Internalization of 125I-human choriogonadotropin in bovine luteal slices. A biochemical study

Various intracellular organelles as well as outer cell membranes of bovine corpora lutea intrinsically contain gonadotropin receptors (Rao et al., J biol chem 256 (1981) 2628 [5]). In order to investigate whether exogenously added human choriogonadotropin (hCG) can internalize and bind to the intracellular sites, bovine luteal slices that had been carefully checked with respect to structural and functional integrity were incubated with 0.1 nM 125I-hCG. Following incubation, specific radioactivity was found to be associated with various intracellular organelles, but not with cytosol. The order of radioactivity uptake by subcellular organelles following a 2-h incubation was: Golgi medium greater than Golgi heavy greater than Golgi light greater than plasma membranes = rough endoplasmic reticulum greater than mitochondria-lysosomes- greater than nuclei. The 5'-nucleotidase activity and electron microscopic examination of the fractions revealed that the presence of radioactivity in the intracellular organelles cannot be attributed solely to plasma membrane contamination. The internalization and intracellular binding of 125I-hCG was time and temperature-dependent. Only excess unlabeled hCG and hLH (but not hCG subunits, FSH and PRL) competed with 125I-hCG for internalization in luteal slices. Very little or no 125I-hCG added was internalized in liver or kidney slices; luteal, liver and kidney slices accumulated neither 125I-BSA nor 125I. The radioactivity eluted from various luteal subcellular organelles was able to rebind to fresh corresponding organelles and came off Sepharose 6B columns in a position corresponding to native 125I-hCG. The gel filtration profile of detergent-solubilized radioactivity revealed that 125I-hCG was macromolecular bound. The degraded and altered 125I-hCG was found in the incubation media.     

Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane.

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.     

Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor

The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves' disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains multiple subunits, linked by noncovalent forces.     

Internalization of I-human choriogonadotropin in bovine luteal slices : A biochemical study

Various intracellular organelles as well as outer cell membranes of bovine corpora lutea intrinsically contain gonadotropin receptors (Rao et al., J biol chem 256 (1981) 2628 [5]). In order to investigate whether exogenously added human Choriogonadotropin (hCG) can internalize and bind to the intracellular sites, bovine luteal slices that had been carefully checked with respect to structural and functional integrity were incubated with 0.1 nM ¹²⁵I-hCG. Following incubation, specific radioactivity was found to be associated with various intracellular organelles, but not with cytosol. The order of radioactivity uptake by subcellular organelles following a 2-h incubation was: Golgi medium > Golgi heavy > Golgi light > plasma MEMBRANES = rough endoplasmic reticulum > mitochondria-lysosomes> nuclei. The 5′-nucleotidase activity and electron microscopic examination of the fractions revealed that the presence of radioactivity in the intracellular organelles cannot be attributed solely to plasma membrane contamination.The internalization and intracellular binding of ¹²⁵I-hCG was time and temperature-dependent. Only excess unlabeled hCG and hLH (but not hCG subunits, FSH and PRL) competed with ¹²⁵I-hCG for internalization in luteal slices. Very little or no ¹²⁵I-hCG added was internalized in liver or kidney slices; luteal, liver and kidney slices accumulated neither ¹²⁵I-BSA nor ¹²⁵I.The radioactivity eluted from various luteal subcellular organelles was able to rebind to fresh corresponding organelles and came off Sepharose 6B columns in a position corresponding to native ¹²⁵I-hCG. The gel filtration profile of detergent-solubilized radioactivity revealed that ¹²⁵I-hCG was macromolecular bound. The degraded and altered ¹²⁵I-hCG was found in the incubation media.     

Covalent Cross-Linking of Radiolabeled Human Chorionic Gonadotropin to Rat Ovarian Luteinizing Hormone Receptor with Glutaraldehyde

Abstract

Crude plasma membranes of pseudopregnant rat ovaries were incubated with ¹²⁵I-labeled human chorionic gonadotropin (¹²⁵I-hCG) and the formed luteinizing hormone (LH)/hCG receptor-¹²⁵I-hCG complex was solubilized, partially purified by Sepharose 6B gel filtration, cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. An apparent molecular weight (mol wt) of 130,000 was obtained for the non-reduced complex. A similar-sized complex was observed, when ³H-hCG (β-subunit labeled) instead of ¹²⁵I-hCG (α-subunit labeled) was used, indicating that the complex contains intact hCG. Upon reduction of the cross-linked receptor-¹²⁵I-hCG complex, a 105,000 mol wt complex in addition to the 130,000 one was observed. No smaller complexes appeared, however, upon reduction of the receptor-³H-hCG complex, suggesting that the α-subunit of hCG predominantly interacts with the receptor. The cross-linking efficiency was dependent on protein concentration, glutaraldehyde concentration, pH, reaction time and temperature. Under optimal conditions (2 mM glutaraldehyde, pH 7.0-8.0, 60 min, 20°C) no nonspecific complexes appeared and the efficiency of cross-linking of the partially purified detergent-solubilized receptor-¹²⁵I-hCG complex was approximately 30%. Glutaraldehyde thus provides a rapid and efficient cross-linking reagent to covalently attach ¹²⁵I-hCG to its receptor and is likely to be highly applicaple to other receptor-ligand systems as well.     

