Analysis of glucagon-receptor interactions on isolated canine hepatocytes. Formation of reversibly and irreversibly cell-associated hormone

We have investigated the interactions of ligand with the canine hepatic glucagon receptor. Whereas time courses for radiolabeled glucagon binding to receptor and dissociation from receptor revealed fast and slow components at both 30 and 4 degrees C, time courses of ligand dissociation revealed a third component of irreversibly cell-associated (nondissociable) ligand only at the higher temperature. Related experiments identified that (a) the initial rate of formation of nondissociable ligand was slower than that of dissociably bound hormone; (b) the fraction of ligand bound to nondissociable sites achieved a plateau during extended incubations, whereas that bound to dissociable sites was seen to rise and then slowly to fall; (c) the kinetics of formation of a nondissociable ligand was consistent with linked, sequential reactions; (d) dissociable ligand-receptor complexes formed at 4 degrees C were converted to nondissociable complexes during subsequent incubation at 30 degrees C, and (e) nondissociable sites were filled by prior incubation of cells with unlabeled ligand. Analysis of receptor-bound hormone resulting from the incubation of cells with 125I-labeled glucagon and selected concentrations of either glucagon or [[127I]iodo-Tyr10]glucagon at steady state revealed in each case four components of receptor-bound ligand: those corresponding to high and low affinity components of dissociably bound ligand and to high and low affinity components of nondissociably bound ligand. Implications of these findings are considered in terms of mechanisms for the formation of irreversibly bound hormone and for the distribution of hormone among the various components of hepatic glucagon-binding sites.     

Stabilization of estradiol-receptor complexes by elimination of cytosolic factors

Treating mature rat uterine cytosol with dextran coated charcoal (DCC) for 2 hrs at 0-4C in the absence of ligand causes the subsequently formed receptor-estradiol complex to be stable at 37C. Receptor binding is increased by the DCC treatment for uteri excised at metestrus or diestrus but remains nearly unchanged for uteri obtained at proestrus or estrus. Results suggest that the DCC removes or inactivates factor(s) present in the cytosol which render the receptor complex thermolabile.     

A monoclonal antibody to the estrogen receptor inhibits in vitro criteria of receptor activation by an estrogen and an anti-estrogen

The effect of an IgM class monoclonal antibody (B36) (Greene, G. L., Fitch, F. W., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 157-161) raised against the calf uterine estrogen receptor was tested in vitro on certain parameters of estrogen receptor activation by estradiol or 4-hydroxytamoxifen, a potent anti-estrogen. The following results were obtained. The antibody prevented the decrease in the dissociation rate of the receptor-estradiol complex which results from activation of the complex, whereas it did not affect the dissociation rate of the receptor-4-hydroxytamoxifen complex, which remains unchanged upon activation. The antibody also increased the dissociation rate of the preactivated receptor-estradiol complex. The antibody protected the naked estrogen receptor against heat-inactivation. B36 partially inhibited the binding of the estradiol- and 4-hydroxy-tamoxifen-receptor complexes to DNA adsorbed onto cellulose, but did not reverse the receptor-DNA binding. This inhibition was not overcome by higher DNA concentrations and was more pronounced for the receptor interacting with estrogen than with anti-estrogen. All these effects were specific since they were related to antibody/antigen recognition and were dose-dependent. These results indicate that the binding of the antibody to the estrogen-activated receptor induces a conformational change in the receptor and that the antibody can prevent and overcome the effect of activation whatever its mechanism. They also confirm that the conformations of the estrogen receptor differ when bound to estradiol or to 4-hydroxytamoxifen.     

Salt extractability of nuclear receptor-estrogen complex from the rat uterus

Estrogen receptors in the nuclei of rat uterus were determined 1 or 6 h after the injection of [³H]estradiol. Ratio of 0.4 M KCl-extractable to -resistant receptor-estradiol complex was constant during this time interval. Three consecutive extractions took out about 95% of receptor-estradiol complex either 1 or 6 h after injection. Extraction with KCl before the exchange assay reduced the amount of salt-resistant nuclear receptor-estradiol complex. However, when exchange incubation with [³H] estradiol was done before KCl extraction, salt-resistant receptor-estradiol complex was significantly increased, even after three consecutive extractions.     

Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies

The specific binding of 125I-PDGF (platelet-derived growth factor) to intact fibroblasts becomes relatively nondissociable during incubation at 37 degrees C. To characterize the interaction of PDGF with its receptors under conditions in which there is no receptor internalization, we have studied the binding of 125I-PDGF to membrane preparations derived from mouse 3T3 cells and rat liver. The binding sites had the affinity and specificity characteristics expected of PDGF receptors. At 37 degrees C (but not at 4 degrees C) the specific binding of 125I-PDGF to membranes gradually became nondissociable as assessed by either dilution or by addition of excess unlabeled PDGF. This tight binding was not due to a covalent interaction since the polyanionic compound suramin readily dissociated specifically bound 125I-PDGF. This property of suramin was used to expose rat liver PDGF receptors which were occupied by endogenous PDGF. Affinity cross-linking studies demonstrated that the formation of the nondissociable state of 125I-PDGF binding was associated with the binding of 125I-PDGF to a 160,000-dalton protein and to a 110,000-dalton species. The cross-linked binding sites could be adsorbed to wheat germ agglutinin and to anion exchange resins. The isoelectric point of both cross-linked species determined by two-dimensional gel electrophoresis was approximately 4.7. These data demonstrate that in membrane preparations, PDGF binds to an anionic 160,000-dalton glycoprotein which is likely to be the receptor. A high affinity state of PDGF binding, which is formed rapidly at 37 degrees C, can be dissociated by suramin.     

Reactions of thrombin-serpin complexes with thrombospondin.

Activated platelets release proteins that form stable complexes with thrombin (J. J. Miller, P. C. Browne, and T. C. Detwiler, Biochem. Biophys. Res. Commun. 151, 9-15, 1988). A working model for the reaction (P. C. Browne, J. J. Miller, and T. C. Detwiler, Arch. Biochem. Biophys. 265, 534-538, 1988) includes a dissociable complex of thrombin with released platelet protease nexin, leading to formation of a nondissociable thrombin-nexin complex that then becomes disulfide linked to thrombospondin. This disulfide-linked complex is converted back to the thrombin-nexin complex by reduction of disulfide bonds. Results that allow elaboration on this model are presented. After longer periods of incubation or after incubation with higher concentrations of thrombin, the amount of thrombin complexed with thrombospondin exceeded the amount of thrombin-nexin complex recovered after reduction of disulfide bonds. When the reaction mixture included inhibitors of formation of the thrombin-nexin complex, a slow formation of the thrombin-thrombospondin complex was observed. It was concluded that there is a nexin-independent as well as the faster nexin-dependent disulfide linkage of thrombin to thrombospondin. Addition of thrombin-antithrombin III complexes to the supernatant solution of activated platelets also led to complexes with thrombospondin, demonstrating that serpins other than platelet protease nexin facilitate incorporation of thrombin into complexes with thrombospondin. By heparin affinity chromatography, it was shown that thrombin-nexin complexes dissociably associate with thrombospondin prior to formation of disulfide-linked complexes. These observations are incorporated into a more detailed model of the reaction.     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Association of the transformed glucocorticoid receptor with a cytoskeletal protein complex

In a recent paper we described a system in which glucocorticoid receptors associate with particulate complexes containing tubulin [Cancer Res. 49 (1989) 2222s–2229s]. When L cell cytosol is mixed with a microtubule stabilizing buffer and heated to 37°C, the receptor becomes associated with a complex that can be centrifuged out of solution at 150,000 g. In this work we show that the glucocorticoid receptor—cytoskeletal protein complex forms in a temperature and glutamate-dependent manner. Molybdate does not affect generation of the cytoskeletal protein complex but it inhibits association of the receptor with the complex. This suggests that transformation of the receptor to its DNA-binding form is required for interaction with the cytoskeletal complex. Colchicine has no effect on generation of the particulate complex or on the association of receptor with it, suggesting that formation of the complex does not represent a classic in vitro process of tubulin polymerization.     

