Cross-talk between ERs and signal transducer and activator of transcription 5 is E2 dependent and involves two functionally separate mechanisms.

Steroid hormone receptors and signal transducers and activators of transcription (STAT) factors constitute two distinct families of transcription factors activated by different signaling pathways. In previous reports, cross-talk between STAT5 and several steroid receptors has been demonstrated. We investigated putative cross-talk between ERalpha and ERbeta and STAT5. ERalpha and ERbeta were found to potently repress PRL-induced STAT5 transcriptional activity on a beta-casein promoter construct in a ligand-dependent manner. This down-regulation was found to rely on direct physical interaction between the ERs and STAT5, mediated via the ER DNA-binding domain (DBD). The contact between the ER DBD and STAT5 is highly specific; the interaction is abolished if the ERalpha DBD is replaced with the DBD of a closely related steroid receptor. The physical interaction, however, is insufficient to confer the repression of STAT5 activity, which in addition requires the ligand-activated C-terminal part of the ERs, although these domains are not in direct contact with STAT5. Negative cross-talk between ERs and STAT5 is thus mediated via several functionally separated domains of the ERs. Our findings may enhance the understanding of mechanisms of regulation of the different hormonal signaling pathways occurring during different functional events in tissues coexpressing ERs and STAT5.

Growth hormone-regulated intracellular signaling in UMR 106 osteosarcoma cells.

Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) are important growth factors for postnatal longitudinal bone growth. Although many effects of GH on bone growth are mediated by IGF-1, GH can directly influence bone cells. Limited knowledge exists regarding specific intracellular signaling pathways and genes activated by GH in bone cells. GH is known to activate several intracellular signaling pathways, among them the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. GH mainly activates JAK2 and both isoforms of STAT5, A and B. STAT5 gene deletion experiments have shown the importance of these transcription factors for growth. To understand the molecular mechanism(s) behind this, different experimental models are needed. The UMR 106 cell line is a rat clonal osteosarcoma cell line with osteoblast-like phenotypic properties, one is the endogenous expression of GH receptor (GHR). The present study focused on whether these cells express a functional GH-responsive JAK2/STAT5 pathway. Analysis of cell extracts by immunoprecipitation and Western blot showed that physiological concentrations of GH activated JAK2. Western blot analysis of nuclear extracts from GH-stimulated UMR 106 cells showed that physiological concentrations of GH induced nuclear translocation of both STAT5 isoforms, but with STAT5A being predominant. Both isoforms displayed similar nuclear turnover after GH stimulation of cells. Gel electrophoretic mobility shift assay (GEMSA) of nuclear extract revealed that both STAT5A and STAT5B obtained DNA-binding capacity after GH stimulation. Thus, we have shown, for the first time, the expression and GH-induced activation of JAK2 and STAT5A/B in UMR 106 osteoblast-like cells. This study also shows that this cell line is a suitable experimental model to study unique GH effects in osteoblasts mediated by STAT5.

Extracellular signal-regulated kinase (ERK) interacts with signal transducer and activator of transcription (STAT) 5a.

Serine phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 modulates their DNA-binding capacity and/or transcriptional activity. Earlier we suggested that STAT5a functional capacity could be influenced by the mitogen-activated protein kinase (MAPK) pathway. In the present study, we have analyzed the interactions between STAT5a and the MAPKs, extracellular signal-regulated kinases ERK1 and ERK2. GH treatment of Chinese hamster ovary cells stably transfected with the GH receptor (CHOA cells) led to rapid and transient activation of both STAT5a and ERK1 and ERK2. Pretreatment of cells with colchicine, which inhibits tubulin polymerization, did not inhibit STAT5a translocation to the nucleus and ERK1/2 activation. In vitro precipitation with a glutathione-S-transferase-fusion protein containing the C-terminal transactivation domain of STAT5a showed GH-regulated association of ERK1/2 with the fusion protein, while this was not seen when serine 780 in STAT5a was changed to alanine. In vitro phosphorylation of the glutathione-S-transferase-fusion proteins using active ERK only worked when the fusion protein contained wild-type STAT5a sequence. The same experiment, performed with full-length wild-type STAT5a and the corresponding S780A mutant, showed that serine 780 is the only substrate in full-length STAT5a for active ERK. In coimmunoprecipitation experiments, larger amounts of STAT5a-ERK1/2 complexes were detected in cytosol from untreated CHOA cells than in cytosol from GH-treated cells, suggesting the presence of preformed STAT5a-ERK1/2 complexes in unstimulated cells. Transfection experiments with COS cells showed that kinase-inactive ERK1 decreased GH stimulation of STAT5-regulated reporter gene expression. These observations show, for the first time, direct physical interaction between ERK and STAT5a and also clearly identify serine 780 as a target for ERK. Furthermore, it is also established that serine phosphorylation of STAT5a transactivation domain, via the MAPK pathway, is a means of modifying GH-induced transcriptional activation.

