Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.     

A single-chain bifunctional gonadotropin analog is secreted from Chinese hamster ovary cells as two distinct bioactive species

One of the major developments in exploring structure activity relationships of the glycoprotein hormone family was the genetic engineering of single chains comprised of the common alpha subunit and one or more of the hormone-specific beta subunits tandemly arranged. These studies indicate that there is a structural permissiveness in the quaternary relationships between the subunits and biological activity. However, the conformational relationships between the ligand and the receptor are unclear. Bifunctional triple-domain analogs represent an ideal model to address this issue. Does a single molecule possess the ability to simultaneously interact with both specific receptors or are there two functionally distinct species in the chimeric population? Here we show, using a preadsorption protocol comprised of Chinese hamster ovary cells expressing either the luteinizing hormone (LH)/chorionic gonadotropin (CG) or follicle-stimulating hormone (FSH) receptor, that at least two distinct bioactive populations of the dually active triple-domain chimera FSHbeta-CGbeta-alpha are synthesized, each corresponding to a single activity (CG or FSH). Furthermore, we show that these bioactive populations form distinct stable heterodimer-like contacts. That there is not a single biologically active species formed during synthesis of the chimera implies that in vivo the heterodimer exists in multiple conformations and is not a static rigid molecule.     

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.     

Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin

Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) from pituitary and chorionic gonadotropin (CG) from placenta are a family of closely related glycoproteins. Each hormone is a heterodimer, consisting of an alpha- and a beta-subunit. Within an animal species, the alpha-subunits of all four glyco-protein hormones have an identical amino acid sequence, whereas each beta-subunit is distinct and confers hormone-specific features to the heterodimer. LH and FSH are synthesized within the same cell, the gonadotroph of the anterior pituitary, but are predominantly stored in separate secretory granules. We have characterized the asparagine-linked oligosaccharides on bovine, ovine and human LH, FSH and TSH. The various pituitary hormones were found to contain unique sulfated oligosaccharides with the terminal sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, sialylated oligosaccharides with the terminal sequence SA alpha Gal beta GlcNAc beta Man alpha, or both sulfated and sialylated structures. Despite synthesis of LH and FSH in the same pituitary cell, sulfated oligosaccharides predominate on LH while sialylated oligosaccharides predominate on FSH for all three animal species. We have examined the reactions leading to synthesis of the sulfated oligosaccharides to determine which steps are hormone specific. The sulfotransferase is oligosaccharide specific, requiring only the sequence GalNAc beta 1----4GlcNAc beta 1----2Man alpha. In contrast, the GalNAc-transferase appears to be protein specific, accounting for the preferential addition of GalNAc to LH, TSH, and free (uncombined) alpha-subunits compared with FSH and other pituitary glycoproteins. The predominance of sulfated oligosaccharide structures on LH may account for sorting of LH and FSH into separate secretory granules. Differences in sulfation and sialylation of LH, FSH and TSH may also play a role in the regulation of hormone bioactivity.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common alpha subunit assembled to a hormone-specific beta subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.     

Site-directed mutagenesis defines a domain in the gonadotropin alpha-subunit required for assembly with the chorionic gonadotropin beta-subunit.

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that share a common alpha-subunit, but differ in their hormone-specific beta-subunits. Using site-directed mutagenesis and gene transfer, we studied the region in the common alpha-subunit that has been implicated in the assembly with the beta-subunits. The wild-type or mutated alpha-gene was cotransfected into Chinese hamster ovary cells with the wild-type hCG beta gene. Deletion of the sequence Pro38-Thr39-Pro40 or a change in Tyr37 or Thr39 in the alpha-subunit eliminated or reduced combination with the beta-subunit. Deletion of the sequence Leu41-Arg42-Ser43 had little effect on hCG dimer formation. Disruption of the disulfide bone in the carboxyl end of the subunit did not affect assembly, which suggests that the disulfide bond of Cys59 and Cys87 is not critical for dimer formation. Based on our data and the previously published results from several laboratories, the region encompassed by amino acids 37-40 is a key determinant in initiating and maintaining alpha:beta assembly.     

