Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.

We have shown that osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that approximately 1.4 microg OP-1/ml is available in the circulation 1 min after intravenous administration of 250 microg/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered 10 min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion. Histochemical and molecular analyses demonstrate that OP-1: (a) minimizes infarction and cell necrosis, and decreases the number of plugged tubules; (b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation and activity of neutrophils; (c) maintains the expression of the vascular smooth muscle cell phenotype in pericellular capillaries; and (d) reduces programmed cell death during the recovery. Collectively, these data suggest that OP-1 prevents the loss of kidney function associated with ischemic injury and may provide a basis for the treatment of acute renal failure.

Structure--function relationships of the glycoprotein hormones and their receptors.

The primary structures of the glycoprotein hormones follitropin (FSH), lutropin (LH), human choriogonadotropin (hCG) and thyrotropin (TSH) have been determined, hCG has been crystallized and initial diffraction data obtained. Studies with synthetic peptides have provided information on regions involved in receptor interaction and signal transduction. The receptors for the glycoprotein hormones have been prepared by gene cloning methods and their primary structures deduced. As Leo Reichert and colleagues discuss here, although cAMP is involved in glycoprotein hormone signal transduction, recent evidence also implicates other second messengers, especially Ca2+ and may include both the phosphatidylinositol pathway and activation of Ca2+ channels.

Regulation of follicle-stimulating hormone binding to receptors on bovine calf testis membranes by cholera toxin-sensitive guanine nucleotide binding protein.

In a previous study we reported that FSH receptors in bovine testes membranes are physically and functionally associated with a guanine nucleotide-binding protein (N protein). In this study we examined the mechanism whereby GTP binding to N protein regulates FSH binding to its receptors. Binding of FSH to receptors decreased in the presence of GTP in a dose-dependent and noncompetitive manner. This effect did not require the presence of Mg+2 and is in contrast to the reported requirement for Mg+2 for GTP effects on human CG binding to ovarian receptors. Equilibrium binding experiments indicated that decreased hormone binding in the presence of GTP was not due to a decrease in the number of FSH receptors per se; rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH. Moreover, the dissociation of [125I]human FSH from preformed FSH-receptor complex was rapid in onset and significantly accelerated in the presence of GTP. In a series of nucleotides, GTP was most effective in causing this effect. Evidently, occupancy of GTP binding sites on the N protein, including low affinity and high capacity sites, is necessary for GTP regulation of FSH binding to receptors. The fact that pretreatment of bovine testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminates the GTP effect on FSH binding to its receptors suggests that the GTP regulatory binding protein mediating the GTP regulation of FSH binding is probably Ns and not Ni. Further characterization of FSH receptor sensitivity to GTP, however, indicated that the N protein involved does not exhibit all of the characteristics reported for Ns. For example, the affinity of GTP for N protein is relatively low even under conditions where GTP hydrolysis has a minimal effect in reducing the total concentration of GTP. Also, the absence of a requirement for Mg+2 in high affinity FSH receptor-N protein coupling is different from the requirement for Mg+2 seen with the beta-adrenergic receptor and Ns. Moreover, the N protein which mediates GTP regulation of FSH-receptor binding appears to be relatively insensitive to N-ethylmaleimide, unlike the N-ethylmaleimide sensitivity of the turkey erythrocyte Ns. These results suggest that differences may exist in the structure-function features of GTP regulatory binding protein associated with different types of hormone ligands and receptors.

Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes.

An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.

Functional properties of polyclonal antibodies raised against the N-terminus region (residues 9-30) of the follicle-stimulating hormone (FSH) receptor: significance of this receptor region in FSH recognition and signal transduction.

