Multimeric structure of the tumor necrosis factor receptor of HeLa cells

The tumor necrosis factor (TNF) receptor of HeLa cells was solubilized in Triton X-100 and characterized by gel filtration, affinity labeling, and ligand blotting studies. Receptors solubilized with Triton X-100 eluted in gel filtration as a major peak of Mr = 330,000 and retained high affinity binding (KD = 0.25 nM). Affinity labeling of soluble receptor/125I-TNF complexes using the reversible, bifunctional bis[2-(succinimidooxycarbonyl-oxy)ethyl] sulfone resulted in the formation of cross-linked species of Mr = 310,000, 150,000-175,000, 95,000, and 75,000. The formation of these complexes was competitively inhibited by unlabeled TNF. Partial reversal of cross-linking in these complexes and their analysis by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 125I-TNF dimers cleaved from the 95,000 band and 125I-TNF monomer cleaved from the 75,000 band, providing evidence for a Mr approximately 60,000 subunit. In addition, the 95,000 and 75,000 bands were resolved as components of larger complexes (Mr = 150,000-175,000), which presumably contain two receptor subunits. The Mr 95,000 and 75,000 bands were also released from the Mr 310,000 complex by reduction with dithiothreitol, suggesting a role for disulfide bond stabilization. To investigate the association of the putative receptor subunits, Triton X-100 extracts from HeLa membranes were fractionated by SDS-PAGE without reduction and transferred electrophoretically to nylon membranes for TNF binding assays. Only two bands of Mr = 60,000 and 70,000 specifically bound TNF, and higher Mr binding activity was not observed. These results indicate that TNF receptors in HeLa cells are high molecular weight complexes containing Mr = 60,000 and 70,000 subunits each capable of binding TNF and that the complexes are primarily stabilized by non-covalent, hydrophobic interactions.     

Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors

A method was developed to label epidermal growth factor (EGF) receptors with 125I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an Mr approximately 180,000 EGF-receptor complex and larger Mr greater than or equal to 360,000 aggregates. The formation of the larger complexes was time and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of 125I-EGF-labeled high- (Kd approximately 0.16 nM) and low- (Kd approximately 1.5 nM) affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the Mr approximately 180,000 125I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant Mr approximately 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the Mr approximately 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S3 cell membranes at 4 degrees C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.     

A binding assay for the solubilized receptors of type beta transforming growth factor: adsorption and removal of free ligand by dextran-coated charcoal

A binding assay was developed for the measurement of solubilized receptors for transforming growth factor type beta (TGF-beta). Solubilized receptors were incubated with 125I-TGF-beta, then the unbound ligand was removed by adsorption to dextran-coated charcoal. The binding of TGF-beta to solubilized receptors was saturable and specific, and increased in a linear manner with respect to the amount of membrane protein present. Crosslinking of radioactive complexes after adsorptive removal of unbound TGF-beta yielded complexes similar to affinity-labeled TGF-beta receptors from whole cells. Treatment of a 20% charcoal suspension with 0.2-0.4% dextran was optimal for the protection of receptors from adsorption to charcoal while allowing free TGF-beta to be removed; Mr approximately 250,000 dextran was most effective. This method can assay receptors from purified membranes and crude extracts of cells and tissues, and was used to demonstrate that TGF-beta receptors are glycosylated and retain a high affinity (Kd approximately 530 pM) for ligand after solubilization.     

Delta 6- and delta 12-desaturase activities and phosphatidic acid formation in microsomal preparations from the developing cotyledons of common borage (Borago officinalis).