Isolation of plasma membranes from bovine corpus luteum possessing adenylate cyclase, 125I-hCG binding and NaK-ATPase activities

Plasma membranes from bovine corpora lutea have been purified by sucrose density gradient centrifugation. The purified membranes, in addition to binding ¹²⁵I-hCG, also possess hCG-stimulated adenylate cyclase and NaK-ATPase. The relative purification of ¹²⁵I-hCG binding, adenylate cyclase and NaK-ATPase on the basis of the specific activities in the whole homogenate were 7.8, 6.4 and 2.6, respectively. The presence of both the hormone sensitive adenylate cyclase and ¹²⁵I-hCG binding activities suggest that these plasma membranes might possess the ‘receptor’ for gonadotropin.     

Characterization of the subunit structure of gonadotropin receptor in luteinized rat ovary

Gonadotropin receptors with specificity, high affinity and low capacity for luteinizing hormone and human chorionic gonadotropin (hCG) have been identified in rat luteal cells. To investigate the nature of the receptor, we have employed disuccinimidyl suberate, a cross-linker noncleavable by reducing agents, and dithiobis(succinimidyl propionate), a cleavable cross-linker, to covalently cross-link the 125I-hCG . receptor complex. The molecular weight of 125I-hCG-linked receptor complex and the receptor subunit structure were determined by electrophoresis in either 10 or 4.5% acrylamide in the presence of 0.1% sodium dodecyl sulfate with or without reducing agents. Autoradiographic analysis of the 125I-hCG-linked receptor separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing condition revealed a single labeled band corresponding to Mr = 305,000 +/- 15,000. However, electrophoresis performed in the presence of 50 mM dithiothreitol and 2% beta-mercaptoethanol resulted in the appearance of four labeled bands corresponding to Mr = 105,000 +/- 4,000, 96,000 +/- 5,000, 74,000 +/- 4,000, and 62,000 +/- 4,000 concomitant with the loss of the labeled band in the Mr = 305,000 region. Further experiments demonstrated that these four labeled bands were derived from the same molecular species. In addition, the 125I-hCG-linked receptor in the absence of reducing agent was not dissociated into subunits even by treatment with strong denaturing agent (8 M urea). The appearance of the cross-linked 125I-hCG . receptor was effectively inhibited by the unlabeled beta-subunit of hCG, intact hCG, and luteinizing hormone and partially inhibited by the alpha-subunit of hCG but not by choleratoxin, gonadotropin-releasing hormone, insulin or bovine serum albumin. These data suggest that 1) the hCG/luteinizing hormone receptor is an oligomeric complex linked by disulfide bonds and 2) that under reducing conditions, the oligomeric receptor dissociates into four nonidentical subunits.     

Purification and characterization of Leydig cell luteinizing hormone receptor

We have purified the testicular luteinizing hormone (LH/human choriogonadotropin (hCG)) receptor by sequential affinity chromatography on hCG-Sepharose. The purified LH/hCG receptor was identified as a single protein of Mr = 90,000 +/- 2,000 on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), showed high affinity binding for hCG, and a binding capacity of 3.8 nmol/mg of protein. Electrophoretically blotted receptor retained the ability to bind 125I-hCG on nitrocellulose membrane, and the Mr of radioactive band was consistent with that revealed by silver staining. Autoradiography after SDS-PAGE analysis of cross-linked purified receptor-hCG complex showed Mr = 145,000 and Mr = 105,000 bands. These results are consistent with a Mr value for the receptor of 90,000 after accounting for contribution by the intact hormone or its alpha-subunit. Analysis of the free receptor by fast protein liquid chromatography on Superose 12 revealed a single peak of binding activity for 125I-hCG which eluted in the position of Mr = 200,000-240,000 in the presence of Triton X-100. Since a single protein species is observed under reducing or nonreducing conditions in SDS-PAGE, the receptor could exist in the membrane as a dimeric form composed of subunits Mr = 90,000 associated through noncovalent interactions. The pure receptor can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (approximately 0.3 mol of phosphate/mol of receptor). This phosphorylation does not affect the binding characteristics of the receptor. The method described is simple and allows rapid purification of microgram amounts of biological active Leydig cell LH/hCG receptor for structural, functional, and immunological studies.     

