The evolution of gonadotropins: some molecular data concerning a non-mammalian pituitary gonadotropin, the hormone from a teleost fish (Cyprinus carpio L.)

The amino acid and sugar compositions as well as long N-terminal sequences and the C-terminal amino acids of the two subunits of carp gonadotropin, SU I and SU II, were determined. An important homology was demonstrated between SU I and alpha-subunits and between SU II and beta-subunits of mammalian gonadotropins. Moreover SU II was more closely related to the beta-subunit of LH than to the beta-subunit of FSH.     

Sequence analysis and expressional regulation of messenger RNAs encoding beta subunits of follicle-stimulating hormone and luteinizing hormone in the red-bellied newt, Cynops pyrrhogaster

Two distinct cDNAs encoding beta subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were cloned from the cDNA library constructed for the pituitary of the red-bellied newt, Cynops pyrrhogaster, and sequenced. The newt FSHbeta and LHbeta cDNAs encode polypeptides of 129 and 131 amino acids, including signal peptides of 20 and 19 amino acids, respectively. The number and position of cysteine and N-glycosylation in each of the beta subunits of FSH and LH, which are considered essential for assembly of the alpha subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the beta subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain alpha subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHbeta, LHbeta, and the alpha subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHbeta and LHbeta in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHbeta mRNA levels, but there was no significant change in LHbeta or alpha subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHbeta is negatively regulated by the ovaries.     

The LHbeta gene of several mammals embeds a carboxyl-terminal peptide-like sequence revealing a critical role for mucin oligosaccharides in the evolution of lutropin to chorionic gonadotropin in the animal phyla

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) beta to the beta-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHbeta gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHbeta gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHbeta gene (boCTP) would be sufficient to generate the LHbeta species of a ruminant with properties typical to the CGbeta subunit. The mutated bovine LHbeta-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGbeta CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGbeta subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGbeta-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGbeta subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHbeta to CGbeta evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHbeta gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.     

Cytoplasmic and transmembrane domain deletions of Na,K-ATPase beta-subunit. Effects on subunit assembly and intracellular transport.

cDNAs encoding Na,K-ATPase beta-subunits containing deletions in the cytoplasmic domain or in the single membrane-spanning domain of the molecule were constructed and expressed in mouse L cells to determine the effect(s) of deletions in these domains on alpha/beta-subunit assembly and intracellular targeting. Avian beta-subunits lacking some or all of the cytoplasmic domain (endodomain) assemble with the endogenous mouse alpha-subunit and are correctly transported to the plasma membrane. Mutants containing deletions in the transmembrane domain were constructed by fusing portions of cDNAs encoding the amino-terminal one-third of human beta-subunit deletion mutants with avian beta-subunit cDNA encoding the carboxyl two-thirds of the molecule. A deletion of 3 amino acids in transmembrane domain resulted in correct alpha/beta-subunit assembly and localization to the plasma membrane. In contrast, deletions of 5 or more amino acids in the transmembrane domain prevented expression of the beta-subunit at the cell surface and resulted in the accumulation of these molecules in the ER. In spite of these targeting differences, all beta-subunit mutants capable of membrane insertion were also able to assemble with the alpha-subunit. These results suggest that the specificity for alpha/beta assembly resides in the ectodomains of the subunits.     

Differential regulation of pituitary gonadotropin subunit messenger ribonucleic acid levels in photostimulated Siberian hamsters

FSH levels begin to rise 3-5 days after male Siberian hamsters are transferred from inhibitory short photoperiods to stimulatory long photoperiods. In contrast, LH levels do not increase for several weeks. This differential pattern of FSH and LH secretion represents one of the most profound in vivo examples of differential regulation of the gonadotropins. The present study was undertaken to characterize the molecular mechanisms controlling differential FSH and LH synthesis and secretion in photostimulated Siberian hamsters. First, we cloned species-specific cDNAs for the three gonadotropin subunits: the common alpha subunit and the unique FSHbeta and LHbeta subunits. All three subunits share high nucleotide and predicted amino acid sequence identity with the orthologous cDNAs from rats. We then used these new molecular probes to examine the gonadotropin subunit mRNA levels from pituitaries of short-day male hamsters transferred to long days for 2, 5, 7, 10, 15, or 20 days. Short-day (SD) and long-day (LD) controls remained in short and long days, respectively, from the time of weaning. We measured serum FSH and LH levels by RIA. FSHbeta, LHbeta, and alpha subunit mRNA levels were measured from individual pituitaries using a microlysate ribonuclease protection assay. Serum FSH and pituitary FSHbeta mRNA levels changed similarly following long-day transfer. Both were significantly elevated after five long days (2.3- and 3.6-fold, respectively; P < 0.02) and declined thereafter, but they remained above SD control values through 20 long days. Alpha subunit mRNA levels also increased significantly relative to SD control values (maximum 2-fold increase after seven long days; P < 0.03), although to a lesser extent than FSHbeta. Neither serum LH nor pituitary LHbeta mRNA levels changed significantly following long-day transfer. The results indicate that long-day-associated increases in serum FSH levels in Siberian hamsters reflect an underlying increase in pituitary FSHbeta and alpha subunit mRNA accumulation.     

