Purification and partial characterization of rat ovarian lutropin receptor

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea and mouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.     

Asparagine-linked oligosaccharides on formyl peptide chemotactic receptors of human phagocytic cells

Formyl peptide chemotactic receptors affinity-labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (where Nle represents norleucine) and ethylene glycol bis(succinimidyl succinate) consist of two isoelectric forms with cell type differences in both apparent size and charge (neutrophils: 55-70 kDa, pI 5.8, and 6.2.; monocytes: 60-75 kDa, pI 5.6 and 6.0; differentiated HL-60 cells: 62-85 kDa, pI 5.6 and 6.0). Endo-beta-N-acetylglucosaminidase F (endo F) cleavage of N-linked oligosaccharides from formyl peptide receptor generates 40-50- and 33-kDa products that can be affinity-labeled. Whereas both pI forms of this receptor from neutrophils are cleaved by endo F to 33-kDa final products, this cleavage does not eliminate pI differences. Tunicamycin decreases expression of formyl peptide receptor on differentiating HL-60 and causes a dose-dependent decrease in size of the major product seen after affinity labeling (0.5 micrograms/ml: 38-48 kDa; 2 micrograms/ml: 32 kDa). Thus, the formyl peptide receptor polypeptide backbone from all three cell types contains at least two N-linked oligosaccharide side chains which contribute to the cell type differences in Mr and are not required for ligand binding. Papain treatment of intact cells generates a membrane-bound formyl peptide receptor fragment that can be affinity-labeled and is of similar size (29-31 kDa) in all three cell types. Endo F treatment of the affinity-labeled papain fragment of formyl peptide receptor does not alter its size, suggesting that this fragment does not contain the N-linked oligosaccharide cleaved by endo F from intact receptor. The results indicate that at least two N-linked oligosaccharide chains are located on the distal 1-3-kDa portion of the receptor polypeptide backbone.     

The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets.

Rat ovarian luteinizing hormone/human choriogonadotropin binding sites were labelled with 125I-choriogonadotropin in vivo, and the resulting 125I-choriogonadotropin-receptor complexes were solubilized by Triton X-100 and purified by use of antibodies to choriogonadotropin immobilized to agarose. The purified 125I-choriogonadotropin-receptor complex was treated with glutaraldehyde to crosslink radiolabelled hormone to the receptor. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the crosslinked product revealed a labelled Mr 130 000 major band in addition to the hormone and its alpha-subunit, indicating that a single receptor component was linked to the hormone. Unoccupied binding sites for luteinizing hormone were also solubilized by Triton X-100 from pseudopregnant rat ovaries, and attached to choriogonadotropin-agarose. The agarose gel was washed, and eluted with 0.1 M-sodium acetate, pH 4. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the pH 4 eluate revealed an Mr 90 000 major band which was abolished when ovaries presaturated with choriogonadotropin were used as starting material. These observations suggest that the hormone-binding component of the luteinizing hormone receptor is a polypeptide of Mr 90 000. This polypeptide was isolated and labelled with Na 125I. The labelled polypeptide showed a single band on sucrose density gradient centrifugation and on gel filtration on agarose.     

Human chorionic gonadotropin. V. Tissue specificity of binding and partial characterization of soluble human chorionic gonadotropin-receptor complexes.

An in vivo human chorionic gonadotropin (hCG)-receptor complex was solubilized from the subcellular fraction of ovarian and testicular tissues of rats that had been injected with 125-I-labeled hCG. The soluble hCG-receptor complex was partially characterized by Sepharose 6B chromatography in the presence of the nonionic detergent, Emulphogene, and was shown to have a molecular size of about 65 A. By this method it was also shown that the in vivo uptake of radioactivity by rat gonadal tissues represents 125-I-hCG and not the dissociated subunits or degradation products of the hormone. A soluble hCG-receptor complex isolated in vitro in approximately the same yield from both rat testicular and ovarian homogenates was shown to be the same size. The hCG-receptor appears to be specifically located in gonadal tissue; a corresponding hCG-receptor complex was not obtained from liver or kidney that incorporated significant levels of 125-I-hCG administered in vivo. Furthermore, a desialyzed hCG-receptor complex was obtained from rat testis but not liver; desialyzed hCG, like other desialyzed glycoproteins, is nonspecifically bound by rat liver homogenates. The binding of hCG and luteinizing hormone (LH) by rat testis receptor exhibits a high degree of specificity. Other glycoprotein hormones without LH activity, such as follicle-stimulating hormone and thyroid-stimulating hormone, and glycoproteins such as fetuin or alpha1-acid glycoprotein do not bind to the hCG/LH receptors. Desialyzed hCG was 2 times more effective in competing for binding to rat testis receptors than "native" hCG, indicating that caution must be exercised when the radioligand receptor assay is utilized to assay hCG preparations varying in sialic acid content.     

