Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell.

We have used bifunctional reagents to examine the subunit composition of the non-DNA-binding form of the rat and human glucocorticoid receptor. Treatment of intact cells and cell extracts with a reversible cross-linker, followed by electrophoretic analysis of immunoadsorbed receptor revealed that three proteins of apparent approximate molecular masses, 90, 53 and 14 kDa are associated with the receptor. The first of these was identified immunochemically as a 90-kDa heat-shock protein (hsp90). The complex isolated from HeLa cells contained 2.2 mol hsp90/mol steroid-binding subunit. Cross-linking of the receptor complex in the cytosol completely prevented salt-induced dissociation of the subunits. The cross-linked receptor was electrophoretically resolved into two oligomeric complexes of apparent molecular mass 288 kDa and 347 kDa, reflecting the association of the 53-kDa protein with a fraction of the receptor. Since no higher oligomeric complexes could be generated by cross-linking cell extracts under different conditions, we conclude that most of the untransformed cytosolic receptor is devoid of additional components.     

Protein components of the nonactivated glucocorticoid receptor.

The nonactivated glucocorticoid receptor (Mr approximately 350,000) of WEHI-7 mouse lymphoma cells was investigated with respect to the stoichiometry of protein subunits. Cross-linking patterns obtained by affinity labeling and denaturing gel electrophoresis revealed a heterotetramer consisting of one receptor polypeptide in association with two 90- and one approximately 50-kDa subunits. The receptor stabilized by molybdate, disulfide bond formation, or chemical cross-linking was purified roughly 6000-fold by immunoaffinity chromatography and analyzed by gel electrophoresis and immunoblotting. The 90-kDa component was consistently detected in a 2:1 ratio with respect to the receptor polypeptide and was identified as the 90-kDa heat shock protein, hsp90. A 70-kDa heat shock protein was found in both stabilized and nonstabilized receptors and bound to the immunomatrix independent of receptor. The additional receptor subunit was unequivocally identified as the 59-kDa protein previously described (Tai, P.-K. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275). This component was found only in complexes cross-linked via amino groups. It was removed from the molybdate-stabilized receptor under our purification conditions, thus leaving behind a trimer composed of the receptor polypeptide and two molecules of hsp90. In the absence of hormone, the receptor had the same subunit composition as in its presence.     

Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells

Mouse lymphoma cells contain a nonactivated glucocorticoid receptor of Mr approximately 330,000 which is heteromeric in nature and is unable to bind to DNA. Following affinity labeling of the steroid-binding subunit and subsequent cross-linking with dimethyl suberimidate at various times either in cell extracts or in intact cells, a series of labeled bands was detected in SDS gels. From the molecular masses of completely and partially cross-linked complexes we conclude that the large nonactivated receptor is a tetramer composed of two 90 kDa subunits, one 50 kDa polypeptide and one steroid-binding subunit.     

Phosphorylation of glucocorticoid receptor-associated and free forms of the approximately 90-kDa heat shock protein before and after receptor activation

Several lines of evidence have suggested that glucocorticoid receptor function may be regulated by phosphorylation-dephosphorylation reactions, and it has been proposed that dephosphorylation accompanies activation to the DNA-binding form. The phosphate content of the approximately 100-kDa steroid-binding protein has been determined directly and was found not to change during activation in intact cells (Mendel, D.B., Bodwell, J.E., and Munck, A. (1987) J. Biol. Chem. 262, 5644-5648). We have now determined the effect of interaction with the receptor and of activation on the phosphate content of the approximately 90-kDa heat shock protein (Hsp 90), which is thought to be a non-steroid-binding subunit of nonactivated glucocorticoid receptors that dissociates on activation. Monoclonal antibodies AC88 and BuGR2 were used to purify free Hsp 90 and cytosolic nonactivated glucocorticoid-receptor complexes, respectively, from WEHI-7 cells grown in the presence of 32Pi and [35S] methionine. Cell-free activation of the nonactivated receptor-antibody complexes immobilized on protein A-Sepharose minicolumns allowed the recovery of the Hsp 90 dissociated from the complexes during activation. Proteins were separated by denaturing polyacrylamide gel electrophoresis, and the 32P/35S ratio, which was used as a measure of the phosphate content relative to protein, was determined for the free, receptor-associated, and dissociated forms of the Hsp 90, as well as for the approximately 100-kDa steroid-binding protein of non-activated and activated receptors. The three forms of the Hsp 90 had the same phosphate contents, as did the approximately 100-kDa steroid-binding protein before and after activation. Based upon these results, we conclude that no net change in the phosphorylation occurs when the Hsp 90 associates with the approximately 100-kDa steroid-binding protein to form nonactivated receptors and that neither protein component of nonactivated complexes is dephosphorylated when they dissociate during thermal activation under cell-free conditions.     