EGF modulates expression of STAT5 in mammary epithelial cells.

HC11 mouse mammary epithelial cells are capable of differentiating in vitro. By growing cells in EGF-containing medium, and upon confluence withdrawing EGF, these cells become competent at responding to lactogenic hormone treatment and expressing milk proteins. We found that during proliferation and at confluence STAT5A and STAT5B proteins were expressed at equal levels or with STAT5B being predominant. In competent cells, expression levels of STAT5A and STAT5B increased markedly with STAT5A now being the predominant form, an expression pattern resembling the expression patterns of STAT5 proteins seen during mammary gland differentiation in vivo. This suggests that EGF has a suppressive effect on STAT5 expression, in particular, STAT5A, which we conclude to be mediated through ras/raf/MEK/MAPK pathway and to a lesser extent through a PI3-kinase-mediated pathway. Furthermore, we also found that EGF regulated a nuclear phosphatase capable of dephosphorylating tyrosine-phosphorylated STAT5. Our data show that HC11 cells have retained the expression patterns of STAT5 proteins seen in vivo. This makes HC11 cells useful for studying molecular mechanisms regulating expression of STAT factors and their participation in differentiation processes of mammary gland.

In vitro interaction between STAT 5 and JAK 2; dependence upon phosphorylation status of STAT 5 and JAK 2.

A working model for haematopoietic cytokine signal transduction has been hypothesised as follows. Binding of cytokines to specific receptor molecules leads to phosphorylation and activation of receptor associated members of the Janus kinase family. This is followed by tyrosine phosphorylation of the associated receptor and members of the STAT (signal transducer and activator of transcription) family of DNA-binding transcription factors. Phosphorylation is accompanied by STAT dimerisation, nuclear transport and activation of gene transcription. Activation of gene transcription is mediated by the binding of STAT dimers to palindromic STAT response elements. A number of areas of confusion remain; not least the mechanism by which multiple cytokines signal via a limited number of STATs. A role has been suggested for phosphorylated receptor tyrosine residues as STAT docking sites on activated receptor-JAK complexes. According to this model the amino acid sequence context of key tyrosine residues confers receptor specificity upon STAT activation. There is some controversy as to whether this model applies to STAT 5. The heterologous expression of STAT 5 in Sf 9 insect cells using the baculovirus expression system is described here. Protein of the correct molecular weight was expressed and found to be phosphorylated on tyrosine residues and to bind to a STAT response DNA element. This binding was dependent upon the phosphorylation status of the STAT protein. DNA binding could be abolished in vitro by treatment with a phosphotyrosine phosphatase and restored in vitro by treatment with activated recombinant JAK 2. The protein was purified to near homogeneity using a simple ion exchange/gel filtration chromatography procedure. The interaction between purified recombinant STAT 5 and JAK 2, either expressed by baculovirus or endogenously expressed in Buffalo rat liver cells, was studied. In both cases STAT 5 in its non-phosphorylated form was found to form a stable complex with activated JAK 2. Non-activated JAK 2 and phosphorylated STAT 5 were unable to participate in complex formation. The results presented provide a mechanistic basis for the activation of STAT 5 by a wide range of cytokines capable of activating JAK 2.

Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C.

Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, Stat5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. However, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity were prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing protein synthesis. Furthermore, inhibition of protein synthesis potentiated GH-induced transcriptional activity in BRL-4 cells transiently transfected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-induced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 and mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Transactivation of SPIGLE1 by GH was potentiated by mepacrine and D609 but not by the phospholipase A2 inhibitor AACOCF3. Thus, a regulatory circuit of GH-induced transcription through the Jak2/Stat5-signaling pathway includes a prompt GH-induced activation of Jak2/Stat5 followed by a negative regulatory response; ongoing protein synthesis and intracellular signaling pathways, where phospholipase C activity is involved, play a critical role to desensitize the GH-activated Jak2/Stat5-signaling pathway.

Mitogen-activated protein kinase kinase inhibition decreases growth hormone stimulated transcription mediated by STAT5.

We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).

Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter.