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.     

Rapid modulation of N-formyl chemotactic peptide receptors on the surface of human granulocytes: formation of high-affinity ligand- receptor complexes in transient association with cytoskeleton

When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4 degrees C. These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively. After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min. Exposure at 15 degrees C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37 degrees C. At 4 degrees C, complex formation was approximately 10% of the maximum amount formed at 37 degrees C. Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation. Electron microscopic autoradiography indicated that after 1 min of incubation at 37 degrees C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton. After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution. We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton.     

Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin

Clostridium perfringens epsilon toxin (ETX) rapidly kills MDCK II cells at 37°C, but not 4°C. The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C. However, the complex formed in MDCK cells treated with ETX at 4°C has the potential to form an active pore, since shifting those cells to 37°C results in rapid cytotoxicity. Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface. Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore. In addition, the ETX complex rapidly dissociated from MDCK II cells at 4°C, but not 37°C; this result is consistent with the ETX complex being resistant to dissociation at 37°C because it has inserted into membranes, while the ETX prepore readily dissociates from cells at 4°C because it remains on the membrane surface. These results support the identification of a prepore stage in ETX action and suggest a revised model for ETX cytotoxicity, i) ETX binds to an unidentified receptor, ii) ETX oligomerizes into a prepore on the membrane surface, and iii) the prepore inserts into membranes, in a temperature-sensitive manner, to form an active pore.     

Degradation and internalization of bound insulin in human erythrocytes.

Internalization and degradation of insulin by human erythrocytes were studied. Erythrocytes were incubated with 125I-insulin at 4 degrees C, 15 degrees C, and 37 degrees C for varying time intervals. These erythrocytes were then subjected to a low pH wash to release bound insulin followed by TCA precipitation. After 4, 22, and 24 hours of insulin binding at 4 degrees C, 92 to 95% of the bound 125I-insulin was dissociable and 92 to 98% of the extractable insulin was undegraded. After 3.5 hours of incubation at 15 degrees, 82% of the bound insulin was dissociable and 60% of this was intact. However, after 60, 90, 120, and 180 minutes of incubation at 37 degrees C, only 42, 34, 24, and 37%, respectively, of the bound insulin was dissociable. The undissociated insulin in the 37 degrees C studies was considered to be intracellular. With increasing time of incubation at 37 degrees C, the extractability of cell bound insulin and the proportion of undegraded dissociable insulin were decreased. When 125I-insulin binding was 95% blocked by preincubating the erythrocytes with anti-insulin receptor antibody, 95% of the degradation of 125I-insulin was also blocked. These studies indicate that mature human erythrocytes degrade internalized insulin and this process is time, temperature, and insulin receptor dependent.     

Circulating oxidized LDL forms complexes with beta2-glycoprotein I: implication as an atherogenic autoantigen

Beta2-glycoprotein I (beta2-GPI) is a major antigen for antiphospholipid antibodies (Abs, aPL) present in patients with antiphospholipid syndrome (APS). We recently reported (J. Lipid Res., 42: 697, 2001; J. Lipid Res., 43: 1486, 2002) that beta2-GPI specifically binds to Cu2+-oxidized LDL (oxLDL) and that the beta2-GPI ligands are omega-carboxylated 7-ketocholesteryl esters. In the present study, we demonstrate that oxLDL forms stable and nondissociable complexes with beta2-GPI in serum, and that high serum levels of the complexes are associated with arterial thrombosis in APS. A conjugated ketone function at the 7-position of cholesterol as well as the omega-carboxyl function of the beta2-GPI ligands was necessary for beta2-GPI binding. The ligand-mediated noncovalent interaction of beta2-GPI and oxLDL undergoes a temperature- and time-dependent conversion to much more stable but readily dissociable complexes in vitro at neutral pH. In contrast, stable and nondissociable beta2-GPI-oxLDL complexes were frequently detected in sera from patients with APS and/or systemic lupus erythematodes. Both the presence of beta2-GPI-oxLDL complexes and IgG Abs recognizing these complexes were strongly associated with arterial thrombosis. Further, these same Abs correlated with IgG immune complexes containing beta2-GPI or LDL. Thus, the beta2-GPI-oxLDL complexes acting as an autoantigen are closely associated with autoimmune-mediated atherogenesis.     