The somatostatin receptors on rat pancreatic acinar membranes were demonstrated by use of a radioiodinated (125I-) analogue of somatostatin (SMS 204-090 or [Tyr3]SMS). The tracer was found to bind to the receptor with a Kd of 58 pM. The number of sites detected by this tracer (4.7 pmol/mg of protein) was 5-10 times higher than the number of sites previously found with other tracers. Since the level of non-specific binding was also very low as compared with findings with other tracers, 125I-204-090 might be of interest in future attempts to characterize the somatostatin receptors in the pancreas. The prelabelled membranes were solubilized with 1% CHAPS, and the solubilized complexes were found to adsorb to wheat-germ-agglutinin-coupled agarose, from which they could be eluted with 4 mM-triacetylchitotriose. The complexes within this eluate were shown by gel filtration on Trisacryl GF-2000 to have an Mr of about 400,000. The dissociation of the complexes was augmented both within the membranes as well as in the solubilized state by incubation with the GTP analogue guanosine 5'-[gamma-thio]triphosphate, indicating that the complexes are probably functionally linked to a guanine-nucleotide-binding regulatory protein. After SDS/slab-gel electrophoresis and autoradiography of cross-linked complexes after treatment with the heterobifunctional reagent N-5-azido-2-nitrobenzoyloxysuccinimide, a broad band occurred at approximately Mr 90,000 both in the membranes and in the eluates of complexes after lectin-adsorption chromatography. We conclude that the augmentation of the number of detectable sites for binding of somatostatin, as well as the very low level of non-specific binding obtained by the use of 125I-[Tyr3]SMS as tracer, has made it possible for us to demonstrate the solubilization of the somatostatin receptor in conjunction with its ligand and a GTP-binding regulatory protein, and we have succeeded in cross-linking 125I-[Tyr3]SMS to a binding subunit of Mr 90,000 in the membranes and in demonstrating the presence of the same labelled binding subunit within complexes solubilized and chromatographed on a lectin column before cross-linking.     

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.     

Evidence that non-covalent forces, thiol and disulphide groups affect the structure and binding properties of the prolactin receptor on hepatocytes from pregnant rats.

Incubation of hepatocytes from pregnant rats with dithiothreitol decreased specific 125I-prolactin (125I-prl) binding to such cells by about 20% relative to control. This was not due to a non-specific effect of dithiothreitol on the cell membrane, since reduction also altered the binding of prl to solubilized partially purified receptor. Exposure of hepatocytes to N-ethylmaleimide (6 mM) for periods as brief as 1 min decreased the subsequent specific binding of 125I-prl by more than 50%. N-Ethylmaleimide was less effective as an inhibitor of binding when applied after hepatocytes had been exposed to 125I-prl, binding being decreased by about 15%. Scatchard analysis demonstrated that the effect of N-ethylmaleimide resulted from loss of receptor-binding capacity without any substantial effect on the affinity of the prl receptor for hormone. Dithiothreitol diminished the affinity of lactogenic sites for prolactin without altering cellular binding capacity. These observations suggest that thiol and disulphide groups are present in the prl receptor and that these functional moieties regulate the formation and properties of prl receptor complexes. The species to which 125I-prl had bound were identified by affinity labelling. 125I-prl was covalently coupled into saturable complexes of Mr 65000 and 50000. 125I-human growth hormone (125I-hGH) was covalently incorporated into complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine growth hormone (bGH), but not prl, competed for 125I-hGH uptake into the 300 000-, 220 000- and 130 000-Mr complexes, indicating that these species were somatogenic. Prl, but not bGH, inhibited 125I-hGH uptake into 65 000- and 50 000-Mr complexes. This demonstrated that 125I-hGH in the presence of bGH could affinity-label lactogenic receptors. 125I-prl aggregates in Triton X-100, whereas 125I-hGH does not. Therefore lactogenic complexes to which 125I-hGH was bound in the presence of excess bGH were solubilized in Triton X-100 and characterized sequentially by gel filtration and affinity labelling. Prl receptors were eluted from columns of Sepharose 6B as a species of Mr380 000. Fractionation of the 380 000-Mr species on sodium dodecyl sulphate polyacrylamide gels resulted in the isolation of complexes of Mr 65 000 and 50 000. Thus non-covalent forces stabilize aggregates of the monomeric prolactin receptor.     

Isolation of a gastrin releasing peptide receptor from normal rat pancreas.

