Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity

OBJECTIVE: To test whether extension of the C-terminus of human chorionic gonadotropin (hCG) alpha-subunit (halpha) alters the bioactivity of the recombined alphabeta heterodimer. DESIGN: The stop codon of halpha was mutated to produce a 24 amino acid extension. METHODS: The extended halpha (alpha(+24)) was co-expressed with hCGbeta in COS-7 cells and the receptor binding and in vivo bioactivity of the secreted hormone was compared with its wild-type counterpart. RESULTS: This extension did not impair the binding of hCG to rat LH/CG receptors and provoked a sixfold reduction in its stimulatory activity of testosterone secretion in rat Leydig cells. CONCLUSIONS: The extension of alpha by itself does not lead to inhibition of the alphabeta heterodimer to LH receptors but the structure of the extension appears to play an important role. It is thus possible that one-chain hCG chimeras with the beta N-terminus fused to the alpha C-terminus might be active.     

A biologically active single chain human chorionic gonadotropin analog with altered receptor binding properties

hCG is a heterodimer consisting of an alpha-subunit common among all members of the glycoprotein hormone family, LH, FSH, and TSH, and a unique beta-subunit responsible for receptor specificity. Biologically active single chain analogs of these hormones have been engineered in which the C-terminus of the beta-subunit was fused to the N-terminus of the alpha-subunit (N-beta-alpha-C) either with or without a linker such as the hCGbeta C-terminal peptide (CTP). This tandem order of subunits was chosen based on studies suggesting that the N-terminal region of hCGbeta and particularly the C-terminal region of the alpha-subunit are important in receptor binding and activation. Single chain hCG (YhCG1) can, in turn, be fused to the LH receptor to yield a hormone-receptor complex that is biologically active in transfected cells. Herein, we report the construction of a new single chain hCG analog (YhCG3) in which the C-terminus of the alpha-subunit is fused to the N-terminus of hCGbeta via a CTP (N-alpha-CTP-beta-C). Compared with YhCG1, this analog binds receptor with a 25- to 30-fold lower affinity, but, surprisingly, is capable of stimulating intracellular cAMP levels to the same extent. Furthermore, YhCG3 can be covalently linked to its receptor to produce a biologically active complex that results in elevated levels of basal cAMP in transfected cells. These results suggest that free N- and C-termini of hCGbeta and the alpha-subunit, respectively, are not essential for receptor binding and activation and that YhCG3 is in a more efficacious conformation for receptor activation than YhCG1.     

Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.

The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.     

Nonassembled human chorionic gonadotropin subunits and alphaalpha-homodimers use fast-track processing in the secretory pathway in contrast to alphabeta-heterodimers

In multimeric glycoproteins, like glycoprotein hormones, mutual subunit interactions are required for correct folding, assembly, and transport in the secretory pathway. However, character and time course of these interactions need further elucidation. The influence of the glycoprotein hormone alpha-subunit (GPHalpha) on the folding of the human chorionic gonadotropin (hCG) beta-subunit (hCGbeta) in hCG alphabeta-heterodimers was investigated in [(35)S]Met/Cys-labeled JEG-3 cells. Completeness of disulfide bridge formation during the time course of folding was estimated by labeling with [(3)H]N-ethylmaleinimide of free thiol groups not yet consumed. Subunit association took place between immature hCGbeta (high (3)H/(35)S ratio) and almost completely folded GPHalpha. Analysis revealed a highly dynamic maturation process comprising of at least eight main hCGbeta folding intermediates (molecular masses from 107 to 28 kDa) that could be micro-preparatively isolated and characterized. These hCGbeta variants developed while being associated with GPHalpha. The 107-kDa variant was identified as a complex with calnexin. In contrast to hCG alphabeta-heterodimers, free nonassociated hCGbeta, free large GPHalpha, and GPHalphaalpha homodimers showed a fast-track-like processing in the secretory pathway. At 10 min before hCG secretion, sialylation of these variants had already been completed in the late Golgi, whereas hCG alphabeta-heterodimers had still not arrived medial Golgi. This shows that the GPHalpha in the hCG alphabeta-heterodimers decelerates the maturation of the hCGbeta portion in the heterodimer complex. This results in a postponed approval of hCG alphabeta-heterodimers by the endoplasmic reticulum quality control unlike GPHalphaalpha homodimers, free hCGbeta, and GPHalpha subunits.     