Subunit structure of the glucocorticoid receptor and activation to the DNA-binding state

Glucocorticoid receptors of S49.1 mouse lymphoma cells were analyzed under a variety of conditions. The complexes with an agonist or a steroidal antagonist can be formed in cytosolic extracts, they are of high molecular weight, Mr approximately 330,000 and have a Stokes radius of 82 A. Cross-linking by several agents stabilized this structure against subunit dissociation which produces the activated receptor form of 60 A and DNA-binding ability. Careful analysis of intermediate cross-linked forms lead to the conclusion that the large receptor structure is a hetero-tetramer consisting of one hormone-bearing polypeptide of Mr approximately 94,000, two 90 kDa subunits and a protein component of Mr approximately 50,000. The 90 kDa subunits are the heat shock protein hsp90. The high molecular weight receptor form also exists in intact cells as revealed again by cross-linking. The cytosolic complex with the antagonist can become activated to the DNA-binding form upon warming but simultaneously looses the ligand. Ligand rebinding does not occur subsequent to receptor dissociation. Upon incubation of intact cells at 37 degrees C with agonist or antagonist the respective receptor-ligand complexes are formed. The agonist complex is immediately activated, however, the antagonist complex remains stable in the undissociated state. This explains the biological effect of the antagonist.     

Activation of cytosolic glucocorticoid-receptor complexes in intact WEHI-7 cells does not dephosphorylate the steroid-binding protein

In order to determine the ratio of phosphates to hormone-binding sites on nonactivated (non-DNA-binding) glucocorticoid receptors in WEHI-7 mouse thymoma cells, we have extracted these receptors from cells grown to a steady state with 32P, labeled them with a saturating concentration of [3H]dexamethasone 21-mesylate, purified them using a monoclonal antibody, and analyzed them by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The complexes contained approximately 5 mol of phosphate/mol of bound steroid. Only half of the phosphates were associated with the approximately 100-kDa protein which is labeled with [3H]dexamethasone 21-mesylate. The remaining phosphates were associated with the approximately 90-kDa non-steroid-binding component of the nonactivated complex. Dual label studies, using [35S]methionine to measure receptor protein and 32P to measure receptor phosphates, have enabled us to determine the phosphate content, relative to receptor protein, of both nonactivated and activated cytosolic complexes generated in intact WEHI-7 cells exposed to triamcinolone acetonide at 37 degrees C. The total amount of phosphate associated with the activated complex is roughly half of that associated with the nonactivated complex, the decrease being accounted for by dissociation of the approximately 90-kDa phosphoprotein which accompanies activation. However, the ratio of 32P to 35S counts associated with the approximately 100-kDa steroid-binding protein is the same for the activated and nonactivated complexes. These results indicate that there is no net change in the phosphorylation of the approximately 100-kDa steroid-binding component of the cytosolic glucocorticoid-receptor complex upon activation in the intact cell.     

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc     

Molybdate-stabilized nonactivated glucocorticoid-receptor complexes contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activation

We have used a monoclonal antibody to purify glucocorticoid-receptor complexes from WEHI-7 mouse thymoma cells. Molybdate-stabilized, nonactivated complexes were found to contain two distinct proteins which could be separated by polyacrylamide gel electrophoresis under denaturing and reducing conditions. One of the proteins, 100 kDa, was labeled when cytosol was incubated with the affinity ligand [3H]dexamethasone 21-mesylate. The second protein, 90 kDa, was not labeled. Several lines of evidence, including Western blot analysis of purified nonactivated complexes, indicate that only the 100-kDa protein is directly recognized by the antibody. The 90-kDa protein appears to be purified as a component of the nonactivated complex due to noncovalent association with the 100-kDa protein. Both the 100-kDa and 90-kDa components of the nonactivated complex become labeled with 35S when cells are grown in medium containing [35S]methionine. Using cells labeled in this manner, we have shown that activated (i.e. DNA-binding) cytosolic complexes, formed by warming either in intact cells or under cell-free conditions, contain only the 100-kDa protein. Complexes extracted from nuclei of warmed cells similarly contain only the 100-kDa protein. These results indicate that the 100-kDa and 90-kDa components of nonactivated complexes separate upon activation. Purification of nonactivated complexes from cells grown in medium containing [32P]orthophosphoric acid indicates that both the 100-kDa and 90-kDa components are phosphoproteins which can be labeled with 32P. Therefore, resolution of the two proteins will be essential in order to determine whether the receptor is dephosphorylated on activation.     