The presence of a putative GRP receptor on rat pancreatic particulate membranes was demonstrated by covalent cross-linking to 125I-gastrin releasing peptide (GRP), which revealed a radioactive band with Mr = 80-90 kDa on reduced SDS-PAGE. Fresh rat pancreatic membranes contained a GRP receptor which was solubilized with Triton X-100 as assessed by its failure to sediment at 100,000 x g for one hour and its ability to pass through a 0.22 mu filter. When 125I-GRP binding was studied using Sephadex G50 gel filtration chromatography to separate bound from unbound ligand, substantial amounts of 125I-GRP binding were observed in rat crude solubilized pancreatic membranes, but essentially no specific binding was observed until the crude solubilized membranes were fractionated by ammonium sulfate precipitation. Specific 125I-GRP binding was 500, 700 and 1400 fmol/mg protein, respectively, in the 0-25%, 25-50% and 50-80% saturated ammonium sulfate fractions (125I-GRP concentration = 1 nM). Specific binding was temperature dependent, saturable and of high affinity, (KD = 2.3 nM). A unique 70 kDa band was visualized by silver staining of the SDS-PAGE of eluates of GRP(14-27) affinity gel compared with eluates of control affinity gels incubated with the 25-50% (NH4)2SO4 fraction. The lower Mr than that observed with covalent cross-linking may represent the binding subunit of a larger receptor protein. This ligand-affinity isolated protein is thus a good candidate for the GRP receptor, or the binding subunit of it, from normal rat pancreas.     

Crosslinking of HCG to its specific receptor of porcine ovarian membranes

HCG receptors of porcine ovarian plasma membranes were loaded with 125I-hCG and covalently crosslinked with glutaraldehyde. The plasma membranes were labeled with tritium. The hCG receptor complex was solubilized in 1% SDS and subsequently chromatographed on Biogel P 300. 40-50% of 125I-radioactivity eluted near the void volume. 3H radioactivity in these fractions indicated the presence of crosslinked hormone receptor complexes. A second minor peak of 125I-radioactivity appeared at 55000 d. A third peak represented subunits of 125I-hCG. Analysis of the Biogel P 300 purified 125I-hCG receptor complex by polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in two radioactivity peaks. The main peak corresponded to 90 000-100 000 d, the second peak was calculated to approximately 140 000 d. As a result the majority of the hormone receptor complex has a molecular weight of 90 000-100 000 d. Existence of subunits is discussed.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% beta-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 microM unlabeled insulin, but not by 1 microM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Functional and immunological evidence for stable association of solubilized vasoactive-intestinal-peptide receptor and stimulatory guanine-nucleotide-binding protein from rat liver

We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP-sensitive form of Mr 150,000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14482-14489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide-binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48,000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I-VIP-receptor complexes solubilized in Triton X-100 were adsorbed on WGA-Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I-VIP and (b) alpha s (Mr 42,000) and beta (Mr 35,000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA-Sepharose. When Triton-solubilized 125I-VIP-receptor complexes were adsorbed on WGA-Sepharose, then retained material was specifically eluted with 0.3 M N-acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I-VIP-receptor complexes of Mr 150,000 contained in the eluate indicating that 125I-VIP-receptor-G protein complexes had been adsorbed to the WGA column. (b) The Mr-42,000 alpha s subunit can be specifically ADP-ribosylated by cholera toxin. (c) Immunoblotting using antisera against the alpha s and beta subunits of Gs protein, reveals Mr-42,000 and Mr-35,000 components corresponding to alpha s and beta subunits, respectively. (d) Affinity cross-linking using dithiobis(succinimidyl-propionate) of 125-I-VIP-receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150,000. Immunoblotting using antisera against the beta-subunit shows the presence of the beta subunit (Mr 35,000) in this Mr-150,000 component. In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP-receptor complexes with alpha s and beta subunits of Gs protein.     

Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K+ channel subunits and target size analysis

Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.     

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently cross-link to galactosyl receptors on isolated rat hepatocytes

We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.     