Effect of individual N-glycosyl chains in the beta-subunit on the conformation of human choriogonadotropin

Human choriogonadotropin is a heterodimeric glycoprotein hormone comprised of noncovalently associated alpha- and beta-subunits. Each of the subunits has two N-glycosyl chains. In our previous communication, we investigated the role of individual carbohydrate chains in the alpha-subunit on the signal transduction function and conformation of the hormone. This paper deals with the effect of individual or both N-glycosyl chains in the beta-subunit on the function and conformation of the monomer as well as of the heterodimer. Three mutants each of hCGbeta and hCG lacking N-glycosyl chains at beta13Asn, beta30Asn and beta13,30Asn were prepared by site-directed mutagenesis by replacing Thr residues in the recognition triplet sequence at beta15 and beta32 positions with Gln. All mutant heterodimers had receptor binding and cAMP and progesterone stimulating activities comparable to wild type hCG. While the loss of carbohydrate at beta13Asn or beta13,30Asn in the case of hCGbeta monomer resulted in a 4-6%, decrease in the ordered structure, the loss of the glycosyl chain at beta 30Asn did not alter the conformation as compared with the wild type hCGbeta. Similarly, all carbohydrate deficient hCG heterodimers had a decrease of 6-8%) in the ordered structure as compared with hCG. Thus, while the individual N-glycosyl chains did not affect the function of the hormone, they did have marked effect on its conformation but the conformational changes were localized and did not perturb the receptor binding and signal transduction sites.     

Diagnosis of hydatidiform mole and persistent trophoblastic disease: diagnostic accuracy of total human chorionic gonadotropin (hCG), free hCG {alpha}- and {beta}-subunits, and their ratios

OBJECTIVE: Human chorionic gonadotropin (hCG) is widely used in the management of hydatidiform mole and persistent trophoblastic disease (PTD). Predicting PTD after molar pregnancy might be beneficial since prophylactic chemotherapy reduces the incidence of PTD. DESIGN: A retrospective study based on blood specimens collected in the Dutch Registry for Hydatidiform Moles. A group of 165 patients with complete moles (of which 43 had PTD) and 39 patients with partial moles (of which 7 had PTD) were compared with 27 pregnant women with uneventful pregnancy. METHODS: Serum samples from patients with hydatidiform mole with or without PTD were assayed using specific (radio) immunoassays for free alpha-subunit (hCGalpha), free beta-subunit (hCGbeta) and 'total' hCG (hCG + hCGbeta). In addition, we calculated the ratios hCGalpha/hCG + hCGbeta, hCGbeta/hCG + hCGbeta, and hCGalpha/hCGbeta. Specificity and sensitivity were calculated and paired in receiver-operating characteristic (ROC) curve analysis, resulting in areas under the curves (AUCs). RESULTS: hCGbeta, hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta show AUCs ranging between 0.922 and 0.999 and, therefore, are excellent diagnostic tests to distinguish complete and partial moles from normal pregnancy. To distinguish partial from complete moles the analytes hCGbeta, hCG + hCGbeta and the ratio hCGalpha/hCGbeta have AUCs between 0.7 and 0.8. Although hCGalpha, hCGbeta and hCG + hCGbeta concentrations are significantly elevated in patients who will develop PTD compared with patients with spontaneous regression after evacuation of their moles, in predicting PTD, these analytes and parameters have AUCs <0.7. CONCLUSIONS: Distinction between hydatidiform mole and normal pregnancy is best shown by a single blood specimen with hCGbeta, but hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta are also excellent diagnostic parameters. To predict PTD, hCGalpha, hCGbeta, hCG + hCGbeta and hCGalpha/hCGbeta are moderately accurate tests, although they are not accurate enough to justify prophylactic chemotherapy treatment for prevention of PTD.     

Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells.

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.     

Internalization of ovine luteinizing hormone/human chorionic gonadotropin recombinants: differential effects of the alpha- and beta-subunits

The rate of internalization and degradation of radioiodinated hCG and ovine LH (oLH) as well as homologous and heterologous recombinants of their subunits was studied in suspensions of ovine luteal cells. Hormone bound to the plasma membrane was quantified by treating the cells with acidic buffer (pH 3.0) and quantification of the radioactivity that was removed from receptor. The radioactivity remaining in the cell pellet was considered to be intracellular. The extent of degradation of the radioactive hormones was determined by subjecting the radioactivity released into the medium during incubation to precipitation with 20% trichloroacetic acid. The non-precipitable radioactivity was considered to have been degraded. Radioactivity was lost from the cell membrane with a t1/2 of 16.8 +/- 2.5 h for radioiodinated hCG, 22.8 +/- 3.8 h for the alpha-subunit of hCG recombined with radioiodinated beta-subunit of hCG, 8.9 +/- 4.5 h for the alpha-subunit of oLH recombined with the radioiodinated beta-subunit of hCG, 0.5 +/- 0.1 h for the alpha-subunit of hCG recombined with the radioiodinated beta-subunit of oLH, 0.5 +/- 0.1 h for radioiodinated oLH, and 0.7 +/- 0.2 h for the alpha-subunit of oLH recombined with the radioiodinated beta-subunit of oLH. In general, the levels of radioactivity that were intracellular and the rate of degradation of the hormone were inversely related to the quantities of hormone that remained on the plasma membrane. The preparations that contained the beta-subunit of hCG were all internalized at a slower rate (t1/2 = 9-22 h) and consequently were degraded more slowly than any of the preparations that contained the beta-subunit of oLH (t1/2 = 0.5-0.7 h). When the radioiodine in oLH was present in the beta-subunit, intracellular levels of radioactivity were higher and degradation occurred more rapidly than when the radioiodine was in the alpha-subunit. Although the differences were less dramatic, hCG with radioiodine in the beta-subunit also reached higher levels intracellularly, but was degraded to a lesser extent at 16 and 24 h than was hCG with radioiodine in the alpha-subunit. These data demonstrated a dramatic difference (approximately 30-fold) in the rate of loss of oLH vs. that of hCG from the membrane of ovine luteal cells. Further, it appears that the beta-subunit of each of the two hormones is the major factor in determining the rate of internalization of the intact hormone. The differences is how the two hormones are processed at the receptor level may help explain the known differences in their steroidogenic potencies.     

Role of amino acid residues at the interface of alpha52asparginyl-N-glycosyl chain of human choriogonadotropin.