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.     

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.     

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.     

Evolution of lutropin to chorionic gonadotropin generates a specific routing signal for apical release in vivo.

One of the fundamental differences among mammals is the mechanism of maintaining the corpus luteum of pregnancy. Placentation in primates is associated with the production of the glycoprotein hormone chorionic gonadotropin (CG), which is secreted into the maternal serum and stimulates progesterone synthesis from the corpus luteum, which is essential for early development of the embryo. CG together with the pituitary hormones lutropin (LH), follitropin, and thyrotropin constitute the family of glycoprotein hormones comprised of a common alpha subunit and a hormone-specific beta subunit. The LHbeta and CGbeta subunits share 85% amino acid sequence identity, and functionally LH and CG are interchangeable. CGbeta evolved by a recent gene duplication event from the LHbeta locus, and despite the close relationship between them, their modes of secretion are quite different. CG release from the placenta is apically directed, whereas LH is released from the basal side of the cell, and the determinant(s) for this redirected trafficking are unknown. Here, using the polarized Madin-Darby canine kidney (MDCK) cell line, we provide evidence for the molecular basis of the different secretory patterns of LH and CG in vivo. The apical targeting of CG is programmed by a carboxyl-terminal sequence, which encodes a novel sorting signal. It is also apparent that the presence of the O-linked oligosaccharides in the CTP sequence contributes to this apical routing. The CTP, which is absent in LH, redirects CG to the maternal serum and permits the unique arrangement for primate placentation. Our data also show that the MDCK cells can distinguish the different secretory pathways for the gonadotropins and will be a valuable model for elucidating the determinants associated with the unique sorting of these functionally related hormones.     

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)     

Pig kidney Na+,K+-ATPase. Primary structure and spatial organization

cDNAs complementary to pig kidney mRNAs coding for alpha- and beta-subunits of Na+,K+-ATPase were cloned and sequenced. Selective tryptic hydrolysis of the alpha-subunit within the membrane-bound enzyme and tryptic hydrolysis of the immobilized isolated beta-subunit were also performed. The mature alpha- and beta-subunits contain 1016 and 302 amino acid residues, respectively. Structural data on the peptides from extramembrane regions of the alpha-subunit and on glycopeptides of the beta-subunit underlie a model for the transmembrane arrangement of Na+,K+-ATPase polypeptide chains.     

The carboxyl-terminal 161 amino acids of the Na,K-ATPase alpha-subunit are sufficient for assembly with the beta-subunit.

Chimeric cDNAs encoding regions of the Na,K-ATPase alpha-subunit and a sarcoplasmic reticulum Ca(2+)-ATPase were constructed and expressed together with the avian Na,K-ATPase beta-subunit cDNA in COS-1 cells to determine which regions of the alpha-subunit are required for assembly with the beta-subunit. Assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the avian beta-subunit. A chimera composed of the amino-terminal two-thirds of the Na,K-ATPase and carboxyl-terminal one-third of the Ca(2+)-ATPase did not assemble with the avian beta-subunit. In contrast, the reciprocal chimera, containing the carboxyl-terminal one-third of the Na,K-ATPase, assembled with the beta-subunit. A third chimera, in which 161 amino acids of the Na,K-ATPase carboxyl terminus replaced the corresponding amino acids of the Ca(2+)-ATPase carboxyl terminus, also assembled with the beta-subunit. These results suggest that the aminoacyl residues of the Na,K-ATPase alpha-subunit critical for subunit assembly lie within the carboxyl-terminal 16% of the sequence.     

Intracellular immature subunits of human chorionic gonadotropin in first trimester placental cells: purification and characterization