The growth hormone regulated serine protease inhibitor (SPI) 2.1 and 2.2 gene promoters have been shown to contain a response element similar to the gamma-interferon activated sequence (GAS) family of signal transducer and activator of transcription (STAT) response elements. We have investigated the STAT and cytokine specificity of the SPI 2.1 STAT responsive element using a luciferase (LUC) reporter construct and a cDNA complementation strategy in the COS 7 cell line. Growth hormone was found to stimulate SPI-LUC reporter gene expression via activation of STAT 5, but not STATs 1 or 3, which indicates that the SPI 2.1 STAT responsive element is STAT 5 specific. In addition to the growth hormone receptor, the receptors for prolactin and erythropoietin enhanced gene transcription via the SPI 2.1 STAT responsive element, which indicates that this element is, on the other hand, not cytokine specific. Activation of STAT 5 was also observed after growth hormone treatment of cells transfected with cDNA expression plasmids for several different truncated growth hormone receptor mutants, although this activation was less efficient than with the wild type receptor. Point mutation of individual tyrosines in the growth hormone receptor intracellular domain to phenylalanines had no significant effect on signal transduction via STAT 5. These data, taken together with results from experiments using the phosphatase inhibitor sodium orthovanadate, suggest that STAT 5 may not have an absolute requirement for specific phosphorylated receptor tyrosine docking sites. That receptor tyrosine residues in a variety of amino acid contexts, or phosphorylated Janus kinase (JAK) 2 alone, can facilitate STAT 5 activation could explain the observed lack of cytokine specificity in STAT 5 activation.

Constitutive nuclear localization of Janus kinases 1 and 2.

Both GH and the GH receptor have been reported to undergo rapid nuclear translocation. Janus kinases (JAK) 1 and 2 have been implicated in GH receptor signaling, and both of these kinases are phosphorylated by GH stimulation. In this report, we have investigated the subcellular distribution of JAK1 and JAK2. Both JAK1 and JAK2 exhibit a nucleocytoplasmic distribution by immunocytochemistry in unstimulated serum deprived CHO cells stably transfected with rat GH receptor complementary DNA (cDNA). The nucleocytoplasmic localization of JAK2 was verified by immunogold electron microscopy in both rat liver hepatocytes and CHO cells stably transfected with rat GH receptor cDNA. Nucleocytoplasmic localization of JAK2 was also verified by transient tranfection of CHO cells with a Haemophilus influenzae haemagglutinin (HA) epitope tagged JAK2 expression plasmid and subsequent localization of HA immunoreactivity. Western blot analysis of purified nuclear extracts revealed the presence of immunoreactive JAK1 at 130 kDa and immunoreactive JAK2 at 128 kDa. No change in the nuclear content of JAK1 or JAK2 was observed upon ligand stimulation of GH receptor cDNA transfected cells with 100 nM human GH for 5, 10, 15, 30, or 60 min. GH stimulation caused, however, the appearance of tyrosine phosphorylated 42- and 44-kDa proteins as well as tyrosine phosphorylated JAK2 in the nucleus. The constitutive nuclear localization of the Janus Kinases is suggestive of a novel nuclear role for JAK family members, in addition to their described cytosolic function and presents an interesting challenge to the subcellular site of hormone action.

STAT-like DNA-binding activity in Spodoptera frugiperda cells.

Nuclear extracts from Spodoptera frugiperda (Sf9) cells were shown to contain a factor binding to DNA elements with gamma interferon activated site-like sequences. The DNA-binding activity was shown to be dependent on tyrosine phosphorylation. Hydrodynamic characterization of this entity revealed a Stokes radius of 8.4 nm and a sedimentation coefficient of 5.9 S. The molecular weight was calculated to 209,000. Estimated frictional (f/to) and axial (a/b) ratios indicated an elongated structure of this DNA-binding entity. This DNA-binding factor could represent a dimer of a Sf9 homolog to the mammalian signal transducers and activators of transcription.

Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity.

The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF-Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF-Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF-Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF-Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF-Stat5 or vectors encoding GH receptor and MGF-Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF-Stat5. The transcriptional induction of a beta-casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF-Stat5; it is not observed in EPO- and GH-treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte-macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF-Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF-Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mediation of growth hormone-dependent transcriptional activation by mammary gland factor/Stat 5.