Insulin-like growth factor I receptor beta-subunit heterogeneity. Evidence for hybrid tetramers composed of insulin-like growth factor I and insulin receptor heterodimers

In both NIH3T3 cells and HepG2 cells, insulin-like growth factor I (IGF-I) receptors possess two beta-subunits that display different electrophoretic mobilities. Increasing concentrations of IGF-I stimulated the phosphorylation of both beta-subunits to a similar extent, whereas insulin stimulated the phosphorylation of both subunits only at elevated concentrations. Both beta-subunits were immunoprecipitated with p5, an insulin receptor-specific anti-peptide antibody, or with A410, a polyclonal anti-insulin receptor antisera. However, if the tetrameric IGF-I receptor was first dissociated into alpha-beta heterodimers with 1 mM dithiothreitol, only the lower molecular weight beta-subunit was immunoprecipitated. These results suggested that p5 and A410 specifically recognized the lower molecular weight beta-subunit but immunoprecipitated the higher molecular weight beta-subunit because it was present in the same disulfide linked tetramer. Similarly, alpha-IR-3, an antibody specific for the alpha-subunit of the IGF-I receptor, immunoprecipitated both types of beta-subunit from the intact tetramer but only the higher molecular weight beta-subunit from the dissociated heterodimers, suggesting that there are two types of alpha-subunits in the same tetramer and that the alpha-subunit recognized by alpha-IR-3 is only associated with the higher molecular weight beta-subunit. Tryptic phosphopeptide maps of the lower molecular weight beta-subunit of IGF-I receptor were different from the higher molecular weight beta-subunit, but were similar to those of the insulin receptor beta-subunit. Thus, by immunochemical cross-reactivity and structural criteria, the lower molecular weight beta-subunit of the IGF-I receptor was similar to the beta-subunit of insulin receptor. These data suggest that there exists a species of IGF-I receptor that is a hybrid composed of an insulin receptor alpha-beta heterodimer and an IGF-I receptor alpha-beta heterodimer. The existence of such a hybrid receptor could have important functional consequences.     

Molecular cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for the beta-subunit of rat follicle stimulating hormone

To provide a hybridization probe for analysis of the regulation of rat gonadotropin subunit mRNA levels, an effort was made to isolate a cloned cDNA for the beta-subunit of rat FSH (FSH beta). Using a cloned bovine FSH beta cDNA as a hybridization probe, a rat pituitary lambda gt10 cDNA library was screened and a single, strongly hybridizing clone identified. The 874 base pair cDNA insert from this clone contains the complete sequence of rat FSH beta including an amino-terminal precursor segment. Hybridization of this cloned cDNA to rat pituitary RNA demonstrated the presence of an approximately 2.0 kilobase RNA species containing FSH beta sequences. Cloned rat cDNA was also used to demonstrate that estrogen treatment of ovariectomized female rats results in decreases in mRNA concentrations for FSH beta and the beta-subunit of LH with somewhat smaller decreases in alpha-subunit mRNA concentrations. Little or no change was detected in the mRNA for the beta-subunit of TSH.     

Partial restoration of lutropin activity by an intersubunit disulfide bond: implications for structure/function studies

Gonadal function is controlled by lutropins and follitropins, heterodimeric cystine knot proteins that have nearly identical alpha-subunits. These heterodimeric proteins are stabilized by a portion of the hormone-specific beta-subunit termed the "seatbelt" that is wrapped around alpha-subunit loop 2 (alpha 2). Here we show that replacing human chorionic gonadotropin (hCG) alpha 2 residue Lys51 with cysteine or alanine nearly abolished its lutropin activity, an observation that implies that alpha Lys51 has a key role in hormone activity. The activity of the heterodimer containing alpha K51C, but not that containing alpha K51A, was increased substantially when beta-subunit seatbelt residue beta Asp99 was converted to cysteine. As had been reported by others, heterodimers containing alpha K51C and beta D99C were crosslinked by a disulfide. The finding that an intersubunit disulfide restored some of the activity lost by replacing alpha Lys51 suggests that this residue is not crucial for receptor binding or signaling and also that hCG and related hormones may be particularly sensitive to mutations that alter interactions between their subunits. We propose the unique structures of hCG and related family members may permit some subunit movement in the heterodimer, making it difficult to deduce key residues involved in receptor contacts simply by correlating the activities of hormone analogs with their amino acid sequences.     