Evidence that dissociation, not intracellular degradation, is the major pathway for removal of receptor-bound 125I-human chorionic gonadotropin in cultured rat luteal cells

The nature of the labeled products released by cultured rat luteal cells pulse-labeled with 125I-human chorionic gonadotropin (hCG) was examined. After pulse labeling in a 3-h incubation, the cells containing receptor-bound 125I-hCG were incubated in fresh medium in the absence of 125I-hCG up to 48 h. The medium was collected at different time intervals and analyzed to determine the extent of degradation of 125I-hCG. The amounts of radioactivity remaining associated with the cells at these time intervals were also determined. Most of the released radioactivity could be precipitated with 10% trichloracetic acid and was identical in molecular weight to intact 125I-hCG as determined by gel filtration chromatography. After 20 h of reincubation, only less than 50% of the initially bound hormone remained on the cells. At this time point the cells were capable of rebinding 125I-hCG at levels comparable to the original when incubated with a fresh dose of the labeled hormone. The rebinding ability was not a result of de novo receptor synthesis since cycloheximide had no effect on this process. The results indicate that dissociation is the major pathway for release of hCG bound to cultured rat luteal cells and that receptors become functional again after dissociation of the hormone by a cycloheximide-independent process.     

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.     

Composition and peptide maps of cross-linked human choriogonadotropin-receptor complexes on porcine granulosa cells

Radioiodinated human choriogonadotropin was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to the hormone receptor on porcine granulosa cells and the solubilized sample was electrophoresed. Cross-linked samples revealed four additional bands of slower electrophoretic mobility in addition to the hormone alpha, beta, and alpha beta dimer bands. The four bands corresponded to masses of 68, 74, 102, and 136 kDa whereas the alpha beta dimer band corresponded to 50 kDa. Formation of the four bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding can be prevented by excess of native hormone but not by follitropin. A monofunctional analog of the cross-linking reagent failed to produce the four bands. They were also produced by cross-linking Triton X-100-solubilized hormone-receptor complexes. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of the cross-linked sample were treated with reagents that cleave covalent cross-links and then electrophoresed in a second dimension gel, 18-, 24-, 28-, and 34-kDa components were released, in addition to the alpha and beta subunits of the native hormone. Simultaneous peptide mapping of the cross-linked complexes in the gel matrix with Staphylococcus V8 protease or papain revealed progressive proteolysis to generate terminal fragments of 30 or 27 kDa, respectively. These fragments were unique to and commonly present in the 74-, 102-, and 136-kDa hormone-receptor complexes but were not produced by proteolysis of the cross-linked human choriogonadotropin (hCG) alpha beta dimer or the hCG alpha subunit. Apparently, the radioactively labeled segment(s) of the alpha subunit of 125I-hCG was cross-linked to the 24-kDa component. The results demonstrate the protein nature of the receptor and suggest that 125I-hCG was initially cross-linked to the 24-kDa component to generate the 74-kDa complex, then the 28- and 34-kDa components were sequentially cross-linked to the 24-kDa component in the 74-kDa complex to generate the 102- and 134-kDa complexes.     

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.     

Evidence that the subunit structure of gonadotropin receptor is preserved during regression of rat corpus luteum

The level of hCG/LH receptor has been shown to undergo marked changes during the life span of rat corpus luteum. To evaluate whether these fluctuations are due to changes in the receptor subunit structure or receptor protein content, the 125I-hCG binding activity and the receptor subunit structure were determined during different time periods of pseudopregnancy. The maximum 125I-hCG binding activity was observed on day 7, after which it decreased by 20 and 45% on day 11 and day 14, respectively. The Scatchard analysis of 125I-hCG binding data showed that the decrease in binding activity was caused by a change in the number of binding sites rather than a change in the binding affinity. The LH/hCG receptor in ovarian membranes obtained on days 7, 11 and 14 were then characterized by the method of affinity cross-linking. All four subunits of the LH/hCG receptor were detected in the ovarian membranes at all stages while the intensity decreased parallel to a decrease in hCG binding from day 7 to day 14. These results suggest that the decrease in 125I-hCG binding activity in rat ovarian membranes from day 7 to day 14 of pseudopregnancy is due to a decrease in receptor concentration rather than a change in the receptor subunit structure.     

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.     