Translation of glucocorticoid receptor mRNA in vitro yields a nonactivated protein

The glucocorticoid receptor is present in cytosol prepared from cell extracts of nonhormone-treated cells as a large nonactivated (i.e. non-DNA binding) 9 S heteromeric complex which contains the Mr approximately 90,000 heat shock protein, hsp90. hsp90 is expressed under physiological conditions in mammalian cells and is also present in reticulocyte lysate, as assessed by Western immunoblotting using specific anti-hsp90 antibodies. We have translated glucocorticoid receptor mRNA in reticulocyte lysates. The receptor synthesized under cell-free conditions also interacts with hsp90 both in the presence and absence of ligand, as determined by sucrose gradient centrifugation. The in vitro synthesized glucocorticoid receptor does not bind to DNA-cellulose but can be converted to a DNA binding form following labeling with dexamethasone and heat treatment. Thus, the glucocorticoid receptor is synthesized in a nonactivated form under cell-free conditions. These data indicate that the 9 S glucocorticoid receptor complex found in cytosol does not represent an artifact due to cell homogenization and supports the existence in vivo of the glucocorticoid receptor-hsp90 complex.     

Cell-free synthesis of rat glucocorticoid receptor in rabbit reticulocyte lysate. In vitro synthesis of receptor in Mr 90,000 heat shock protein-depleted lysate

The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.     

The non-DNA-binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59-kilodalton protein

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.     

Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IFN-gamma. One single 90-kilodalton IFN-gamma receptor can lead to multiple cross-linked products and isolated proteins

We analyzed the high affinity receptor for IFN-gamma of Raji cells and human placenta by combining Scatchard analysis, cross-linking experiments, and receptor purification. Only one high affinity binding site was found, Kd 2.1 X 10(-10). The receptor is a 90-kDa glycoprotein. However, multiple cross-linked products of 110 kDa to about 250 kDa could be generated and proteins of 90, 70, and 50 kDa could be obtained upon purification. These proteins all contained the same 90-kDa receptor, or part of it. We suggest that extensive cross-linking and/or proteolysis may explain many of the conflicting results published thus far. The extracellular domain of the 90-kDa receptor protein was highly resistant to digestion with trypsin or proteinase K. Trypsin digestion neither affected the number of binding sites per cell, nor the Kd for IFN-gamma. A cluster of sites for different proteases was found in the intracellular domain. The 50-kDa fragment created by trypsin digestion had the same characteristics as the isolated 50-kDa receptor fragment. It contained the IFN-gamma binding site and the receptor's extracellular and amino-terminal domain. N-linked glycosylation contributed about 15 kDa to its molecular mass, of which 4 kDa were attributable to sialic acid residues. O-Linked glycosylation was not detected. The number of binding sites per cell and the Kd for IFN-gamma were not affected by the presence or absence of N-linked glycosylation. The receptor contained at least one critical disulfide bridge and the reduced receptor could be reactivated in vitro.     

Phosphorylated sites within the functional domains of the approximately 100-kDa steroid-binding subunit of glucocorticoid receptors