An examination of methods used to assay potato tuber invertase and its naturally occurring inhibitor

Confirming an earlier report, it was shown that the endogenous inhibitor of potato tuber invertase forms an essentially undissociable complex with the enzyme. Consequently, several previous analyses of potato tuber invertase which were based on equations derived for highly dissociable enzyme-inhibitor complexes are presumed to be in serious error. The complex formation proceeded slowly, requiring approximately 1 day to reach completion at 2 C, and 1 hr at 37 C. Allowing complex formation to reach completion before assaying enzyme activity did not affect the noncompetitive nature of the inhibition.     

Mapping on the calf estrogen receptor of the binding domain for an antibody interfering with receptor activation

The localization on the calf estrogen receptor of the binding domain for B36 (an IgM antibody which prevents and reverses the effects of receptor activation) has been studied by means of controlled proteolysis of the receptor-estradiol complex using trypsin, chymotrypsin, and papain. We successively determined for intact and proteolyzed receptor-estradiol complex (i) the abilities of estradiol-binding species to aggregate in low salt medium, to bind to nonspecific DNA absorbed onto cellulose, and to interact with B36 antibody in sucrose gradients; (ii) the hydrodynamic properties of estradiol-binding species, by gel permeation chromatography and sucrose gradient centrifugation in high salt media and (iii) the molecular weights of B36-reactive species, by immunoblot analysis. Three tryptic receptor fragments of Mr 36,000, 34,000, and 33,000 and two chymotryptic fragments of Mr 36,000 and 33,000 included both the hormone- and B36-binding domains but did not interact with DNA, whereas at least two receptor fragments resulting from the action of chymotrypsin and papain bound estradiol with high affinity but interacted neither with DNA nor with B36. Taking into account these results and assuming that structure of the calf estrogen receptor is similar to those of sequenced estrogen receptors (which show a highly conserved organization with considerable homologies in the functional domains), we propose that the B36-binding domain is located either between the DNA- and hormone-binding domains (model I) or at the C-terminal end of the estrogen receptor (model II). The regions that include the main proteolytic cleavage sites of the receptor are also specified, and the abilities of the two models of the calf estrogen receptor to account for the effect of B36 on receptor activation are discussed.     

Receptor for sex steroid-binding protein of endometrium membranes: solubilization, partial characterization, and role of estradiol in steroid-binding protein-soluble receptor interaction.

The sex steroid-binding protein (SBP) receptor was solubilized from the membranes of human premenopausal endometrium with the zwitterionic detergent CHAPS. The binding activity of the soluble receptor was studied, allowing it to interact with [125I]SBP and precipitating the complex with polyethylene glycol 8,000. The interaction of SBP with the soluble receptor was specific, saturable, and at high affinity. Indeed, the specific binding was definitely improved on the solubilized form of the receptor. The effect exerted by sex steroids on the interaction of SBP with receptor was also examined on both the soluble and membrane-bound forms. At physiologic doses (10(-8) M) estradiol inhibits the binding at a significant extent on the soluble receptor, but not on membrane-bound form. The dose of estradiol required to significantly inhibit the SBP-specific binding was dependent on the form of receptor. In membrane-bound receptor the inhibiting dose of estradiol was higher than its physiologic concentration. Thus, it is likely that, while soluble receptor cannot recognize the complex steroid-SBP, membrane-bound receptor can interact both with "unliganded" SBP and with the estradiol-SBP complex (but not with androgen-SBP complexes) in an estrogen-dependent tissue like human endometrium.     

