxyl-terminal extension arose by a read-through event and was not in the ancestral protein, it contributes little to the tertiary structure and biological action of hCG. These hypotheses are reinforced by studies showing that antibodies to the carboxyl-terminal extension react similarly to either denatured or native hCG/3 (18). Thus, the results described here and those of others imply that the carboxyl-terminal extension of hCG/? does not interact directly with the LH/hCG receptor. Furthermore, the carboxyl-terminal extension does not apparently influence the tertiary structure or folding of the j8-subunit, since the CG/3AT mutant combines efficiently with the a-subunit (28, 29), and absence of the hCG/3 carboxyl-terminal extension does not alter signal transduction. Since both receptor binding and subsequent activation events are not altered by absence of the carboxyl-terminal extension, the carboxyl-terminal extension is probably not responsible for the increased binding of hCG to the LH/hCG receptor compared to LH from different species (13, 35, 36). Since

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BIOLOGICAL ROLE OF hCG/3 CARBOXYL-TERMINAL EXTENSION

truncated hCG and wild-type hCG have similar steroidogenic effects, it is also unlikely that absence of the hCG/3 carboxyl-terminal extension has any effect on the lateral mobility of the hormone-receptor complexes (36), because presumably this movement is intricately involved in signal transduction via receptor microaggregation (13, 37, 38). Furthermore, since wild-type hCG and truncated hCG have similar receptor binding and biopotencies in vitro and since the rate of internalization is directly related to receptor binding and signal transduction (12), the hCG/3 carboxyl-terminal extension is apparently not responsible for the different endocytic pattern of the hCG-receptor complex us. those of the other glycoprotein hormone-receptor complexes (12, 25, 39, 40) as suggested previously (12). Use of the truncated hCG permitted us to assess the in vivo role of the carboxyl-terminal extension. In contrast to what is seen in vitro, injection via either intrabursal or iv routes showed that the truncated hCG is approximately 3-fold less potent than wild-type hCG. Since in vitro studies show that both forms bind equally to the LH/hCG receptor and stimulate cAMP and steroidogenesis to similar extents, the different biological effects elicited in vivo must occur before hormone-receptor interaction. Several studies have examined the clearance of injected hCG (7-10) and the other glycoprotein hormones (8, 41-43). These studies have shown that hCG injected into rats is cleared with a half-life of 48141 min (7, 10) which approximates the 1-2 h half-life in humans (9). In contrast, other studies demonstrated that ovine LH (41) and rat and bovine TSH (42) are cleared very rapidly from the circulation, with half-lives of 5-15 min. Human LH injected into ewes has a halflife of 23 min (8), and human FSH injected into humans is cleared with a half-life of 24-38 min (43). Thus, LH, FSH, and TSH, which lack the carboxyl-terminal extension found in hCG, are cleared at a faster rate than hCG. Furthermore, whereas only approximately 10% of hCG is cleared via the kidney (9, 10), the kidney is the main site of clearance of LH (41), TSH (42), and FSH (43), where more than 35% is excreted. Previous studies also suggest that the size and charge of these hormones may limit renal clearance. More negatively charged forms of human FSH {i.e. forms with increased sialic acid content) have longer half-lives, which is postulated to be due to decreased glomerular filtration (44). Other studies also suggest that the O-linked oligosaccharides play an important role in prolonging plasma half-life; the absence of the two hCGa JV-linked oligosaccharides does not affect hCG clearance, whereas absence of the hCG/3 Nlinked and O-linked oligosaccharides increases the renal clearance 5-fold and decreases the plasma half-life of the derivative to 15 min (7). Thus, the above studies and those presented here suggest that the presence of the

Endo • 1990 Voll26«No 1

hydrophilic carboxyl-terminal extension containing the four negatively charged O-linked oligosaccharides plays an important role in hCG bioactivity by prolonging the circulating half-life of the hormone secondary to a decrease in renal clearance. In addition, the carboxyl-terminal extension may minimize metabolism of hCG by preventing serum proteases from acting on the hCG. Furthermore, the presence of the hydrophilic carboxylterminal extension (45) may increase the solubility of the hormone in the circulation and thereby allow it to remain in the plasma longer, with a greater likelihood of interacting with its receptor. Alternatively, the carboxylterminal extension may influence the stability of the dimer by altering the processing of the iV-linked oligosaccharides. There is some heterogeneity of the truncated hCG when analyzed on native gels, which may be due to subtle alterations in the oligosaccharides (data not shown). However, we (46) have shown that subtle changes in the hCG iV-linked oligosaccharides will cause significant effects on in vitro intracellular cAMP accumulation which is not observed here (Fig. 3). Although studies using both intrabursal and iv injections demonstrate that truncated hCG is 3 times less potent than intact hCG, the iv route of hormonal administration is more effective than the intrabursal route for inducing ovulation. Also, no major differences in ovulation rate were found between the two ovaries after unilateral intrabursal administration of the hormones. These findings suggest that iv injection readily allows access of hCG to ovarian cells, and a prolonged exposure to a high local concentration of hCG is not needed for induction of ovulation. Since equine LH/3 and CG/3 also contain glycosylated carboxyl-terminal extensions (47, 48), our studies would suggest that the in vivo biological effects of the dimers containing these equine subunits behave similarly to hCG. Indeed, PMSG is a potent gonadotropin in rodents and is used routinely for induction of follicle maturation. Furthermore, equine LH/3 derived from the pituitary also contains a carboxylterminal extension similar to hCG/3, suggesting that this extension is not unique to placental function. Thus, the carboxyl-terminal extension may serve as a critical determinant for maintaining high circulating levels of hCG during the critical physiological period of synthesis in the first trimester of pregnancy.

Acknowledgments We thank Dr. Monty Krieger for helpful comments and advice on the use of the IdlD cells. We also thank Dr. Mario Ascoli for the gift of the MA-10 cells, Drs. William Moyle and Ermie Cogliani for performing Western blotting experiments, and Dr. M. Tamoto for help with the rat receptor binding experiments. We appreciate the help of Carol Patterson, Barbara Baiter, and Vickey Farrue in the expert preparation of this manuscript.

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