The glycoprotein alpha-subunit is critical for secretion and stability of the human thyrotropin beta-subunit

TSH is a member of a family of heterodimeric glycoprotein hormones which have a common alpha-subunit but differ in their hormone-specific beta-subunit. To study the posttranslational processing and assembly of human TSH, eukaryotic expression vectors were constructed that contained either the human TSH beta gene only or both the TSH beta and alpha-genes. These vectors were transfected into Chinese hamster ovary cells and stable cell lines synthesizing TSH beta or TSH dimer were isolated. The kinetics of secretion of TSH beta and the rate of assembly of TSH dimer were compared to the known secretion and assembly of human LH and human CG. In the absence of the alpha-subunit, CG beta is secreted efficiently, but TSH and LH beta-subunits are slowly degraded intracellularly (t1/2 approximately equal to 6 h) and less than 10% is secreted into the medium. In the presence of the alpha-subunit CG beta was also secreted efficiently as dimer but only 50% of the LH beta appeared in the medium as LH dimer. However, unlike LH beta, the alpha-subunit efficiently combines with TSH beta since greater than 95% was secreted as TSH dimer. Thus, the determinants for human TSH beta secretion and assembly are unique from the other human glycoprotein hormone beta-subunits.     

Engineering a potential antagonist of human thyrotropin and thyroid-stimulating antibody

Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, alpha and beta. We have recently converted the hTSH heterodimer to a biologically active single chain (hTSHbeta.CTPalpha) by fusing the common alpha-subunit to the C-terminal end of the hTSH beta-subunit in the presence of a approximately 30-amino acid peptide from hCGbeta (CTP) as a linker. The hTSHbeta.CTPalpha single chain was used to investigate the role of the N-linked oligosaccharides of alpha- and beta-subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the alpha-subunit (hTSHbeta.CTPalpha(1+2)) and the other lacking the oligosaccharide chain on the beta-subunit (hTSHbeta.CTPalpha(deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSHbeta.CTPalpha wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSHbeta.CTPalpha(1+2) variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroid-stimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma.     

The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin.

The glycoprotein hormones are heterodimeric and contain a common alpha-subunit, which is noncovalently associated with a hormone-specific beta-subunit. The alpha-subunit has been highly conserved throughout evolution; for example, the five amino acid residues of the carboxy-terminus, Tyr-Tyr-His-Lys-Ser-COOH, are identical in nine of the 10 available amino acid sequences. It has been shown that enzymatic removal of these five amino acid residues, while not affecting holoprotein formation, results in a heterodimer that exhibits very little, if any, binding to the CG/LH receptor. Using site-directed mutagenesis on the human alpha-subunit, we have prepared two deletion mutants, Des-(88-92)alpha and Des-(89-92)alpha, and two point mutants, where each of the two tyrosines, 88 and 89, was replaced with phenylalanine, in order to delineate more specifically the contributions of these aromatic side-chains to receptor binding. The cDNAs for wild-type hCG alpha and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCG beta was inserted into a pRSV plasmid; both were transiently cotransfected into DUXB-11 cells. The media were collected, and RIAs showed that all mutants formed heterodimers; moreover, there was no discernable difference in subunit assembly between wild-type hCG alpha and the various mutant alpha-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [125I]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin and free alpha-subunit secretion in patients during GnRH-agonist treatment.