Of all the four N-glycosyl chains present in hCG, only one of them at alpha52Asn is located at the alpha/beta subunit interface and is crucial for the biological function of the hormone. The other three are exposed on the surface of the molecule and play only a minor role in the function of the hormone. The alpha52Asn oligosaccharide interacts with five amino acid residues in the beta-subunit, Tyr59, Val62, Phe64, Ala83, and Thr97. The present studies were undertaken to determine the role of the residues at the alpha52Asn-oligosaccharide and the beta-subunit interface in the mechanism of subunit association and downstream signaling events. Ten mutants, two of the alpha-subunit by the replacement of Asn52 and Thr80 with Gln and eight of the beta-subunit by multiple or single amino acid mutations, were prepared. These mutants included, hCGbeta59,62,64,97Ala, hCGbeta59,62,64Ala, hCGbeta62,64Ala, hCGbeta59Phe, hCGbeta62Ala or Thr, hCGbeta83Ile and hCGbeta97Ala. The mutation of the Asn52 to Gln resulted in a drastic change in its conformation and as a consequence in its weak affinity with the wild type beta as compared with that of the wild type hCGalpha and hCGalpha80Gln. The mutants with mutations in the four or three amino acids as well as both mutants of hCGbeta62Val almost failed to combine with hCGalpha again as a result of conformational changes shown by circular dichroism (CD) analysis and not due to their direct involvement in the subunit association. The double mutant combined with hCGalpha and the heterodimer behaved more like the wild type hCG. The mutation of Tyr to Phe resulted in a drop of 20% in the receptor binding and cAMP stimulation although Tyr is considered to be involved directly in subunit association. HCGbeta with mutations in the other amino acids, Phe64, Ala83, and Thr97, combined with the alpha subunit forming heterodimers with biological activity comparable to that of the wild type hCG. Thus, it appears that among the five amino acids in the vicinity of alpha52Asn carbohydrate, only beta59Tyr and beta62Val may be involved directly or indirectly in the alpha/beta beta dimer formation.     

Human chorionic gonadotropin beta-subunit affects the folding and glycosylation of alpha-cys mutants

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of beta-subunits is rigid, whereas that of alpha-subunit is flexible and can be molded by a beta-subunit. Previously, we reported that secreted forms of a mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCGbeta-subunit affects the conformations of alpha mutants, alpha-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGbeta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha-subunit were assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or -hCGbeta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that a homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of beta-subunit. This finding indicated that hCGbeta-subunit rescued the a half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the alpha mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive alpha-subunits was less than 5% of total alpha-subunits. In contrast to free alpha-subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCGbeta-subunit affects the folding and glycosylation of the alpha-subunit mutants.     

The biological role of the carboxyl-terminal extension of human chorionic gonadotropin [corrected] beta-subunit

hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (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 beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.     

Variations in the oligosaccharides on free and combined alpha-subunits of human choriogonadotropin in pregnancy

The glycoprotein hormone hCG and a free alpha-subunit are secreted by trophoblastic cells during pregnancy. We have purified the alpha-subunit of hCG and the free alpha-subunit population from pregnancy urine. Dissociation of hCG with 10 M urea at 37 C, followed by chromatography on DEAE-Sephacel, resulted in separation of alpha- from beta-subunit, as hCG alpha does not bind to DEAE in the presence of urea, while beta-subunit does bind. Similar treatment of the free alpha population resulted in fractionation into two populations, a nonbinding fraction of free alpha and a population which was retained by DEAE in the presence of urea (free alpha 2). The three populations, hCG alpha, free alpha, and free alpha 2, were further purified by affinity chromatography using anti-alpha antisera linked to Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed that hCG alpha consisted of a single component with an apparent mol wt of 22,000, while free alpha and free alpha 2 consisted of multiple components. Radioactive labeling of sialic acid by limited periodate oxidation and NaB[3H]4 reduction resulted in a higher specific activity for free alpha than for hCG alpha, suggesting that free alpha contains more sialic acid per immunoreactive molecule than does alpha dissociated from hCG. [3H]hCG alpha, but not 3H-labeled free alpha, was able to combine with native hCG beta-subunit. The oligosaccharide moieties were released from the different labeled subunits by alkaline hydrolysis and then compared with respect to Concanavalin A (ConA)-binding and DEAE-binding properties. Most of the oligosaccharides from dissociated hCG alpha bound to ConA-Sepharose (72%), while less material from free alpha (40%) and even less from free alpha 2 (25%) were capable of binding to ConA. DEAE chromatography of the oligosaccharides suggested that hCG alpha contained primarily monosialylated forms (greater than 60%), while free alpha and alpha 2 contained primarily (greater than 70%) di- and trisialylated forms. Thus, the ConA and DEAE binding data indicated that the oligosaccharide contents of free alpha and free alpha 2 were quite different from that of hCG alpha. These results also suggest that some of the oligosaccharide structures contained on hCG alpha and most of those on free alpha and free alpha 2 differ substantially from the structures that have been previously described.     