In this study, we raised polyclonal antibodies in rabbits against a synthetic peptide corresponding to a unique region of the FSH receptor, residues 9-30, with no sequence homology to receptors for LH and TSH, and examined their characteristics relevant to receptor function. Binding of [125I]human (h) FSH to membrane-bound receptors was inhibited in a concentration-dependent manner by the anti-FSH receptor-(9-30) peptide antibody. Preimmune serum had no effect. Lineweaver-Burke plot analysis of [125I]hFSH binding to membrane receptors in the presence or absence of antireceptor peptide antibody indicated that the antibody effectively competed with FSH at a hormone-binding site on the receptor. Also, antireceptor peptide antibody, but not preimmune serum, inhibited the ability of FSH to stimulate the conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulation of estradiol synthesis by Sertoli cells caused by cholera toxin or forskolin (which are known to act through the Gs-protein and catalytic unit of adenylate cyclase, respectively) was not inhibited by antireceptor peptide antibody. Indirect immunofluorescence staining of cultured rat Sertoli cells showed binding of antibody to plasma membrane receptor. No fluorescent staining of receptor was observed when cells were incubated with preimmune serum or antireceptor peptide antibody in the presence of excess receptor-(9-30) peptide or hFSH. These results were consistent with specific labeling of membrane-bound FSH receptors by anti-receptor-(9-30) peptide antibody. When detergent-solubilized membrane preparations from rat Sertoli cells were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and then subjected to Western blot analysis, antireceptor peptide antibody, but not preimmune rabbit serum, specifically recognized intact FSH receptor. Although the antireceptor peptide antibody occupied the N-terminus 9-30 epitope region in the FSH receptor, it did not induce postbinding events, such as receptor patching (aggregation), as shown by indirect immunofluorescence staining of rat Sertoli cells and the estradiol response. In contrast, a polyclonal antibody against the FSH holoreceptor capable of interacting with multiple epitopes on the receptor could induce FSH-like effects, such as receptor patching and estradiol response in Sertoli cells. In conclusion, antibody raised against the N-terminus region (9-30) of the FSH receptor recognized intact FSH receptor, inhibited FSH binding, and behaved as an antagonist, suggesting that this N-terminus epitope region of the receptor is involved in hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrate moiety of follitropin receptor is not required for high affinity hormone-binding or for functional coupling between receptor and guanine nucleotide-binding protein in bovine calf testis membranes.

Previously, we have extensively studied FSH-receptor interactions using bovine calf testis membranes, and demonstrated that the high-affinity FSH binding to receptors and coupling of FSH receptors with guanine nucleotide-binding protein (Gs protein) in a GTP-sensitive state are important initial events in FSH action. In this study, using the same plasma membrane system, we examined the glycoprotein nature of the FSH receptor and determined the contribution of carbohydrate moieties to these functions of the FSH receptor. Our approach involved enzymic deglycosylation of FSH receptors present in calf testis plasma membranes and then removal of incompletely deglycosylated FSH receptors by lectin affinity chromatography. Following treatment of testis membranes with peptide N-glycosidase, the receptor, as identified by ligand-blot analysis, had a higher electrophoretic mobility indicating a decrease in M(r) from 240-200K. Treatment of testis membranes with neuraminidase caused a reduction (to approximately 225K) in the size of the receptor consistent with desialylation. However, digestion with O-glycosidase (endo-alpha-N-acetylgalactosaminidase) did not affect the mobility of the FSH receptor. These results suggest that bovine testis FSH receptor contains predominantly N-linked oligosaccharide chains, a finding which is consistent with recent predictions that N-linked glycosylation, but not O-linked glycosylation sites are present in cloned FSH receptor from rat testis. Moreover, calf testis membranes after treatment with peptide N-glycosidase F, were solubilized with Triton X-100 under optimum conditions that preserve the physical and functional coupling of FSH receptors with guanine nucleotide-binding protein, and then subjected to lectin affinity chromatography. Scatchard analysis indicated that intact and deglycosylated FSH receptors bound 125I-human FSH with similar affinities. In the presence of GTP, the binding of 125I-human FSH to intact and deglycosylated receptors decreased similarly and in a noncompetitive manner. Treatment of testis membranes with NAD plus cholera toxin, but not NAD plus pertussis toxin, eliminated the GTP effect on FSH binding to enzymic deglycosylated as well as intact receptors, suggesting that the guanine nucleotide binding protein mediating GTP regulation of FSH binding in these membranes is probably Gs protein. Our results suggest that the bovine testis FSH receptor contains predominantly N-linked oligosaccharide chains consistent with recently predicted N-linked glycosylation sites of cloned FSH receptor of rat testis. The bovine testis FSH receptor does not require N-linked carbohydrate for high-affinity hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential roles of high and low affinity guanosine 5'-triphosphate binding sites in the regulation of follicle-stimulating hormone binding to receptor and signal transduction in bovine calf testis membranes.