Previous observations have shown that binding of growth hormone to its receptor leads to activation of transcription factors via a mechanism involving phosphorylation on tyrosine residues. In order to establish whether the prolactin-activated transcription factor Stat 5 (mammary gland factor) is also activated by growth hormone, nuclear extracts were prepared from COS-7 cells transiently expressing transfected Stat 5 and growth hormone receptor cDNA. Gel electrophoresis mobility shift analyses revealed the growth hormone-dependent presence of specific DNA-binding proteins in these extracts. The complexes formed could be supershifted by polyclonal anti-Stat 5 antiserum. In other experiments nuclear extracts from growth hormone-treated Chinese hamster ovary cells stably expressing transfected growth hormone receptor cDNA and liver from growth hormone-treated hypophysectomized rats were used for gel electrophoresis mobility shift analyses. These also revealed the presence of specific DNA-binding proteins sharing antigenic determinants with Stat 5. Stat 5 cDNA was shown to be capable of complementing the growth hormone-dependent activation of transcription of a reporter gene in the otherwise unresponsive COS-7 cell line. This complementation was dependent on the presence of Stat 5 tyrosine 694, suggesting a role for phosphorylation of this residue in growth hormone-dependent activation of DNA-binding and transcription.

Growth hormone pretranslationally regulates the sexually dimorphic expression of the prolactin receptor gene in rat liver.

The female-specific expression of the rat liver PRL receptor (PRL-R) gene was investigated by Northern analysis of hypophysectomized rats after two alternative human GH treatments that were to mimic either 1) the continuous female-specific or 2) the discontinuous male-specific serum GH patterns. The former (female-specific) pattern was shown to result in a dramatic increase in PRL-R mRNA in both males and females, while the latter (male-specific) pattern failed to evoke this response. A similar inductive effect in hypophysectomized females was shown after continuous administration of bovine GH and was found to constitute an approximately 60-fold increase in PRL-R mRNA levels. This effect by bovine GH, which, unlike the human isoform, is devoid of lactogenic properties, thus indicates the somatogenic origin of the signal resulting in this inductive response. These observations in conjunction with previous data obtained for other GH-regulated nonreceptor genes are interpreted to support the proposal of GH serum patterns being an early signal in a more general mechanism for pretranslational regulation of sex-specific gene expression. In contrast to GH, only a slight elevation of PRL-R mRNA was evoked by the ligand ovine PRL, while coadministration of ovine PRL with bovine GH failed to enhance the mRNA level found with bovine GH alone. The detection of previously unreported PRL-R mRNAs in liver of approximately 3.0, 3.8, and 5 kilobases in addition to the major 2.2-kilobase form was also evident after continuous GH administration.

Binding and cross-linking of iodinated rat prolactin to rat hepatic prolactin receptor.

Basal parameters for binding and cross-linking of 125I-rat prolactin (rPRL) to lactogenic (PRL) binding species present in crude membrane fraction (CMF) or detergent-solubilized preparations of rat liver have been investigated. (1) The highest specific binding to CMF was obtained with an incubation time of 50 h at 20 degrees C and with a 50 mM potassium phosphate buffer adjusted to pH 8.5. (2) Cross-linking of 125I-rPRL to binding sites in CMF with disuccinimidyl suberate (DSS) showed the autoradiographic appearance of an Mr 40,000 binding species. (3) No specific binding or cross-linking of rPRL was seen in Triton X-100-solubilized CMF. This is probably due to Triton X-100-induced changes in the physical properties of rPRL. (4) Specific binding of 125I-rPRL was detected in CHAPS-solubilized CMF. Following cross-linking the autoradiographic appearance of a binding species with an Mr value of 40,000 was shown. 125I-hGH was cross-linked to three PRL binding species with Mr 82,000, 40,000 and 35,000 in CHAPS-solubilized preparations. (5) In Golgi-enriched low-density membrane preparation 125I-rPRL was cross-linked to Mr 82,000, 40,000 and 35,000 species. It is proposed that the inability of rPRL to be cross-linked to Mr 82,000 and 35,000 species present in CHAPS-solubilized preparation is the result of CHAPS-induced changes of rPRL binding properties and low solubilizing capacity of CHAPS. (6) In conclusion, this study shows that also the iodinated endogenous hormone, rat prolactin, and not only hGH identifies high and low molecular forms of the rat liver prolactin receptor.

Detection of glycosylated growth hormone-binding proteins in rat serum.

