Regulation of nuclear uptake of "activated" receptor-glucocorticoid complex by pyrophosphate

The desialylated human chorionic gonadotropin α- and β-subunits were combined with their native complementary subunits and the thyrotropic activities of the recombinants were compared to those of native and desialylated human chorionic gonadotropin using human thyroid membranes. All the combined forms interacted with the thyrotropin receptor-adenylate cyclase system, but only those with sialic acid residues present on the α-subunit were able to activate the enzyme. These data support the concept that the α-subunit contains the domain through which this hormone activates adenylate cyclase.     

Disulfide bonds Cys9–Cys57, Cys34–Cys88 and Cys38–Cys90 of the β-subunit of human chorionic gonadotropin are crucial for heterodimer formation with the α-subunit: experimental evidence for the conclusions from the crystal structure of hCG

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α- and β-subunits of hCG are highly cross-linked internally by disulfide bonds that seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This paper describes the results of our studies on the role of the disulfide bonds of hCG-β in heterodimer formation with the α-subunit. Six disulfide peptides incorporating each of the six disulfide bonds of hCG-β were screened, along with their linear counterparts, for their ability to competitively inhibit the recombination of α- and β-subunits. The disulfide peptides Cys (9–57), Cys (34–88) and Cys (38–90) were found to inhibit the α/β recombination whereas the remaining three disulfide peptides viz. Cys (23–72), Cys (26–110) and Cys (93–100) did not exhibit any inhibition activity. Interestingly, none of the linear peptides could inhibit the α/β recombination. Results clearly demonstrate that the disulfide bonds Cys⁹–Cys⁵⁷, Cys³⁴–Cys⁸⁸ and Cys³⁸–Cys⁹⁰ of the β-subunit of hCG are crucial for heterodimer formation with the α-subunit thus providing experimental confirmation of the conclusions from the crystal structure of the hormone.     

Tetanus toxin conformation

Summary The circular dichroic spectrum of highly purified tetanus toxin has been determined between 200–310 nm. A comparison of the ellipticity between 207–243 nm and of the rotational strengths of the major resolved bands between 200–250 nm with the corresponding values from proteins of known conformation indicates that tetanus toxin contains about 20% -helix and 23% -structure. Above 250 nm the resolved spectrum showed contributions from tryptophanyl, tyrosyl, and phenylalanyl groups. The rotational strengths of the major near ultraviolet circular dichroic bands were significantly higher in the toxin than in low molecular weight peptides containing aromatic residues. This indicates that tetanus toxin has a stable tertiary structure.Andrew W. Mellon Foundation Teacher-Scholar Awardee.Camille and Henry Dreyfus Foundation Teacher-Scholar Awardee.     

Circular dichroism of human pituitary luteinizing hormone and its glycopeptides. Curve resolution and band assignments to the peptide chromophore, aromatic residues, disulfides, and N-acetylated amino sugars.

The circular dichroic (CS) spectrum of the glycoprotein hormone, human pituitary luteinizing hormone (hLH), has been determined between 195-320 nm and resolved into gaussian constituents. Below 230 nm the CD spectrum is characterized by a negative extremum at 207 nm with a shoulder at 217 nm. Resolution into gaussian constituents of the 200-230 nm CD spectrum resulted in two resolved negative bands, one at 206 nm and the other at 215 nm. The latter band is assigned to beta-structure which is estimated to be about 25%. The 206 nm resolved band is assigned to the N-acetylated carbohydrate groups (e.g. N-acetyl glucosamine, galactosamine, and neuraminic acid). This is based partly on the evidence that the CD spectrum of the hLH glycopeptide fraction (prepared by a pronase digestion of s-carboxymethylated hLH) exhibited a negative extremum at 207.5 nm, which is close to the resolved 206 nm band in hLH. Above 230 nm the CD spectrum is characterized by a negative extremum at about 275 nm. Most of the ellipticity in this region is attributed to the disulfides in hLH. Both strong acid (0.1 N hcl) and concentrated guanidine hydrochloride (4 M) affect the ellipticity in the vicinity of 275 nm, but only the latter (as well as concentrated urea) has a major effect on the CD spectrum below 230 nm indicating extensive conformational changes. There is, however, some loss of beta-structure in 0.1 N hcl. Thus, it appears that the conformation of the hLH subunits in these subunit-dissociating agents is rather different. There was no dramatic change in the magnitude of the 207 nm extremum of native hLH between 10-50C.     