We have previously shown that FSH receptors are physically and functionally associated with a guanine nucleotide regulatory protein (Gs) in membranes of calf testis. Using N-ethylmaleimide (NEM), forskolin, and cholera toxin as probes, we have investigated the role of low and high affinity GTP-binding sites of stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs) in the activation of adenylate cyclase. When calf testis membranes were exposed to NEM (1 mM), FSH binding to receptors was slightly (30%) decreased, but the receptors showed continued sensitivity to GTP, resulting in a further decrease in [125I]human FSH binding to receptors. Pretreatment of membranes with NEM (up to 20 microM) produced no effect on GTP-binding. A dose-dependent decrease in high affinity GTP-binding sites, however, was observed at higher (greater than 50 microM) NEM. Adenylate cyclase activity was reduced in response to GTP gamma S or NaF concomitant to a decrease in high affinity GTP-binding sites in membranes treated with 50-100 microM NEM, or completely abolished in membranes exposed to 300 microM NEM. Stimulation by forskolin indicated that the significant inhibition of adenylate cyclase activity occurring in membranes exposed to low NEM (50-100 microM) was not due to inactivation of catalytic unit of adenylate cyclase by NEM. Pretreatment of membranes with 100 micrograms/ml cholera toxin and NAD slightly (18%) reduced specific FSH binding but did not affect Gpp(NH)p-binding. However, adenylate cyclase stimulation by GTP plus FSH in these membranes was significantly enhanced. When membranes were treated with higher concentration of cholera toxin (250 micrograms/ml), the adenylate cyclase stimulation by GTP plus FSH was abolished due to uncoupling of FSH receptors from Gs and a significant decrease in high affinity GTP-binding sites. Our results suggest that high affinity GTP-binding sites of Gs coupled to FSH receptors are essential for FSH and guanine nucleotide activation of adenylate cyclase. The low affinity binding sites bind GTP and thereby regulate FSH binding but are not involved in the activation of adenylate cyclase.

Polyclonal antibodies against follitropin (FSH) receptor interfere with hormone binding, but mimic the effects of FSH.

We have raised polyclonal antibodies in rabbits against the FSH receptor, purified from calf testis and isolated the IgG fraction from the immune serum (immune IgG) by protein A affinity chromatography. When the immune IgG was incubated with purified, radioiodinated FSH receptor, the resulting complex could be immunoprecipitated by goat anti-rabbit gamma globulin. The immunoprecipitate, after dissociation of receptor from antibody, separation by SDS-PAGE under reducing conditions, and autoradiography, showed the presence of a approximately 60 kDa protein previously identified as a component of the FSH receptor. Binding of 125I-hFSH to membrane-bound receptors was inhibited in a concentration-dependent manner by immune IgG (Ed50 = 90 micrograms/ml). Nonimmune serum or IgM/IgA fractions from immune serum had no effect. 125I-labeled immune IgG bound specifically to testis membranes and the binding could be inhibited in a concentration-dependent manner by ovine FSH. These results suggest that the FSH-binding site and the antibody-binding site on the receptor are proximate or identical. Immune IgG mimicked the ability of FSH to stimulate basal adenylate cyclase activity and conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulatory but submaximal effects of FSH were augmented by immune IgG. Rat Sertoli cells treated with IgG fractions from immune serum showed an intense fluorescent staining of plasma membrane receptor. No fluorescent staining of receptor was seen when preimmune IgG was used or in the presence of excess ovine FSH. These observations suggest that the polyclonal receptor antibody capable of recognizing FSH receptor behaved as an FSH binding competitor, but was also active as an agonist producing the biological effect of FSH in vitro. The effectiveness of antibodies against FSH receptor in stimulating estradiol synthesis suggests that the information needed for FSH signal transduction resides in the membrane receptor rather than in the hormone molecule. Such antibodies may offer a useful probe for further study of FSH receptor structure and mechanism of hormone action.

Transglutaminase activity in bovine calf testicular membranes: evidence for a possible role in the interaction of follicle-stimulating hormone with its receptor.