Identification and localization of cholecystokinin-binding sites on rat pancreatic plasma membranes and acinar cells: a biochemical and autoradiographic study

Using the combined approaches of affinity labeling and light and electron microscopic autoradiography, we investigated the identification and localization of cholecystokinin (CCK)-binding sites on rat pancreatic acinar cells. To define the molecular properties of the CCK-binding site, we incubated rat pancreatic plasma membranes with 125-I-CCK-33 for 15 min at 23 degrees C followed by washing and cross- linking with disuccinimidyl suberate. Specific labeling of a major Mr 85,000 component was revealed as assessed by SDS PAGE under reducing conditions and autoradiography of the dried gels. Components of Mr greater than 200,000, Mr 130,000-140,000, and, Mr 55,000 were labeled under maximal cross-linking conditions. The labeling of all components was specifically inhibited by CCK-8 in a dose-dependent manner (Kd approximately 9 nM). The Mr 85,000 component had identical electrophoretic mobilities under reducing and nonreducing conditions indicating that it likely does not contain intramolecular disulfide bonds. The larger labeled species may be cross-linked oligomers of this binding protein or complexes between it and neighboring polypeptides. For studies on the distribution of CCK-binding sites, pancreatic acini were incubated with 125I-CCK-33 (0.1 nM) in the absence or presence of CCK-8 (1 microM) for 2 or 15 min at 37 degrees C, washed, and fixed in 2% glutaraldehyde. Quantitative autoradiographic analysis indicated that approximately 60% of the total grains were located within +/- 1 HD (1 HD = 100 nm) of the lateral and basal plasmalemma with little or no labeling of the apical plasmalemma. From these data, it was estimated that each acinar cell possesses at least 5,000-10,000 CCK-binding sites on its basolateral plasmalemma. The remaining grains showed no preferential concentration over the cytoplasm or nucleus. Together, these data indicate that CCK interacts with a Mr 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell.     

A transforming growth factor beta (TGF-beta) receptor from human placenta exhibits a greater affinity for TGF-beta 2 than for TGF-beta 1

Affinity-labeling techniques have been used to identify three types of high-affinity receptors for transforming growth factor beta (TGF-beta) on the surface of many cells in culture. Here we demonstrate that membrane preparations from tissue sources may also be used as an alternative system for studying the binding properties of TGF-beta receptors. Using a chemical cross-linking technique with 125I-TGF-beta 1 and 125I-TGF-beta 2 and bis(sulfosuccinimidyl)suberate (BS3), we have identified and characterized two high-affinity binding components in membrane preparations derived from human term placenta. The larger species, which migrates as a diffuse band of molecular mass 250-350 kDa on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, is characteristic of the TGF-beta receptor type III, a proteoglycan containing glycosaminoglycan (GAG) chains of chondroitin and heparan sulfate. The smaller species of molecular mass 140 kDa was identified as the core glycoprotein of this type III receptor by using the techniques of enzymatic deglycosylation and peptide mapping. Competition experiments, using 125I-TGF-beta 1 or 125I-TGF-beta 2 and varying amounts of competing unlabeled TGF-beta 1 or TGF-beta 2, revealed that both the placental type III proteoglycan and its core glycoprotein belong to a novel class of type III receptors that exhibit a greater affinity for TGF-beta 2 than for TGF-beta 1. This preferential binding of TGF-beta 2 to placental type III receptors suggests differential roles for TGF-beta 2 and TGF-beta 1 in placental function.     

The subunit structure of the follitropin receptor. Chemical cross-linking of the solubilized follitropin-receptor complex