Use of α- and β-subunit specific antibodies in studying interaction of hCG with Leydig cell receptors

Antisera (a/s) raised to individual α- and β-subunits of human chorionic gonadotropin (hCG) have been characterized for specificity using immunoaffinity procedures and used to study the disposition of the two subunits when intact hCG is complexed with luteinizing hormone (LH) receptor of the Leydig cells. Three kinds of experiments were done. (a) The ability of the preformed hormone-antibody (H-Ab) complex to bind to receptor and stimulate a response; (b) the ability of the a/s to dissociate hCG from its complex with the receptor and thereby terminate response; and (c) the ability of the premixed antibody and receptor to compete for binding of labeled hCG. Although the subunit specific a/s used here were equipotent in binding hCG (capacity to bind and K_a being very similar), their behavior once the receptor preparation or Leydig cell is introduced into the system was drastically different. The β-subunit antibody relative to the α-subunit antibody, appeared to be poorly effective in preventing hCG from either binding to the receptor or inhibiting the continuation of response. The results suggest that hCG upon interaction with the receptor loses the determinants specific to the β-region more rapidly compared to those specific to the α-region suggesting thereby that the initial interaction of hCG with the receptor should be occurring through sites in the β-subunit. Although the α-subunit portion of the hCG molecule is available for binding to the antibody for a relatively longer time, the biological response of the cell seems very sensitive to such binding with the antibody as it invariably results in loss of response. In the Leydig cell system, the ability of the a/s to bind hCG that is already complexed to the receptor appears to be dependent upon the time of addition of the antibody to the incubation medium. The antisera were totally ineffective in inhibiting steroidogenic response to hCG if added 60 min after addition of hCG. This would suggest that the hormone-receptor complex once formed perhaps continues to change its orientation with the result that with time relatively less and less of antigenic determinants become available for antibody binding.     

Structural studies of human chorionic gonadotropin and its subunits using tyrosine fluorescence.

The fluorescence properties of the tyrosyl residues of human chorionic gonadotropin (hCG) and its α and β subunits have been examined. The effects of pH, guanidine, and disulfide cleavage on the intensity and polarization of the fluorescence suggest that the isolated subunits possess little, if any, tertiary structure beyond that which is stabilized by the disulfide bonds. Essentially all of the fluorescence of hCG and its subunits was accessible to quenching by iodide ions. Similar results were observed for several other proteins whose fluorescence originates from tyrosyl residues. Thus, we have confirmed and extended the conclusion of R. W. Cowgill ((1966) Biochim. Biophys. Acta120, 196) that the buried tyrosyl residues in ribonuclease fluoresce with a much lower quantum yield than those which are exposed. The dissociation of hCG into its subunits was accompanied by an increase in fluorescence, suggesting the exposure of tyrosyl residues. This was confirmed by difference absorption measurements which indicate a net exposure of two to three tyrosyl residues upon dissociation of the subunits. An additional 0.6 tyrosine was exposed when the disulfide bonds of the β-subunit were cleaved. The polarization of the fluorescence of hCG-β was high (P = 0.19) and, unlike several other proteins with high polarization, could not be lowered by denaturing conditions. Only by cleavage of the disulfide bonds could the fluorescence polarization of either subunit be lowered to a value (P = 0.08) characteristic of a random polypeptide. It appears that the disulfide bonds play an important role in maintaining the rigidity of the fluorescent tyrosyl residues, located at or near the surface of the protein.     

Chemical and biological properties of the subunits of pregnant mare serum gonadotropin

The two subunits (α and β) of pregnant mare serum gonadotropin have been dissociated and partially characterized. Recombination of the biologically inactive subunits results in the restoration of both the follicle stimulating and leuteinizing activities of pregnant mare serum gonadotropin. In addition, the α subunit of pregnant mare serum gonadotropin can be combined with the β subunit of either ovine luteinizing hormone, human chorionic gonadotropin, or follicle stimulating hormone with generation of the specific activity expected of the β subunit.     

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 α-subunits and betwen SU II and β-subunits of mammalian gonadotropins. Moreover SU II was more closely related to the β-subunit of LH than to the β-subunit of FSH.     