Transglutaminase (TGase) activity, determined by incorporation of [1,4-14C]putrescine into casein, was demonstrated in a light membrane fraction derived from bovine calf testicular homogenates. In common with other TGases thus far identified, testicular TGase is calcium dependent. A concentration-dependent relationship was observed between decreasing TGase activity and increasing concentrations of N-ethylmaleimide and bacitracin, inhibitors of TGase, and the TGase substrate monodansylcadaverine. Although NEM at all concentrations tested had no effect on [125I]human (h) FSH-receptor binding, dissociation of [125I]hFSH from its receptor was enhanced when [125I]hFSH-receptor complexes were formed in the presence of NEM. Dissociation of [125I]hFSH from its receptor also increased in the presence of bacitracin or monodansylcadaverine. Reduced TGase activity always paralleled increases in hormone-receptor dissociation. The inverse relationship between TGase activity and [125I]hFSH-receptor dissociation observed in this study suggests that TGase activity may be involved in the interaction of FSH with its receptor and that protein cross-linking (via peptide bond formation) may be a mechanism whereby some FSH-receptor complexes are stabilized in the bovine testis.

Nature of cross-reaction between hCG and anti-oLH serum and development of a radioimmunoassay to measure hLH specifically in the presence of hCG.

Immunological cross-reaction between hCG and anti-oLH sera has been demonstrated using radioimmunoassay techniques. The results indicate that this cross-reaction is incomplete and that the anti-oLH sera used have the ability to distinguish between LH and hCG. Following absorption with purified hCG, anti-oLH serum was used to develop a heterologous radioimmunoassay "[125I]iodo-hLH + anti-oLH serum" (H-O, RIA) which specifically and selectively measures hLH in serum samples containing both hLH and hCG. In this radioimmunoassay hCG and subunits of hCG do not cross-react with hLH, in the range in which these hormones are present in human serum under physiological conditions. Other hormones such as hPL, hPRL, hGH, hFSH, hTSH, and GnRH do not interfere with the measurement of LH by radioimmunoassay. The sensitivity of the assay was 1.5 mIU (25 ng) per ml (LER 907 standard), and the inter- and intra-assay coefficients of variations for samples were 10.83% and 8.4%, respectively. The recoveries of hLH added to pregnancy serum containing an hCG concentration of 8.55 IU/ml were in the range 95-108%. Determination of LH content of human pituitary extracts by H-O RIA gave values which were in close agreement with those derived by bioassay (indices of discrimination 0.72-1.12). Serum LH patterns in women during normal menstrual cycles as well as in amenorrheic patients who received GnRH treatment are comparable to those reported by other investigators using other radioimmunoassay systems. Serum samples obtained during the first trimester of pregnancy, when analyzed by H-O RIA, showed basal LH levels.

Identification in human ovarian follicular fluid of proteins that share an epitope region unique to the extracellular domain of the follicle-stimulating hormone receptor.

Recently we identified a unique region, residues 9-30 in the extracellular domain of the FSH receptor, capable of binding FSH but not LH or TSH. We have shown that polyclonal antibodies raised against this region specifically recognized intact FSH receptors present on plasma membranes of cultured rat Sertoli cells. In the present study, plasma membranes from human granulosa-lutein cells were solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by Western blot analysis. Antireceptor peptide antibody, but not preimmune serum control, recognized intact human FSH receptors, suggesting that human and rat FSH receptors share this unique N-terminus epitope region. Recent cloning studies have identified, in addition to full length receptor, the presence of FSH receptor-spliced messenger RNA variants, which encode receptor proteins with variable lengths of hydrophilic extracellular domains, but lacking transmembrane domains. Such proteins could theoretically represent secreted forms of the receptor. In this study, we used a polyclonal anti-FSH receptor (residues 9-30) peptide antibody to investigate whether FSH receptor-related soluble proteins might also be present in human ovarian follicular fluid (FF). In an enzyme-linked immunosorbent assay specific for the FSH receptor, antireceptor peptide antibody, but not preimmune serum serving as control, identified significant immunoreactivity in several human ovarian FF samples, suggesting that protein(s) present in FF share a common epitope with the extracellular domain of the FSH receptor. The apparent levels of FSH receptor-related activity in FF samples, expressed relative to the FSH receptor (residues 9-30) peptide, ranged from 31-55 ng/mL. Passing of the samples through 0.22-micron filters or subjecting the samples to high speed centrifugation did not alter the activity profiles of the samples ruling out effects due to contamination with plasma membranes from granulosa cells. When human FF samples were subjected to gel permeation chromatography, at least four distinct protein peaks were resolved, in the molecular mass range between 70-460 kilodaltons, each of which was recognized by the FSH receptor 9-30 peptide antibodies. Our results provide initial evidence for the presence in human ovarian FF of proteins sharing epitope with the extracellular domain of the FSH receptor and presumably derived from the granulosa cell. Since we have previously shown that the epitope region, represented by residues 9-30 in the extracellular domain of the FSH receptor specifically binds FSH, the proteins in human FF sharing this epitope may have functional significance.