As we previously reported [Sakakibara et al. (1986) Biochem. Biophys. Res. Commun. 137, 443-452; and Tominaga et al. (1989) J. Biochem. 105, 992-997], subunits of human chorionic gonadotropin (hCG) containing immature N-linked sugar chains (immature subunits), i.e., the 21 kDa form of alpha-subunit and the 23 and 19 kDa forms of beta-subunit, are present predominantly in first trimester placental cells. The molecular mass of intracellular hCG consisting of these subunits, based on gel filtration, was approximately 200 kDa, suggesting homo- or hetero-oligomerization of intracellular hCG. In the present study, we purified the 21 kDa form of alpha-subunit as well as the 23 and 19 kDa forms of beta-subunit from fresh normal first trimester placental tissues by gel filtration and reverse-phase high-performance liquid chromatography. Purified subunits were hydrolyzed (with a decrease in their molecular weighs) by endoglycosidase H and alpha-mannosidase but not by sialidase or sialidase followed by O-glycanase, indicating that those forms have presumably only high-mannose-type N-linked sugar chains but not O-linked sugar chains of the type present in mature beta-subunit. Fifteen cycles of Edman degradation of the purified forms of the subunits were performed. Only one phenylthiohydantoin amino acid, which was the same amino acid as in the urinary beta-subunit, was detected at each step for the mixture of 23 and 19 kDa forms of beta-subunit, indicating that the protein backbones of both forms are identical to each other as well as to the urinary beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human chorionic gonadotropin alpha-subunit of normal placenta: characterization of synthesis and association with beta-subunit

The forms of human chorionic gonadotropin (hCG) alpha-subunit synthesized and released by normal placental tissue were examined in explants of first trimester placenta that had been incubated for 30 min with [35S]methionine and then incubated for 6 h in medium containing unlabeled methionine. The media and tissue extracts collected at 0, 0.5, 1, 2, 4, and 6 h after the exposure to [35S]methionine were chromatographed on Sephadex G-100 and the amounts of radioimmunoassayable alpha-subunit and immunoprecipitable 35S-labeled alpha-subunit were determined. In tissue extracts, a single form of alpha-subunit was observed at 0 h that had an apparent molecular weight smaller (Ve/Vo = 1.90) than that of a urinary hCG alpha reference preparation (Ve/Vo = 1.81). With chase times of 0.5, 1, 2, and 4 h, a second peak of alpha-subunit was detected that had a larger apparent molecular weight (Ve/Vo = 1.68), and the ratio of large to small forms increased progressively with incubation time. In contrast to that in the extracts, the 35S-labeled alpha-subunit in the culture medium consisted entirely of the large form. Large and small intracellular forms of free alpha-subunit exhibited less than 2% recombination with beta-subunit, as evidenced by gonadotropin receptor binding activity. These studies suggest that normal placental tissue synthesizes a small precursor form of free hCG alpha-subunit that is converted to a larger form prior to secretion and that the free forms of alpha-subunit do not bind to purified hCG beta-subunit.     

Regulation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gene expression by gonadotropin-releasing hormone (GnRH) and sexual steroids in the Mediterranean Sea bass

The secretion of gonadotropins, the key reproductive hormones in vertebrates, is controlled from the brain by the gonadotropin-releasing hormone (GnRH), but also by complex steroid feedback mechanisms. In this study, after the recent cloning of the three gonadotropin subunits of sea bass (Dicentrarchus labrax), we aimed at investigating the effects of GnRH and sexual steroids on pituitary gonadotropin mRNA levels, in this valuable aquaculture fish species. Implantation of sea bass, in the period of sexual resting, for 12 days with estradiol (E2), testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT), almost suppressed basal expression of FSHbeta (four to 15-fold inhibition from control levels), while slightly increasing that of alpha (1.5-fold) and LHbeta (approx. twofold) subunits. Further injection with a GnRH analogue (15 microg/kg BW; [D-Ala6, Pro9-Net]-mGnRH), had no effect on FSHbeta mRNA levels, but stimulated (twofold) pituitary alpha and LHbeta mRNA levels in sham- and T-implanted fish, and slightly in E2- and DHT-implanted fish (approx. 1.5-fold). The GnRHa injection, as expected, elevated plasma LH levels with a parallel decrease on LH pituitary content, with no differences between implanted fish. In conclusion, high circulating steroid levels seems to exert different action on gonadotropin secretion, inhibiting FSH while stimulating LH synthesis. In these experimental conditions, the GnRHa stimulate LH synthesis and release, but have no effect on FSH synthesis.     

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

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.     

Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin alpha-subunit genes.

Primate and equine species are thought to be unique among mammals in synthesizing placental gonadotropin glycoprotein hormones. Human chorionic gonadotropin (CG) and equine pregnant mare's serum gonadotropin (PMSG) are produced in placenta by the specific activation of a glycoprotein hormone alpha-subunit gene and a corresponding beta-subunit gene. The evolutionary mechanisms for the apparently independent acquisition of tissue specificity were investigated by cloning the 5' flanking region of the equine alpha-subunit gene and comparing the DNA elements and trans-acting factors involved in placental expression. We find that though the equine gene is expressed and induced by cAMP, it does not contain the elements known to confer tissue-specific expression to the human gene, the cAMP response element (CRE) and the trophoblast-specific element (TSE), nor does it bind to the trans-acting factors CREB and TSEB. Instead, an additional factor (alpha-ACT) is found which binds to the equine and human, but not the murine, alpha-subunit genes in a region between the positions of the CRE and TSE and confers cAMP responsiveness.