Contribution of subunits to the function of luteinizing hormone/human chorionic gonadotropin recombinants

The alpha- and beta-subunits of hCG and of ovine and porcine LH were used to prepare all nine homologous and heterologous alpha beta-recombinants. EAch purified recombinant was assayed in vitro, using dispersed Leydig tumor cells derived from the M5480P tumor, for its ability to stimulate steroidogenesis and to inhibit [125I]iodo-hCG binding. It was found that the potency of a given recombinant in both assays was most similar to that of the hormone from which the beta-subunit was derived. For example, hCG and hCG beta-containing recombinants were invariably more potent than LH and LH beta-containing recombinants. However, within groups of recombinants containing a common beta-subunit, the alpha-subunit exhibited modulatory effects on the biological potencies. The different observed potencies did not result from alpha beta dissociation since the recombinants were stable in dilute solution at 37 C for periods greatly exceeding that of the assay conditions. LH and the LH beta-containing recombinants were found to dissociate more readily from the Leydig tumor cell gonadotropin receptor than hCG and hCG beta-containing recombinants. (These experiments were performed under conditions where internalization was minimal.) However, analogous to the potency measurements, the alpha-subunit contributed to the rate of dissociation. For example, in recombinants with a common beta-subunit, hCG alpha conferred the greatest stability to the hormone-receptor interaction. These results emphasize a positive relationship between receptor occupancy and biological potency. Whereas the beta-subunit of these gonadotropins seems to exhibit the predominant influence in determining potency, it is clear that both subunits contribute to biological activity. This could involve direct effects as well as induced conformational changes in the complementary subunit.     

Differential processing of the two subunits of human choriogonadotropin by granulosa cells. II. In vivo studies

Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.     

Analysis of the three dimensional structure of the CXGXC motif in the CMGCC and CAGYC regions of alpha-and beta-subunits of human chorionic gonadotropin: importance of glycine residue (G) in the motif

The three dimensional structures of the C(1)X(2)G (3)(X(3))X(4)C(5) motif of hCG, which is considered to be important for noncovalent assembly of the alpha- and beta-subunits of glycoprotein hormones were analyzed to assess the importance of glycine (Gly) (G) at site X(3) in the motif by the conformational energy calculation using computational procedures. In the C(1)M(2)G (3)(X(3))C(4)C(5) motif of the alpha-subunit, Ramachandran plot analysis showing the allowed area of the dihedral angles demonstrated that only a Gly residue was allowed at site X(3). In calculating collision with surrounding atoms as a monomer the possible main chain models of the C(1)A(2)G(3)(X(3))Y(4)C(5) motif in the beta-subunit showed that only alanine (Ala) (A) or Gly at site X(3) is allowed to alleviate the collision with the cysteine (Cys) (C) residues which form a disulfide bridge. A mutant of the beta-subunit with the C(1)A(2)A(3)(X(3))Y(4)C(5) motif (Ala at site X(3)) may not compose a heterodimer with the alpha-subunit because of interference of intermolecular hydrogen bond formation. These findings indicate that the Gly residue at site X(3) (G(3)) in the motif is essential for heterodimer formation of glycoprotein hormones. The significance of similar motifs found in various human proteins other than glycoprotein hormones was suggested.     