Purification of a high molecular weight follicle-stimulating hormone receptor-binding inhibitor from human follicular fluid.

In a previous study we reported the presence in human follicular fluid (hFF) of a FSH receptor-binding inhibitor (hFSH-BI) with FSH agonist activity, which was immunologically similar to FSH but could be distinguished from FSH on the basis of its greater stability in acid. We have now purified hFF-derived hFSH-BI after molecular sieving on Sephracyl S-100 ion exchange chromatography using Diethyl-aminoethyl-cellulose followed by polyacrylamide gel electrophoresis (PAGE). The purified hFSH-BI had a potency approximately 12,000-fold greater than that of dialyzed hFF, based on its ability to inhibit the binding of [125I]hFSH to its membrane receptor. The purified hFSH-BI also had FSH agonist activity, stimulating estradiol synthesis in cultured rat Sertoli cells. Upon sodium dodecyl sulfate (SDS)-PAGE, hFSH-BI migrated as two bands of almost identical mobility, with an estimated mol wt of 57,000, compared with 30,000 for pituitary FSH run simultaneously. A monoclonal antibody to hFSH that also recognizes hFSH-BI was used for Western blot analysis of the SDS-PAGE fraction. The Western blot confirmed the detection of two bands with very similar mobilities and estimated mol wt of 57,000, which were clearly distinguishable from that of immunologically reactive hFSH run in parallel. The hFSH-BI bands showed similar profiles upon cyanogen bromide cleavage and had indistinguishable amino acid compositions. The amino acid composition of hFSH-BI was clearly distinct from those of hFSH, hLH, hCG, and the alpha-subunit of human inhibin. Our studies confirm the presence in hFF of a unique agonist protein which is of potential importance in the regulation of gonadal function.

The size of the mature membrane receptor for follicle-stimulating hormone is larger than that predicted from its cDNA.

A 240 kDa protein isolated from bovine calf testis has been shown to have properties characteristic of an FSH receptor. However, rat testis FSH receptor has, on the basis of cloning experiments, been found to have a much lower molecular mass of 75 kDa (peptide only). To examine this point, the size of the FSH receptor in membranes obtained from cultured Sertoli cells of immature rats was determined after polyacrylamide gel electrophoresis under non-reducing conditions, followed by transfer to polyvinylidene difluoride membranes and direct identification of the FSH receptor by ligand blot analysis utilizing radioiodinated human FSH. In this system, the rat Sertoli cell membrane FSH receptor also showed a molecular mass of 240 kDa. Bovine testis contains LH and FSH receptors. We compared the sizes of FSH and LH receptors present in the same bovine testis membrane preparation by ligand blot analysis. The FSH receptor again showed a molecular mass of 240 kDa, whereas the LH receptor showed a molecular mass of 90 kDa. The latter value is similar to that deduced by cloning techniques (75 kDa, peptide only). The evidence seems to suggest that, whereas the molecular mass deduced for the LH receptor on the basis of its cDNA is similar to that of the mature membrane receptor, the size of the FSH membrane receptor is considerably different from that deduced on the basis of its cDNA, presumably as a result of post-translational processing. The marked difference in size between mature FSH (240 kDa) and LH (90 kDa) receptors may reflect significant structural differences of importance with regard to mechanisms of signal transduction.