Homobifunctional cross-linkers were utilized to characterize high affinity (Ka = 2.2 X 10(-10) M-1) follitropin (FSH) receptors in immature bovine testis. Following the formation of radioiodinated human FSH (125I-hFSH)-receptor complexes, the membranes were solubilized with Triton X-100 or beta-octyl glucoside and the supernatants from ultracentrifugation (220,000 X g) subjected to gel filtration (Sephadex G-200) to separate the labeled hormone-receptor complexes from the unbound 125I-hFSH. The appearance of a high molecular weight (greater than or equal to 200,000) radioactive component in the elution profile was abolished when an excess of unlabeled hFSH was included in the initial incubation. After concentration by ultrafiltration, the 125I-hFSH-receptor complex, as well as the free hormone, was treated with a variety of chemical cross-linkers and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Bands of Mr = 65,000 and 83,000 observed in the autoradiograph of the hormone-receptor complex was not present in autoradiographs of free 125I-hFSH, nor were they present when an excess of unlabeled hFSH was included in the initial binding incubation mixtures. The 65,000 and 83,000 Mr bands were, therefore, considered to represent cross-linked complexes of labeled hFSH (Mr = 38,000) or its subunits (hFSH alpha, Mr = 16,000; hFSH beta, Mr = 21,000) and components of the FSH receptor. The bands were observed on autoradiographs when the extraction of the membranes was performed with either Triton X-100 or beta-octyl glucoside and when cross-linking was accomplished with disuccinimidyl suberate, ethylene glycol bis(succinimidyl succinate), or bis[2-(succinimido oxycarbonyl)oxyethyl]sulfone. The Mr of the native FSH receptor in the calf testis has been estimated at 146,000. Our studies demonstrate the multimeric nature of the FSH receptor. However, FSH is also composed of subunits, so that due to the complexity of the system, it was not possible to arrive at a precise assessment of the Mr or quaternary structure of the receptor subunits.     

Isolation of mitochondrial succinate: ubiquinone reductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using nonionic detergent.

The electron transfer complexes, succinate: ubiquinone reductase, ubiquinone: cytochrome c reductase, and cytochrome c: O2 oxidase were isolated from the mitochondrial membranes of Neurospora crassa by the following steps. Modification of the contents of the complexes in mitochondria by growing cells on chloramphenicol; solubilisation of the complexes by Triton X-100; affinity chromatography on immobilized cytochrome c and ion exchange and gel chromatography. Ubiquinone reductase was obtained in a monomeric form (Mr approximately 130 000) consisting of a flavin subunit (Mr 72 000) an iron-sulfur subunit (Mr 28 000) and a cytochrome b subunit (Mr probably 14 000). Cytochrome c reductase was obtained in a dimeric form (Mr approximately 550 000), the monomeric unit comprising the cytochromes b (Mr each 30 000), a cytochrome c1 (Mr 31 000), the iron-sulfur subunit (Mr 25 000), and six subunits without known prosthetic groups (Mr 9000, 11 000, 14 000, 45 000, 45 000, and 52 000). Cytochrome c oxidase was also isolated in a dimeric form (Mr approximately 320 000) comprising two copies each of seven subunits (Mr 9000, 12 000, 14 000, 18 000, 21 000, 29 000, and 40 000). The complexes were essentially free of phospholipid. Each bound one micelle of Triton X-100 (Mr approximately 90 000). After isolation, the bound Triton X-100 could be replaced by other nonionic detergents such as: alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers and acyl polyoxyethylene sorbitan esters.     

Subtypes of betaglycan and of type I and type II transforming growth factor-beta (TGF-beta) receptors with different affinities for TGF-beta 1 and TGF-beta 2 are exhibited by human placental trophoblast cells.

Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.     

Multiple growth hormone-binding proteins are expressed on insulin-producing cells

The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as disulfide-linked multimers of Mr 270-350K (300K). In addition, a minor Mr 180K GH-binding protein is identified which does not appear to be associated with other proteins by disulfide bridges. A plasma membrane-enriched fraction accounts for 86% of the RIN-cell GH-binding activity while cytosol and intracellular organelles are low in GH-binding activity. The plasma membrane-bound activity is soluble in Triton X-100 with intact hormone binding characteristics. The apparent KD in detergent solution is estimated to 18 ng/ml (8 x 10(-10) M). 125I-hGH-affinity cross-linking to intact and detergent-solubilized membranes as well as hGH-affinity purified protein reveals labeled proteins of Mr 180K and Mr 285-350K. In contrast to the cross-linked Mr 300K complexes of intact cells those of disintegrated cellular material are resistant to reduction with dithiothreitol, and it is speculated that this is due to intersubunit cross-linking of the disulfide-linked Mr 110K GH-binding subunits. The GH-binding proteins are purified approximately 100-fold by one cycle of hGH-affinity chromatography and five major proteins of Mr 180K, 94K, 86K, 64K, and 54K are identified by silver staining in the purified fraction. It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production.     

Solubilization of functional calcitonin receptors.

Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.