Affinity cross-linking technique revealed the presence of three growth hormone-binding proteins (GHBP) in dealbuminized rat serum. The apparent molecular weights, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 52,000, 44,000 and 39,000. By use of carbohydrate chain cleaving enzymes it was found that the binding proteins contain N-linked complex carbohydrate chains, representing 8000, 4000 and 5000 in apparent molecular weight, respectively. Gel permeation chromatography and sucrose density gradient centrifugation revealed a growth hormone-binding entity with Stoke's radius 94.5 A (+/- 2.8, n = 5) and an s-value of 10.8 S (+/- 0.31, n = 5). The molecular weight could be calculated to 413,000. Both for gel chromatography and sucrose density gradient experiments unlabelled growth hormone reduced the radioactive peaks from 0 to 50%. The relation of this binding entity to the GHBP's described with affinity cross-linking technique and the nature of this binding entity is at present unclear. No serum binding protein(s) for prolactin was detected by affinity cross-linking technique, gel permeation chromatography or sucrose density gradient analysis.

Characterization of hepatic lactogen receptor. Subcellular distribution and characterization of N-linked carbohydrate chains.

The types of carbohydrate chains present in a rat liver lactogenic hormone-binding receptor species with an Mr of 82,000, and in its hormone-binding subunits with Mr values of 40,000 and 35,000, were characterized using carbohydrate-chain-cleaving enzymes and affinity cross-linking. The subcellular distribution of lactogenic hormone-binding species was studied in organelle-enriched fractions. The monomeric Mr-40,000 and Mr-35,000 species contain N-linked tri- or tetra-antennary complex and high-mannose chains respectively. The Mr-82,000 species exists in two forms, where the Mr-40,000 and Mr-35,000 subunits are each combined with unglycosylated and, with the technique used, unlabelled subunit(s). Studies with organelle-enriched fractions revealed that the Mr-35,000 species was found in an endoplasmic reticulum-enriched fraction. The Mr-40,000 species was the predominant monomeric binding species in Golgi/endosome- and plasma membrane-enriched fractions. It is suggested that the Mr-35,000 species is a precursor to the Mr-40,000 species. In lysosome/endosome- or lysosome-enriched fractions, a broad distribution in Mr (35,000-40,000) was characteristic of the hormone-binding species. The Mr-82,000 species was only found in a Golgi/endosome-enriched fraction. Labelling of endosome lactogen receptor by injection of 125I-labelled ovine prolactin in vivo and cross-linking yielded only the Mr-40,000 species. Thus, the Mr-40,000 and Mr-35,000 lactogenic hormone-binding species each appear to be combined with the unglycosylated receptor subunit(s) in the Golgi complex to form Mr-82,000 heterodimeric complexes.

Detection of specific binding of [1,6-3H]2,3,7,8-tetrachlordibenzo-p- dioxin ([3H]TCDD) in human leukocytes using electrofocusing in polyacrylamide gel.

The binding of [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) in human leukocyte cytosol has been studied using electrofocusing in polyacrylamide gel. One single peak of bound [3H]TCDD is found after completed focusing with an isoelectric point of 6.0. The binding capacity for 1.5 nM [3H]TCDD in the leukocyte cytosol was completely saturated by incubation in the presence of 150 nM unlabelled 2,3,7,8-tetrachlorodibenzofuran. [3H]TCDD also binds to a single binding species in human serum and plasma which focuses at pH 5.2 and which is not saturated by the addition of a 100-fold excess of unlabelled 2,3,7,8-tetrachlorodibenzofuran to the incubation. The levels of specific binding of [3H]TCDD in leukocytes from healthy blood donors ranged from 0 to 50 fmol/mg cytosolic protein.

Lactogenic and somatogenic binding sites in intact and detergent-solubilized membrane preparations of female rat liver.

The presence of lactogenic and somatogenic binding sites in intact microsomal membranes and in detergent-solubilized microsomal membrane preparations of female rat liver has been studied by affinity cross-linking-SDS/polyacrylamide-gel electrophoresis. In microsomal membrane preparations an Mr 40,000 lactogenic binder is present which is not disulphide-linked to another protein. Triton X-100 solubilization of membranes results in the appearance of three lactogenic 125I-human growth hormone (125I-hGH) binders with Mr values of 87,000, 40,000 and 35,000, and one somatogenic 125I-hGH binder with Mr 32,000. Treatment of rats with oestrogen increased the amount of lactogenic and somatogenic binding species in liver. The lactogenic binding sites are present as one entity in Triton X-100-solubilized preparations, clearly separated from the somatogenic binder as analysed by gel chromatography. Furthermore, 125I-hGH interacts with an Mr 95,000 somatogenic binder in membrane preparations to which the hormone can be cross-linked only following Triton X-100 solubilization.

Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking.

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.