Identification of regions of the follitropin (FSH) beta-subunit that interact with the N-terminus region (residues 9-30) of the FSH receptor.

We have recently identified a region, N-terminus residues 9-30, in the extracellular domain of the follicle-stimulating hormone (FSH) receptor capable of binding FSH, but not luteinizing hormone (LH) or thyroid-stimulating hormone (FSH) (Dattatreyamurty and Reichert (1992) Mol. Cell. Endocrinol. 87, 9-17). The objectives of the present study were to examine the interaction between a synthetic peptide corresponding to this receptor sequence and the beta-subunit of FSH, and to identify which FSH-beta regions are involved in the interaction. FSH-beta subunit and synthetic FSH-beta peptides 1-15, 71-85 and 101-111 effectively bound 125I-labeled FSH rec-(9-30) peptide, and binding was inhibited by excess unlabeled FSH receptors. Scatchard analysis indicated that the synthetic FSH-beta peptides had affinities for FSH rec-(9-30) peptide in the order of 10(6) M-1 (Ka), with the sum of individual peptide affinities (Ka = 1.21 x 10(7) M-1) closely approximating that of the intact beta-subunit (1.02 x 10(7) M-1). Polyclonal antibodies raised against FSH rec-(9-30) peptide completely inhibited the binding of 125I-labeled receptor peptide to hFSH, hFSH-beta, and hFSH-beta peptides 1-15, 71-85 and 101-111. Our results indicate that recognition of FSH-beta by N-terminus region (9-30) of the FSH receptor involves contact with residues in three discontinuous binding regions on FSH-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic peptide corresponding to amino acids 9-30 of the extracellular domain of the follitropin (FSH) receptor specifically binds FSH.

As deduced on the basis of cloning experiments, the putative extracellular domain of pituitary glycoprotein hormone (lutropin (LH), thyrotropin (TSH), and FSH) receptors (rec) is sufficiently large to suggest involvement in hormone binding. Comparison of the amino acid sequences of the extracellular domains of the glycoprotein hormone receptors indicates that the FSH receptor has a peptide sequence in the external domain close to the amino terminus (residues 9-30) which has no sequence homology to receptors for LH or TSH. To examine whether this region is involved in FSH-receptor interaction, we studied the hormone-binding properties of a corresponding synthetic peptide in several systems. (1) Binding of 125I-hFSH to receptor-containing bovine testis membranes was inhibited by preincubation with FSH rec-(9-30) peptide amide in a concentration-dependent manner. (2) 125I-labeled rec-(9-30) peptide amide bound to ovine, bovine, or human FSH preparations, and the binding was inhibited by solubilized bovine FSH receptor. 125I-labeled rec-(9-30) peptide amide, however, did not bind to LH or TSH. (3) 125I-hFSH bound to unlabeled rec-(9-30) peptide amide, and the binding was inhibited by excess unlabeled FSH, but not by LH or TSH. (4) Scatchard analysis indicated that the FSH rec-(9-30) peptide amide contained a single class of FSH binding sites with a Ka = 1.1 x 10(6) M-1. (5) The binding of 125I-labeled rec-(9-30) peptide amide to hFSH, bFSH or oFSH was effectively inhibited by rabbit polyclonal antibodies raised against rec-(9-30) peptide amide but not by preimmune rabbit serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of elevated circulating levels of LH during the pre-menstrual period in some copper IUD users and the possibility of their confusion with hCG.

A heterologous radioimmunoassay (RIA) capable of discriminating between LH and hCG was used to measure LH levels in sera obtained during the pre- and post-menstrual periods from 80 women bearing copper intrauterine contraceptive devices (IUD). hCG levels in these samples were also estimated by use of a homologous beta-hCG radioimmunoassay and a radioligand-receptor assay. Only two IUD users during the pre-menstrual period had detectable, but low hCG levels. However, LH levels, as estimated by specific RIA, in the serum of these two women were elevated to a level that would cause detection at low levels in the hCG assays. The data thus provide direct evidence to indicate that the positive hCG levels observed by others during the pre-menstrual period in some copper IUD users could be due to the interference in hCG assays by elevated LH.