Coordinate regulation of the messenger ribonucleic acids encoding the alpha- and beta-subunits of human chorionic gonadotropin in HeLa cells and butyrate-resistant variants

Short term exposure of HeLa S3 cells to sodium butyrate induces accumulation of the mRNAs encoding both the alpha- and beta-subunits of hCG. Both mRNAs accumulate with the same kinetics and reach maximal levels with the same concentration of butyrate, suggesting that the levels of alpha and hCG beta mRNAs are coordinately regulated. In addition, induction of both mRNAs is tightly coupled with a similar increase in secreted levels of hCG subunit protein, further suggesting that regulation of CG biosynthesis in HeLa cells occurs before translation. To determine whether HeLa cells which have overcome the growth inhibitory properties of butyrate show uniformly high levels of alpha and hCG beta mRNAs, we isolated clonal variants by stepwise selection with increasing concentrations of butyrate. Of 69 isolated variants, at least 2 (AO and B2) display different patterns of CG gene expression. In AO cells, alpha-subunit mRNA was not detectable, while in B2 cells, the level of alpha-subunit mRNA was similar to that of wild-type HeLa S3 cells. Conversely, hCG beta mRNA levels in both variant cell lines approximated levels found in butyrate-treated HeLa S3 cultures, suggesting that the mRNAs for alpha and hCG beta are regulated independently. Analysis of genomic DNA by blot hybridization indicated that the alpha-subunit gene was present as a single unrearranged copy in HeLa S3 cells and in both variants, indicating that differences in alpha-subunit gene expression are not associated with major genomic alterations, but probably reflect more subtle changes.     

Immunoreactive beta-core-like material in normal postmenopausal urine: human chorionic gonadotrophin or LH origin? Evidence for the existence of LH core.

Material with the immunochemical properties of the beta-core of human chorionic gonadotrophin (hCG) can be found in the urine of normal postmenopausal women. However, we have been unable to detect intact hCG (using an assay which is specific for the alpha-beta heterodimer of intact hCG) in serum of such subjects. The levels of serum LH and urinary beta-core were compared in matched samples from 28 women (serum LH: median 27 U/l, range 4-70 U/l, urinary beta-core: median 0.27 microgram/l, range less than 0.05-0.645 microgram/l). Urine (4 litres) from three postmenopausal women was concentrated, dialysed and subjected to gel exclusion chromatography on Sephadex G-100. Fractions were analysed by specific assays for LH, intact hCG, total beta-hCG (free beta-subunit and intact hCG), free alpha-subunit and beta-core. Material eluting at the expected position of the beta-core fragment of hCG was detected in all three samples by the beta-core, beta-hCG and LH assays, despite the fact that the LH antibody does not recognize the authentic beta-core of pregnancy. Electrophoresis and Western blotting of the concentrated urines revealed that material of the same molecular size as beta-core was recognized by the antibody to LH but not by a monoclonal antibody raised to free beta-hCG which also recognizes the beta-core molecule of hCG. We conclude that the predominant core-like material identified in postmenopausal urine is probably derived from the beta-subunit of LH.     

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.     

A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes.

A unique characteristics of thyrotrope-specific gene expression is the coordinated expression and regulation of the alpha- and beta-subunits of TSH. A cell line (alpha TSH) derived from the transplantable mouse thyrotropic tumor MGH101A, which no longer expresses the TSH beta-subunit gene but continues to secrete large amounts of alpha-subunit, was used as a model to study alpha-subunit gene expression independent from the TSH beta-subunit gene and was compared with the expression in TSH-secreting TtT97 tumors. Transient transfection studies showed a striking similarity in the activity of 5' deletions of the mouse alpha-subunit gene promoter in both alpha TSH and TtT97 cells and localized two regions important for expression that spanned 100 base pairs, from -480 to -417 and from -417 to -381. These regions were found to have no activity in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Analysis of the alpha-subunit 5' flanking DNA interactions with alpha TSH and TtT97 nuclear extracts showed two DNase I protected sequences, from -474 to -452 and from -447 to -400, both of which colocalized with the functionally important regions. Gel retardation analysis demonstrated the specificity of these interactions, and a similar migration of the DNA-protein complexes suggested that protein factors were similar in the two cell types. We conclude that the nuclear factors necessary for alpha-subunit expression in thyrotropes are retained in alpha TSH cells. Moreover, since alpha TSH cells do not express the TSH beta-subunit gene, the factors that determine the expression of the alpha-subunit may not be sufficient for TSH beta-subunit gene expression.