Comparison of the effects of human chorionic gonadotropin and luteininzing hormone on phosphofructokinase activity in isolated rat ovaries.

Phosphofructokinase activity in prepubertal rat ovaries is elevated by in vitro treatment with human chorionic gonadotropin or luteinizing hormone. The stimulatory effects of the two gonadotropins are additive. Puromycin and actinomycin D do not affect the enzyme increase induced by human chorionic gonadotropin, but the stimulatory action of luteinizing hormone is completely abolished by these antibiotics. These data suggest that the two hormones have different mechanisms of action and probably occupy different receptor sites on the ovarian cells.     

Tissue specific compartmental analysis of gonadotropin stimulation of ovarian ornithine decarboxylase

Luteinizing hormone is known to stimulate the enzyme ornithine decarboxylase in the ovary. Highly purified human follicle stimulating hormone that is devoid of significant biologically active luteinizing hormone can also induce ornithine decarboxylase activity in intact immature rats with a time course of induction similar to that reported for luteinizing hormone. A maximum of 8–10-fold stimulation above controls was observed 4 h following intravenous administration of human follicle stimulating hormone. This stimulation followed a strict dose response relationship. Ovine luteinizing hormone and human chorionic gonadotropin always induced more ovarian ornithine decarboxylase activity than that achieved by maximally effective doses of follicle stimulating hormone. This could not be attributed solely to the ability of specific cell population to respond to the respective gonadotropins. Although granulosa cells contained little receptor for luteinizing hormone/human chorionic gonadotropin and the residual tissue contained little receptor for follicle stimulating hormone, each tissue responded to these gonadotropins in a manner suggestive of the mediation by one or more diffusable factors. A relationship between gonadotropin induced 3’5’-cyclic adenosine monophosphate (cyclic adenosine monophosphate) concentration and ornithine decarboxylase activity suggests that the mediation of gonadotropin stimulated ovarian ornithine decarboxylase is not solely through cyclic adenosine monophosphate, indicating the presence of other factors in the induction of gonadotropin increased ornithine decarboxylase activity.     

The Pharmacology of the Benzodiazepine Site of the GABA-A Receptor is Dependent on the Type of γ-Subunit Present

Abstract

The pharmacology of native and recombinant GABA-A receptors containing either γ1, γ2 or γ3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individual γ-subunits and analysing their radioligand binding characteristics. Receptors containing a γ1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either a γ2 or γ3 subunit bind [³H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types of α-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of composition α₁β3γ2 followed by immunoprecipitation of individual subunits revealed that the α and γ but not the β-subunit could be irreversibly labelled by [³H]flunitrazepam.

The properties of recombinant receptors have been investigated in oocytes expressing γ1, γ2, or γ3 subunits in combination with an α and a β-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing different γ-subunits. Others such as CL 218,872 show no selectivity between receptors containing different γ-subunits but exhibit selectivity for receptors containing different α-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both the α and γ-subunits.     

Identification of heterotrimeric G proteins in human sperm tail membranes

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein α-subunits, and A 86, which detects all known pertussis toxin-sensitive α-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein α-subunits failed to detect any specific antigens in enriched tail membranes AS 36, recognizing the ã2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa β₁-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive α-subunits. However, membrane preparations of nonpurified human spermatozoa contained α₂ subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein α-subunits; the putative β-subunit was identified as a β₂-subunit. © 1995 Wiley-Liss, Inc.     

Purification,properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of α-subunit (702 amino acid residues) and β-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX α- and β-subunits share as high as 87 and 95% sequence identity with the SNTX α- and β-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX β-subunit with the reported VTX β-subunit is 90%, being lower than that with the SNTX β-subunit.     