Sheep testicular gonadotropin binding sites: characterization and changes with surgical shortening of the scrotum

Gonadotropin receptors in previously frozen (-70 degrees C) sheep testicular tissue were characterized, and methods of assessment of receptor binding activity were established and applied to an investigation of testicular function in the short scrotum ram. Binding of 125I-labelled ovine luteinizing hormone (125I-oLH) and 125I-labelled ovine follicle-stimulating hormone (125I-oFSH) to testicular membranes was highly specific and saturable. Uptake of labelled gonadotropins was proportional to the amount of membrane protein, with 125I-oFSH showing greater specific binding. Initial association of 125I-oLH with binding sites was comparable at 4, 25, and 34 degrees C; with prolonged incubation, maximal binding occurred at 4 degrees C. Equilibrium was achieved in 8 h at 34 degrees C and in 16 h at 25 and 4 degrees C. In contrast, the temperature-dependent association of LH with rat testicular membranes was greater at 25 than at 4 degrees C. The rate of association of 125I-oFSH to binding sites was proportional to incubation temperature, with equilibrium being achieved in 2 h at 34 degrees C and in 16 h at 25 degrees C; binding at 4 degrees C; was slow and still increasing by 48 h. Binding of radioactive and nonradioactive oLH and oFSH was hormone specific and increased in a dose-dependent manner until saturation occurred. Shortening the scrotum of adult rams led to reductions (p less than 0.05) in testicular weight (60%) and in the number of LH (55%) and FSH (90%) binding sites per testis, with no apparent change in serum testosterone concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of specific FSH binding sites in the testes of adult monkeys of different species

The interaction of 125I-labelled hFSH with primate testicular tissue from 4 species of adult monkeys (Macaca mulatta, M. nemestrina, M. fascicularis and Papio cynocephalus) was investigated. 125I-labelled hFSH binding to a particulate fraction (P1, 40 000 g) of frozen testes was highly specific and saturable. Displacement curves generated using the P1 fraction of testes from the 4 species and 125I-labelled hFSH and unlabelled FSH were very similar. The binding of FSH to the monkey testicular receptor was not species specific because purified FSH from heterologous species such as horse, sheep, pig and rat were very effective in competing with 125I-labelled hFSH for binding. The equine FSH was about 10 times more active than hFSH in this respect. Similarly, 125I-labelled ovine FSH bound as well as labelled hFSH to the testes fractions of all 4 monkey species. In marked contrast to the high binding of 125I-labelled hFSH, binding of 125I-labelled hCG with rhesus monkey testis homogenates and P1 fractions was very low. The FSH receptor in the adult rhesus monkey testis was present in much larger quantity than the LH receptor and was more readily detectable. Our studies show that frozen primate testis can be utilized for investigating testicular-FSH interactions.     

The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common alpha and a hormone specific beta subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the alpha and beta subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (alpha, FSHbeta and hCGbeta) asparagine-linked (N-linked) oligosaccharides. CGbeta subunit is distinguished among the beta subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure-function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

Evidence for a cryptic lectin site in the cell-binding domain of plasma fibronectin

Various proteolytic fragments from the central region of the fibronectin subunit chains containing the main cell-affinity site were applied in cell binding studies using peritoneal macrophages of guinea pigs. A 125I-labelled 23-kDa peptide was relatively well bound by the cells. Attachment to cells was partially inhibited by wheat germ lectin, suggesting a lectin-like site in the cell-binding domain which recognizes oligosaccharide groups with terminal N-acetylglucosamine or N-acetylneuraminic acid. Binding was inhibited by N-acetylneuraminic acid with half-maximal effect at 2 X 10(-3) M. Other inhibitors were a sialic acid rich ganglioside preparation and fetuin, a sialic acid-containing glycoprotein. In contrast to the 23-kDa peptide a 125I-labelled 125-kDa fragment was only weakly bound, although it included the sequence of the 23-kDa peptide on its C-terminus. The residual binding was weakly inhibited by low concentrations of wheat germ lectin and was remarkably improved by higher concentrations. The behavior of the peptide was explained by the presence of a sialic acid-containing oligosaccharide side chain localized outside of the 23-kDa region and interacting with the lectin-like site in the cell-binding sequence. In accord with this suggestion a 95-kDa fragment representing the oligosaccharide-containing part of the 125-kDa peptide was capable of inhibiting at least partially the cell attachment of the 23-kDa piece. The results indicate a lectin-like affinity site in the cell-binding region of fibronectin which is accessible in the 23-kDa peptide, but is masked in the 125-kDa fragment and in fibronectin by a sialic acid-containing oligosaccharide moiety.     