The steroid-binding subunit of the glucocorticoid receptor is known to be a approximately 100-kDa phosphoprotein composed of an immunogenic, DNA-binding, and steroid-binding domain. When isolated from WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel et al., 1987). To identify the domains that contain these phosphorylated sites, we have analyzed the phosphate content of selected proteolytic fragments of the approximately 100-kDa steroid-binding protein from nonactivated and activated receptors. The approximately 100-kDa steroid-binding protein from WEHI-7 cells grown in the presence of [32P]orthophosphate was covalently labeled with [3H]dexamethasone 21-mesylate, purified with the BuGR2 monoclonal antibody, digested with chymotrypsin or trypsin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin digestion of this protein yields a approximately 45-kDa fragment containing both the steroid-binding and DNA-binding domains, which contained both 32P and 3H. Trypsin digestion of the protein yields a approximately 29-kDa fragment encompassing the steroid-binding domain but not the DNA-binding domain of the approximately 100-kDa protein, which also contained both 32P and 3H. The 32P/3H ratio of each fragment provides a measure of phosphate content per steroid-binding site and indicated that each fragment has approximately 30% of the phosphate content of the intact protein. This is sufficient to account for one of the three receptor phosphoryl groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies to distinctive epitopes on the alpha and beta subunits of the fibronectin receptor

Monoclonal antibodies (MAbs) have been developed that can recognize epitopes that are unique to either the alpha or beta subunit of the fibronectin receptor (FnR). MAbs 11B4 and 7A8 immunoblot the alpha subunit of FnR either in purified form from Chinese hamster ovary (CHO) cells or in nonionic detergent extracts of cells of human and rodent origin electrophoresed under reducing or nonreducing conditions. The MAbs seem to be more reactive to the subunit when it has been electrophoresed under reducing conditions, suggesting that the epitope may be partially masked by the conformation conferred by disulfide bonding. A second set of MAbs, 7E2 and 7F9, is directed to an epitope on the beta subunit that is conformationally dependent upon disulfide bonding, as reduction of the subunit leads to loss of reactivity with both MAbs. Further, 7E2/7F9 immunoblots of nonionic detergent extracts of CHO cells, run under nonreducing conditions, reveal the presence of a third band (90-kDa), immunologically related to the beta subunit, which is not surface-labeled with 125I in intact cells and which does not copurify with the alpha and beta subunits isolated by immunoaffinity purification of FnR using the MAb PB1. The 90-kDa component is not found associated with a plasma membrane fraction prepared by crude cell fractionation, but is abundant in a low-speed pellet containing nuclei and intracellular membranes. This finding suggests that the 90-kDa component is a precursor to the beta subunit. Finally, the epitope of 7E2/7F9 is unique to CHO cells, as cross-reactivity to other cell types cannot be demonstrated by either immunoblotting or immunoprecipitation.     

A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain.

The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Three-step purification method and characterization of the bovine brain 90-kDa heat shock protein

A protein that cross-reacted with antibody against the 90-kDa heat shock protein (HSP90) of a mouse lymphoma cell line was purified from bovine brain by three steps. Fifty milligrams of the 90-kDa protein was recovered from 350 g of the brain cortex. The sedimentation coefficient and Stokes radius of the purified protein were 6.0 s and 6.7 nm, respectively. The molecular weight was calculated to be 170,000. The molecule was composed of two identical 90-kDa subunits. A partial amino acid sequence (23 residues) of this protein was homologous (96%) to human HSP90 (the sequence of 174-196). These facts led to the identification of the 90-kDa brain protein with HSP90. In bovine tissues, the brain contained this protein at a remarkably high concentration. The brain HSP90 was separable from glucocorticoid receptor by heparin-agarose and DNA-cellulose columns. It is concluded that HSP90 is present in brain cytosol and mostly as free molecules. Immunohistochemical studies showed that the protein was localized in nerve excitable cells. It was not found in nuclei but in cytosol.     

Chick heat-shock protein of Mr = 90,000, free or released from progesterone receptor, is in a dimeric form