In female patients gonadotropin and free alpha-subunit serum levels during GnRH-agonist (GnRH-a) treatment were investigated. Using two different immunoassays (RIA, IRMA) as well as LH in-vitro bioassay evidence was found for pituitary secretion of incomplete LH molecular forms which are detected false positive in RIA measurements but neither in IRMA nor in bioassay. In the GnRH-a induced desensitization state suppression of LH serum levels was more profound as compared to FSH. In contrast to gonadotropins free alpha-subunit serum levels were shown to increase in a biphasic course leading to ninefold higher levels persisting during GnRH-a treatment. A further increase was achieved by exogenous GnRH but not by estradiol. In conclusion these in-vivo data give evidence for a gradually different suppression of gonadotropic secretion by the use of GnRH-a. The mechanisms of inhibition are discussed to be directed to steps in the processing of beta-subunit synthesis but not of alpha-subunit.     

Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone beta by inhibin from primate Sertoli cells

We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein alpha-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P less than 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the alpha-subunit was decreased to 89% of control at 72 h (P less than 0.05). Also by 72 h, cell content of both FSH (73% of control) and alpha-subunit (81% of control) were significantly suppressed (P less than 0.001, P less than 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for the rat alpha-, LH beta-, and FSH beta-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)     

The biological action of choriogonadotropin is not dependent on the complete native quaternary interactions between the subunits

Human CG (hCG) is a member of the glycoprotein hormone family characterized by a heterodimeric structure consisting of a common alpha-subunit noncovalently bound to a hormone-specific beta-subunit. The two subunits are highly intertwined and only the heterodimer is functional, implying that the quaternary structure is critical for biological activity. To assess the dependence of the bioactivity of hCG on the heterodimeric interactions, alpha- and beta-subunits bearing mutations that prevent assembly were covalently linked to form a single chain hCG. Receptor binding and signal transduction of these analogs were tested and their structural integrity analyzed using a panel of monoclonal antibodies (mAbs). These included dimer-specific mAbs, which react with at least four different epitope sites on the hormone, and some that react only with the free beta-subunit. We showed that there was significant loss of quaternary and tertiary structure in several regions of the molecule. This was most pronounced in single chains that had one of the disulfide bonds of the cystine knot disrupted in either the alpha- or beta-subunit. Despite these structural changes, the in vitro receptor binding and signal transduction of the single chain analogs were comparable to those of the nonmutated single chain, demonstrating that not all of the quaternary configuration of the hormone is necessary for biological activity.     

Alternatively folded choriogonadotropin analogs. Implications for hormone folding and biological activity

Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their beta-subunits that is wrapped around alpha-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the beta-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the alpha-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of alpha-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10-20% the activity of hCG. Attachment of the seatbelt to alpha-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the beta-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of alpha-subunit loop 2, and the end of the alpha-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.     

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.     

Molecular cloning and histological localization of LH-like substances in a bottlenose dolphin (Tursiops truncatus) placenta

All mammals exhibit pituitary-specific expression of LH and FSH, whereas placental expression of gonadotropins has been reported only in primates and equids. Some cetaceans, such as dolphins, have a long gestational period and a sexual cycle of about 27 days almost comparable with that of humans. Histologically, dolphins have an epitheliochorial placentae that resembles placentas of Perissodactyla including horses. In the present study, we cloned cDNAs encoding gonadotropins and observed their immunohistochemical localization in the placenta of bottlenose dolphin. The cDNAs obtained encoded 120 amino acids for the alpha-subunit (including 96 amino acids of mature proteins), and 141 amino acids for the beta-subunit (including 121 amino acids of mature proteins). The sequence of the alpha-subunit was similar to that in the pig (Artiodactyla) pituitary glycoprotein hormone [96.7% homology at amino acids (aa) level], and the sequence of the beta-subunit was similar to that of luteinizing hormone (LH) in the pig [94.3% homology at aa level] and white rhinoceros (Perissodactyla) [93.3% homology at aa level]. Of interest, dolphin LHbeta lacks carboxyl-terminal-peptides (CTP). This fact suggests that CTP are not essential for placental expression of gonadotropin in dolphins. Immunohistochemical observations employing anti-ovine LHbeta antibody revealed positive staining in the villositycal tissue. Our observations suggest placental expression of gonadotropin homologues in cetaceans and possible evolutionary conservation of placentae-derived hormonal control of ovarian functions during pregnancy.