Estrogen receptor in chicken oviduct: receptor dissociation kinetics and transformation

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22 degrees C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate (k = 120 X 10(-3) min-1) decreases as a function of the pre-incubation at 22 degrees C; after prolonged pre-incubation only the slowly dissociating (k = 12.3 X 10(-3) min-1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30 degrees C is similar, showing a transition of a fast dissociating form (k = 120 X 10(-3) min-1) to a slowly dissociating form (k = 7.6 X 10(-3) min-1) as a result of pre-incubation at 30 degrees C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0-35%) shows monophasic dissociation kinetics of estradiol (k = 39.5 X 10(-3) min-1) and for moxestrol (k = 10.8 X 10(-3) min-1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0 degree C at 4.8 S. Only after subsequent incubation at 30 degrees C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22 degrees C (k = 70 X 10(-3) min-1; k = 14.0 X 10(-3) min-1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22 degrees C. Moxestrol dissociates from the nuclear receptor at 30 degrees C monophasically with a slow rate (k = 6.1 X 10(-3) min-1), suggesting that it is extracted as an activated hormone-receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor

Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor complex that accompanies transformation to the DNA-binding state. When steroid-receptor complexes that are prebound to DNA-cellulose are exposed to MMTS, the steroid rapidly dissociates, but the receptor remains bound to DNA. Thus, the presence of steroid is not required for the receptor to remain bound to DNA in a high affinity manner. Treatment of cytosol containing transformed glucocorticoid-receptor complexes at 0 degrees C with 20 mM hydrogen peroxide also inactivates the DNA-binding activity of the receptor. The peroxide-induced inactivation is reversed by DTT. Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25 degrees C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. The presence of peroxide during heating of the cytosol also prevents dissociation of the receptor complex as assayed both by reduction in sedimentation value of the receptor and by dissociation of the 90-kDa heat shock protein from the steroid-binding protein. These results strongly suggest that critical sulfur moieties in the receptor complex must be in a reduced form for the temperature-mediated dissociation of the receptor to occur.     

Characterization of nonactivated and activated glucocorticoid-receptor complexes from intact rat thymus cells

In cells exposed to glucocorticoids at 37 degrees C activated glucocorticoid-receptor complexes (complexes with affinity for nuclei and DNA) are formed after nonactivated complexes. Activation thus appears to be an obligatory physiological process. To investigate this process we have characterized cytoplasmic complexes formed in rat thymocytes at 0 and 37 degrees C. Complexes in cytosols stabilized with molybdate were analyzed by sucrose gradient centrifugation and by chromatography on DNA-cellulose, DEAE-cellulose, and agarose gels. Two major complexes were observed: the nonactivated complex, eluted from DEAE at approximately 200 mM KCl, was formed at 0 and 37 degrees C, gave S20,w = 9.2 S, Stokes radius = 8.3 nm, and calculated Mr = 330,000; the activated complex, eluted from DEAE at approximately 50 mM KCl, appeared only at 37 degrees C, gave S20,w = 4.8 S, Stokes radius = 5.0 nm, and Mr = 100,000. A third, minor complex, probably mero-receptor, which appeared mainly at 37 degrees C, bound to neither DNA nor DEAE, and gave S20,w = 2.9 S, Stokes radius = 2.3 nm, and Mr = 27,000. With three small columns in series (DNA-cellulose, DEAE-cellulose and hydroxylapatite), the three complexes can be separated in 5-10 min. By this method we have examined the stability of complexes under our conditions. We conclude that in intact thymus cells glucocorticoid-receptor complexes occur principally in two forms, nonactivated and activated, and that activation is accompanied by a large reduction in size. The origin of the mero-receptor complex remains uncertain.