Gonadotropin receptor of a mouse luteoma: interactions with luteinizing hormone (LH) and its and subunits

A radioligand-receptor system for luteinizing hormone (LH), USING transplantable mouse luteoma, was used to investigate the interactions of LH, other peptide hormones, and LH subunits. Since tumor size decreased as did production of androgenic hormones following hypophysectomy, the luteoma is believed to have been dependent on pituitary tropic hormones; posthypophysectomy histologic changes supported this conclusion. An homogenate was prepared from 1-4 gm luteomas, which had been borne by mice for 4-10 months. Ovine LH, bovine LH, and human chorionic gonadotrophin reduced the binding of iodine-125 human luteinizing hormone (125-I-hLH). Growth hormone, adrenocorticotrophic hormone, and prolactin had no capacity to interfere with binding of 125-I-hLH. Though follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) reduced the binding somewhat, the reductions were consistent with the known presence of contaminating amounts of LH in the FSH and TSH. The accumulated results of a number of experiments suggest that binding to the luteoma LH receptor requires a particular polypeptide structural conformation, one found in the native hormone but found in neither alpha nor beta subunit alone.     

Purification, characterization, and amino-terminal sequence of rat ovarian receptor for luteinizing hormone/human choriogonadotropin

The luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor of rat ovary was solubilized with Lubrol PX in the presence of 20% glycerol and protease inhibitors, and purified by one-step affinity chromatography. Purified receptor had a specific hCG binding capacity of 4900 pmol/mg protein, and displayed a single class of high affinity binding sites (Ka = 6.20 X 10(9) M-1). An 11,200-fold purification over the starting crude homogenate was achieved. The purified LH/hCG receptor was identified by sodium dodecyl sulfate-gel electrophoresis and silver staining as a single protein of 92 kDa. The ability of the purified 92-kDa protein to specifically bind hormone was demonstrated by electroblotting onto Immobilon P membrane, incubation with 125I-labeled hCG, and autoradiography of the blot. In addition to a 92-kDa band, ligand blotting also yielded a 170-kDa band representing receptor dimer. Covalent cross-linking of hCG, with isotope in either the alpha- or beta-subunit, to membrane-bound receptor produced complexes that contained a single receptor component of approximately 92 kDa. The cross-linking studies indicated that both subunits interact with receptor and also suggested receptor dimer formation. Following sodium dodecyl sulfate-electrophoresis, purified receptor was electroblotted onto polyethylenimine-treated glass fiber filters for direct microsequencing in a gas-phase sequenator. Eleven cycles of sequence analysis yielded the unique sequence: NH2-Arg-Glu-Leu-Ser-Gly-Ser-Leu-XXX-Pro-Glu-Pro-COOH. These results indicate that the rat ovarian LH/hCG receptor is a protein of 92 kDa which can be easily purified in microgram amounts. This study also describes a relatively simple technique for electroblotting and microsequencing that should be applicable to other membrane-bound hormone receptors.     

Receptor- and non-receptor-mediated uptake and degradation of insulin by hepatocytes

Native insulin inhibits the binding and degradation of ¹²⁵I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of ¹²⁵I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of ¹²⁵I-labelled insulin (1nm) with the insulin receptor. In contrast, 20μm-glucagon inhibits 75% of the uptake of insulin (0.1μm) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37°C. During dissociation, fragments of ¹²⁵I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione–insulin transhydrogenase, with which both insulin and glucagon interact.     

Purification and the effect of peptide N-glycosidase F on lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts.

Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme.     

Retention of glucose on oligosaccharide chains linked to the LH/hCG receptor prevents cell surface expression

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [(125)I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.     

The vasoactive intestinal peptide receptor on intact human colonic adenocarcinoma cells (HT29-D4). Evidence for its glycoprotein nature.

We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.     