Gastric inhibitory polypeptide receptor in hamster pancreatic beta cells. Direct cross-linking, solubilization and characterization as a glycoprotein

125I-labelled gastric inhibitory polypeptide (125I-GIP) is directly cross-linked to its specific receptor in hamster pancreatic beta cell membranes by using an ultraviolet irradiation procedure. This approach results in the identification of a GIP-protein complex of apparent Mr 64,000. The labelling of this protein species is specific since it is inhibited when incubating the membranes with increasing doses of native GIP (0.1 nM-1 microM) together with 125I-GIP, half-maximal inhibition being elicited by 5 nM peptide. Reduction of the GIP-protein complex by 100 mM dithiothreitol induces a decrease of the electrophoretic mobility of the complex. Alternatively pretreatment of membranes with dithiothreitol (up to 1 M) does not prevent the binding of 125I-GIP to its receptor. When prelabelled membranes are extracted by 0.5% Triton X-100 (v/v) and the extract is layered on a Sephadex G-50 column, a high peak of radioactivity is eluted with the void volume of the column. Treatment of this peak by 10 min ultraviolet irradiation followed by SDS-PAGE leads to identification of a major band of Mr 64,000. When the peak is further layered on Sephacryl S-200 it yields a single peak of radioactivity corresponding to a protein species with a Stokes radius of 3.2 nm and an apparent Mr of 65,000. The solubilized GIP-receptor complex is specifically adsorbed by Sepharose coupled to wheat germ agglutinin and concanavalin A and eluted from these lectins by their respective sugars. In conclusion the GIP receptor in pancreatic beta cells is a protein monomer of apparent Mr 59 000; its structure is maintained by intrachain disulfide bridges, these bonds being, however, not involved in the interaction of GIP with its receptor; the GIP receptor is a glycoprotein containing N-acetylglucosamine, mannose and probably sialic acid in its carbohydrate moiety.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with ¹²⁵I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of ¹²⁵I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed ¹²⁵I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Photoaffinity labeling of the canine renal receptor for parathyroid hormone

Studies were undertaken to identify and characterize components of the parathyroid hormone receptor. An analog of the bovine parathyroid hormone(1-34) sequence was derivatized at the 23-tryptophan position with the photoreactive reagent 2-nitro-5-azidophenylsulfenyl chloride. 2-Nitro-5-azidophenylsulfenyl-bovine parathyroid hormone (NAPS-bPTH) analog retained full biological activity with respect to receptor binding and activation of adenylate cyclase in canine renal cortical plasma membranes. 125I-bPTH(1-34) analog was also derivatized without loss of receptor-binding activity. When 125I-NAPS-bPTH(1-34) analog was incubated with canine renal plasma membranes and then photolyzed, at least two 125I-labeled membrane components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation of 125I radioactivity into one of these components (Mr congruent to 60,000) was inhibited when incubation and photolysis were performed in the presence of excess, unlabeled bPTH(1-34). Furthermore, photolysis of membranes in the presence of NAPS-bPTH(1-34) analog led to activation of adenylate cyclase which persisted following washing to remove noncovalently bound peptide, suggesting a functional, covalent hormone-receptor complex had been formed. We conclude that the M r congruent to 60,000 membrane component may be a part of the renal receptor for parathyroid hormone.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum.

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 X g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI. U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Structure and properties of the cellular receptor for transforming growth factor type beta

Swiss 3T3 cells respond to picomolar concentrations of type beta transforming growth factor (TGF-beta) with a dose-dependent increase in the formation of colonies in soft agar, a decrease in the growth of cells in monolayer culture, and changes in morphology. This indicates that these cells have functional TGF-beta receptors able to mediate a biological response. Binding analysis revealed a single class of TGF-beta binding sites (80 000 per cell) with a Kd approximately 50 pM. Receptors were affinity-labeled by covalent attachment to 125I-TGF-beta with bis(sulfosuccinimidyl) suberate (BS3). The complexes formed were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 100 mM dithiothreitol and migrated as Mr approximately 180 000 complexes in 3-10% linear gradient gels. The apparent size of these complexes was larger in gels with a higher percentage of acrylamide. The labeling of the 125I-TGF-beta-receptor complexes was inhibited by the presence of excess unlabeled TGF-beta but was unaffected by other growth factors. These complexes could be formed by cross-linking whole cells, intact membranes, or solubilized membranes, demonstrating that the TGF-beta receptor is located on the plasma membrane and can be solubilized without destruction of its ability to bind TGF-beta. A larger Mr approximately 360 000 complex was present in 3-10% linear gradient gels without reduction or after extensive cross-linking, suggesting that the receptor consists of two subunits of similar size attached by disulfide bonds. Since BS3 is membrane-impermeable, at least a portion of both subunits is located on the outer surface of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)