Purification of equine gonadotropins and comparative study of their acid-dissociation and receptor-binding specificity

A method for the simultaneous purification of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from equine pituitaries is briefly described. Different forms of each hormone were obtained. The total yield of LH was 24.2 mg·kg^?1 with a recovery of 22% and the yield of FSH was 26 mg·kg^?1 with a recovery of 34%. The specific activities of both hormones, measured in homologous equine radio-receptor assays are equal to or higher than those of the preparations described so far. In all species studied so far the acid-dissociation curves of LH and FSH are similar; this is an agreement with the view that the binding of the common α-subunit and the specific β-subunits involves polypeptide regions which are identical in both hormones. In contrast, the acid-dissociation pK_a of equine LH was found to be considerably lower (3.9) than that of equine FSH (5.8). The equine gonadotropins exhibit a much lower specificity with receptors of a porcine testicular fraction compared with an equine fraction. Equine LH exhibited a binding activity on FSH receptors from a porcine testicular fraction equal to 20% that of equine FSH instead of only 1% for an equine binding fraction. Similarly, all the equine FSH preparations tested exhibited a five-fold higher binding-activity on porcine LH receptors than on equine LH receptors. In the porcine system, pregnant mare serum gonadotropin behaved like equine LH towards LH and FSH receptors. In contrast, on equine binding fraction, pregnant mare serum gonadotropin was only 4% as active as equine LH and was devoid of FSH activity. All the data we have obtained are consistent with the ‘negative specificity’ model we proposed recently.     

Different asparagine-linked sugar chains on the two polypeptide chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) purified from placenta, like urinary hCG, is shown to have the sialylated forms of three neutral oligosaccharides: Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4(Fucα1→6)GlcNAc (N-1), Galβ1→4GlcNAcβ1→2Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-2) and Manα1→6(Galβ1→4GlcNAcβ1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc (N-3). Gel permeation chromatographic analysis of oligosaccharides released from α- and β-subunits of placental hCG has revealed that the α-subunit has one each of sialylated N-2 and N-3, while the β-subunit has one each of sialylated N-1 and N-2.     

Role of PCSK5 expression in mouse ovarian follicle development: identification of the inhibin α- and β-subunits as candidate substrates

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and β(B)-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability.     

Immunological properties of the β-subunit of human chorionic gonadotropin

The β-subunit of human chorionic gonadotropin (hCG-β) has been modified to a varying degree by the cleavage of the disulfide bonds by reduction with dithioerythritol followed by S-carboxamidomethylation. The resulting derivatives of hCG-β show a preferential loss in their immunological cross-reactivity with human luteinizing hormone (hLH). The immunological specificity of the partially reduced and S-alkylated derivatives can be further enhanced by conjugation with tetanous toxoid using glutaraldehyde. Neither conjugation of hCG-β with the toxoid nor its treatment with anti-hLH immunoadsorbent affects its cross-reactivity with hLH.     

Thermodynamic analysis reveals that GTP binding affects the interaction between the alpha- and gamma-subunits of translation initiation factor 2

Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of α-, β-, and γ-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the α- and γ-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The α-subunits bound to the γ-subunit with large heat capacity change (ΔC_p) values. The ΔH and ΔC_p values for the interaction between the α- and γ-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the α-subunit and GTP changes the conformation of the switch region of the γ-subunit and increases the affinity of the γ-subunit for tRNA.     

Circular dichroism of corticotropin, fragment 1-24, and model compounds. An assessment of the contributions of the peptide chromophore and armoatic residues.

The far-ultraviolet circular dichroic spectrum of the 39-residue peptide hormone porcine corticotropin and the biologically active fragment corticotropin 1–24 is negative from 250 nm to 195 nm in water, but in 6M guanidinium chloride a positive band appears at about 225 nm. The temperature and guanidinium chloride dependence of this spectral transition indicates the absence of any stable ordered secondary structure in corticotropin and the spectrum is seen to be in only partial agreement with results using the model peptide chromophore, Ala-Ala-Ala. Using oligopeptides containing aromatic amino acid residues sandwiched between glycyl residues, it is shown that the shape and intensity of the corticotropin 225 nm positive band which appears in 6M guanidinium chloride is in agreement with the far-ultraviolet transitions of the aromatic chromophores in the hormone. Curve resolution of the near-ultraviolet circular dichroic spectrum of corticotropin and comparison of the rotational strengths of the phenylalanyl and tyrosyl bands reveals no evidence for increased rotational freedom in 6M guanidinium hydrochloride. Spectral changes are observed, however, in the transitions arising from the single tryptophan. This study suggests that corticotropin in aqueous solution may serve as a better model for the circular dichroic spectrum of the aperiodic regions in globular proteins than either synthetic homopolypeptides or reference proteins for which spectral and X-ray diffraction data are available.