A quantitative analysis of glucagon binding to isolated intact neonatal and adult rat hepatocytes on the basis of two different binding models

The interaction of glucagon with specific receptors has been studied in isolated intact neonatal and adult rat hepatocytes. The hormone binding measured directly with ¹²⁵I-labelled glucagon was saturable and reversible. The ¹²⁵I-labelled glucagon binding was inhibited by unlabelled homologous hormone at concentrations ranging from 0.5 nM to 50 μM. Two different binding models were assumed to analyse the binding data by a nonlinear least-squares procedure: (I) a single class of independent sites and (II) two classes of independent sites. The comparison of the fitted theoretical curves reveals that both binding models are in fact compatible with these data. Adult hepatocytes have a considerably higher affinity for glucagon than neonatal hepatocytes; the binding capacity of neonatal liver cells from 1–7-days-old rats proved to be markedly reduced compared with the cells from adult rats. The glucagon-induced intracellular cyclic AMP production was measured at various hormone concentrations under conditions identical to those for the determination of extracellular hormone binding. The correlation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation. These results suggest that a decrease receptor concentration in neonatal hepatocytes is responsible for the decreased cyclic AMP production.     

The role of O-linked and N-linked oligosaccharides on the structure–function of glycoprotein hormones: Development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common α and a hormone specific β subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the α and β subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (α, FSHβ and hCGβ) asparagine-linked (N-linked) oligosaccharides. CGβ subunit is distinguished among the β subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure–function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Y2 receptor proteins for peptide YY and neuropeptide Y. Characterization as N-linked complex glycoproteins.

Affinity labeling using [125I-Tyr36]PYY and homobifunctional affinity crosslinking reagents of the rabbit Y2 receptor for peptide YY(PYY) results in specifically labeled proteins of both M(r) = 50,000 to 60,000 and M(r) = 96,000 to 115,000 [1,2]. In this work the glycoprotein nature of affinity labeled Y2 receptor proteins were investigated by enzymatic deglycosylation using neuraminidase, endoglycosidase F (endo F), N-glycosidase F (PNGase F), and O-glycanase treatment. Only N-glycosidase F and neuraminidase increased the electrophoretic mobility of the radiolabeled receptor bands, whereas all other glycosidases did not. PNGase F treatment of both radiolabeled receptor bands electroeluted from gel slices reduced the apparent molecular mass of by 16-17 kDa units, that is M(r) = 96,000 to 79,000 and M(r) = 60,000 to 44,000, indicating removal of N-linked oligosaccharide chains of similar size from both species. Neuraminidase treatment caused slight increases in the electrophoretic mobilities suggesting the presence of terminal sialic residues. It is concluded that the Y2 binding proteins are N-linked complex (sialo)glycoproteins with a minimal core protein size of M(r) = 44,000. Furthermore, based on this sensitivity pattern of the glycosidases, the Asn-linked carbohydrate may be of the tri- or tetra-antennary complex type containing terminal sialic acid residues.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormone and labelled antibody binding techniques.

The parathyrin receptor in renal cortex has been investigated by studying the binding of 125I-labelled parathyrin, or of unlabelled parathyrin detected with 125I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 degrees C but this phenomenon was not detectable at 37 degrees C. Specific displacement of lactoperoxidase labelled 125I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was 2.3-10(8) M(-1) and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0-7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1-34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glucagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.     

Human chorionic gonadotropin, its receptor and mechanism of action.

Human chorionic gonadotropin (HCG) is a glycoprotein hormone consisting of two noncovalently bonded subunits, alpha and beta. The hormone can be dissociated and reassociated. Whereas the individual subunits do not show any receptor binding activity, the reconstituted molecule is almost fully active. The amino acid and carbohydrate sequences in hCG-alpha and hCG-beta are described. There are in all seven carbohydrate units, four complex asparagine-linked and three serine-linked short oligosaccharide chains. The sequential removal of monosaccharides from the carbohydrate moiety of the hormone results in derivatives that bind to the cell surface receptors but inhibit the hCG-induced accumulation of cAMP. The derivatives, however, still are able to produce steroidogenesis maximally. The data raise the possibility of other mediator(s) of the hormone action in addition to cAMP. The hCG/LH (luteinizing hormone) receptor has been labeled by the incorporation of N-acetyl-D-1-[14C] glucosamine and also by the selective incorporation of 125I or 131I. Using 131I-labeled bovine corpus luteal plasma membranes, a method for the purification of the receptor to homogeneity has been developed. The purified receptor has properties similar to the membrane-bound receptor. Availability of the purified receptor offers newer approaches to the study of molecular mechanisms of polypeptide hormone action.