A monoclonal antibody (BF4) has been used to characterize and purify the heat-shock protein of Mr approximately 90,000 (hsp 90) present in the chick oviduct. In low salt cytosol, the sedimentation coefficient of hsp 90 is approximately 6.8 S, the Stokes radius approximately 7.1 nm, and the calculated Mr approximately 204,000, thus suggesting a dimeric structure. In 0.4 M KCl cytosol, only slightly smaller values were determined (approximately 6.5 S, approximately 6.8 nm, and approximately 187,000). Following purification by ion exchange and immunoaffinity chromatography, hsp 90 migrated as a single silver-stained band at Mr approximately 90,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient 6.2 S, the Stokes radius approximately 6.8 nm, and the Mr approximately 178,000 confirmed the dimeric structure. However, in both antigen or antibody excess conditions, only one molecule of monoclonal antibody could be bound to the hsp 90 dimer. Whether steric hindrance in a homodimer or the presence of two different 90-kDa proteins in a heterodimer explains this result cannot yet be decided. The dimer is not dissociated by high salt (1 M KCl) or the chaotropic agent (0.5 M NaSCN), but is disrupted by 4 M urea, suggesting a stabilization of the structure by hydrogen bonds. The molybdate-stabilized progesterone receptor hetero-oligomer form of approximately 8 S sedimentation coefficient was purified, and its hsp 90 component was then released by salt treatment. It was found to sediment at approximately 5.8 S and have a Stokes radius approximately 7.1 nm, giving Mr approximately 174,000. This observation is consistent with a previous report suggesting from specific activity determination, scanning of polyacrylamide gels, and cross-linking experiments that each purified nontransformed progesterone receptor molecule includes one progesterone binding unit per two 90-kDa protein molecules (Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., and Baulieu, E. E. (1984) Biochemistry 23, 6016-6023). This work brings direct evidence that both free hsp 90 and the non-hormone binding hsp 90 component released from the nontransformed steroid receptor in the cytosol are in a dimeric form.     

Structural studies on membrane-bound and soluble growth-hormone-binding proteins of rabbit liver.

Covalent cross-linking techniques have been used to investigate the structural characteristics of the growth-hormone (GH) receptor in a variety of rabbit liver cell membrane preparations (particulate and soluble). Two classes of GH-binding protein have been identified which differ in their Mr by gel filtration and susceptibility to precipitation with poly(ethylene glycol) (PEG). The first, a PEG-precipitable (Mr approximately 300,000) protein, contained Mr-65,000 and Mr-40,000 binding proteins linked by disulphide bonds. It was present in aqueous extracts derived from microsomal membranes but was not present in cytosol preparations. The second, a PEG-non-precipitable protein (Mr approximately 100,000) was composed of a non-disulphide-linked primary GH-binding subunit of Mr 60,000-66,000. This binding protein was present in all rabbit liver cell fractions and/or preparations. Both binding-protein classes contained intramolecular disulphide bonds. It is not clear whether the Mr-approximately 100,000 form, or perhaps higher-Mr species which have not been identified by cross-linking studies, represents the native, endogenous, form of the GH receptor present in particulate microsomal or plasma membranes. Accordingly, although these data have identified two classes of GH-binding protein, especially a primary GH-binding subunit of Mr 60,000-66,000, they indicate that, unlike studies on the insulin receptor, covalent cross-linking techniques alone are not sufficient to delineate the complete subunit structure of the native and endogenous form of the GH receptor.     

A mitogenic fibrinogen receptor that differs from glycoprotein IIb-IIIa. Identification by affinity chromatography and by covalent cross-linking

The mitogenic effect of human fibrinogen on the hemopoietic cell lines Raji and JM is mediated by a specific receptor with biochemical and functional properties different from those of the platelet fibrinogen receptor, the glycoprotein complex IIb-IIIa (Levesque, J.P., Hatzfeld, A., and Hatzfeld, J. (1986) Proc. Natl. Acad. Sci. U.S. A. 83, 6494-6498; Levesque, J.P., Hatzfeld, A., Hudry-Clergeon, G., Wilner, G.D., and Hatzfeld, J. (1987) J. Cell. Physiol. 232, 303-310). This work describes the identification of the mitogenic fibrinogen receptor (MFR) by two independent methods, affinity chromatography and covalent cross-linking. Affinity chromatography of surface-labeled cell extracts on fibrinogen-Sepharose revealed a 94 +/- 2-kDa membrane protein that bound specifically to fibrinogen-Sepharose only on cells that expressed the MFR. Its molecular mass was not modified after reduction. This was confirmed by cross-linking fibrinogen to surface-labeled Raji cells using the cleavable cross-linkers, ethyleneglycobis(succinimidyl succinate) and dithiobis(succinimidyl propionate). Complexes between fibrinogen and iodinated cell membrane proteins were immunoprecipitated by anti-fibrinogen antibodies. The biochemical cleavage of these immunoprecipitated conjugates gave rise to a 92 +/- 3-kDa membrane protein whose molecular mass was not modified after reduction. We conclude that fibrinogen binds specifically to a 92-94-kDa MFR which does not belong to the integrin family.