Hormone Binding Studies Research Articles

Regulation of Oestrogen Receptor Gene Expression: New Insights and Novel Mechanisms

17Beta-oestradiol is a pleiotropic hormone with organisational, activational and protective actions in both male and female mammals. It is responsible for numerous aspects of brain development, including sexual differentiation of the brain. The organisational effects of early oestrogen exposure are essential for long-lasting cognitive and behavioural functions. Oestradiol mediates its effects through several intracellular or membrane-associated receptor proteins. In the rodent cerebral cortex, oestrogen receptor (ER) expression, as demonstrated by hormone binding studies, is high early in postnatal life and declines precipitously as the animal approaches puberty. This decline is caused by a decreased expression of ERalpha mRNA. An understanding of the mechanisms involved in the regulation of ERalpha gene expression is critical for understanding the developmental as well as postpubertal expression of the ER. Despite recent data indicating the current hormone replacement therapies can be detrimental in older women, numerous animal studies have shown that the endogenous oestrogen, 17beta-oestradiol, is neuroprotective. Specifically, low levels of oestradiol protect the cortex from cell death caused by middle cerebral artery occlusion (MCAO). The attenuation of cell death by oestradiol in this model is mediated through an ERalpha-dependent mechanism. To this end, ERalpha expression is rapidly increased after MCAO, suggesting a return to the developmental programme of gene expression within neurones. One mechanism of suppressing gene expression is by the epigenetic modification of the promoter regions, which results in gene silencing. Of the epigenetic modifications utilised by cells, DNA methylation has been intensively studied as a mechanism by which genes are both permanently and reversibly silenced. Little is known about the mechanisms of ER gene regulation in the brain; however, in breast cancer cells, both ERalpha and ERbeta are down-regulated by promoter methylation, and subsequent binding of the methyl-CpG-binding protein, MeCP2. Data from our laboratory demonstrate that the promoters of the ERalpha gene are also methylated during development and after neuronal injury, suggesting a role of DNA methylation in regulating ER expression in the brain.

Transcriptional regulation of human insulin receptor gene by the high-mobility group protein HMGI(Y).

We have previously identified two closely related nuclear binding proteins that specifically interact with two unique functional AT-rich sequences of the 5' regulatory region of the human insulin receptor gene. Expression of these nuclear binding proteins increases during myocyte and adipocyte differentiation, and in other tissues appears to correlate with insulin receptor content. We have hypothesized, therefore, that insulin receptor expression in the insulin target tissues is regulated at least in part by these nuclear proteins. Here we show data on purification and biochemical characterization of these DNA binding proteins. Using a conventional chromatographic purification procedure combined with electrophoresis mobility shift assay and immunoblot analyses, a unique approximately 15 kDa protein, either identical to or highly related to the architectural transcription factor HMGI(Y), has now been identified, suggesting an essential role for HMGI(Y) in regulating insulin receptor gene transcription. Direct evidence of HMGI(Y) insulin receptor promoter interactions is provided by functional analysis with the CAT reporter gene and by hormone binding studies in cells expressing HMGI(Y) antisense RNA. In these experiments, antisense HMGI(Y) specifically inhibits insulin receptor promoter function and insulin receptor protein expression, indicating that HMGI(Y) is required for proper transcription of insulin receptor gene. Moreover, our data consistently support the hypothesis that a putative defect in this nuclear binding protein may cause insulin receptor dysfunction with subsequent impairment of insulin signaling and action.

Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones β1 and β3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a ∼3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37°C, OaER requires ∼80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37°C to 25°C or 20°C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25°C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER β3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRα apo, RARγ holo and ERα LBDs. Unliganded and 17β-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone β3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.

Selective Interaction of hsp90 with an Estrogen Receptor Ligand-binding Domain Containing a Point Mutation

The 90-kDa heat shock protein (hsp90) has been implicated in modulating steroid receptor function in vitro and in vivo. Previous studies have suggested that hsp90 interacts with large portions of the estrogen receptor (ER) ligand-binding domain and sequences of the receptor required for stable DNA binding. To characterize the interaction of the ER ligand-binding domain with hsp90, we have compared the properties of chimeras created by coupling the ligand-binding domain to the constitutive transactivator VP16-GAL. Two types of chimeras were created: VP16-GAL-ERG, containing the wild-type ligand-binding domain derived from the cDNA HEG0, and VP16-GAL-ERV, containing the substitution G400V derived from the ligand-binding domain of the original ER cDNA isolate, HE0. The G400V mutation alters the physical properties of VP16-GAL-ERV by rendering it hormone-dependent for DNA binding and more strongly dependent on estradiol for transactivation compared with VP16-GAL-ERG. Glycerol gradient analyses and chemical cross-linking/coimmunoprecipitation showed that, unlike VP16-GAL-ERG, VP16-GAL-ERV formed stable complexes with hsp90 in vitro. These data show that hsp90 selectively recognizes the altered ER ligand-binding domain containing the G400V substitution and indicate that the wild-type ER ligand-binding domain of VP16-GAL-ERG does not interact with hsp90 in vitro. Hormone binding studies showed that the ligand-binding domain of VP16-GAL-ERV was destabilized by incubation in the presence of high concentrations of salt or in the absence of sodium molybdate, conditions that disrupt its interaction with hsp90. The ligand-binding domain of the Val-400 ER thus behaves similarly to that of the wild-type glucocorticoid receptor, which has previously been shown to interact with hsp90 in vitro. These results provide evidence for the action of hsp90 as a molecular chaperone by selectively recognizing destabilized proteins.

Hsp90 Regulates Androgen Receptor Hormone Binding Affinity in Vivo

The regulation of human androgen receptor (AR) by the molecular chaperone Hsp90 was investigated using the yeast Saccharomyces cerevisiae as a model system. These studies were performed in strains expressing a conditional temperature-sensitive mutant allele of the hsp82 gene, which encodes Hsp90 protein. At the restrictive temperature in the mutant, there is a decrease in hormone-dependent transactivation by the AR, although steady state levels of AR protein are unchanged. Quantitative hormone binding studies at the permissive temperature revealed the presence of both high affinity and low affinity hormone binding states. At the restrictive temperature in the hsp82 mutant, the high affinity state was abolished, and only the low affinity state was observed. The change in hormone binding affinity was further investigated by a competition assay with the anti-androgen hydroxyflutamide. Under permissive conditions, hydroxyflutamide competes poorly for the synthetic androgen R1881, but under restrictive conditions in the hsp82 mutant strain, hydroxyflutamide was shown to be a potent competitive inhibitor. Our findings indicate that Hsp90 participates in the activation process by maintaining apoAR in a high affinity ligand binding conformation which is important for efficient response to hormone.

Acute effects of thyroid hormone on vascular smooth muscle.

The enhanced cardiovascular hemodynamics associated with triiodo-L-thyronine (T3) treatment is in part mediated by a decrease in systemic vascular resistance. To determine the molecular mechanisms for the vasoactive properties of T3, we studied primary cultures of aortic endothelial and vascular smooth muscle (VSM) cells. Active tension development by the VSM cells was measured by deformation lines within a siloxane matrix on which the cells were grown. Exposure to T3 (10(-10) M) resulted in cellular relaxation within 10 min. Hormone binding studies to purified VSM cell plasma membranes identified two binding sites specific for T3 with Kd of 1 x 10(-11) and 6.1 x 10(-8) M. L-Thyroxine and reverse T3 did not compete for the L-T3 binding sites. To determine an intracellular signaling pathway of T3 action, cAMP and cGMP content were measured in VSM cell cultures treated with T3. No quantitative changes were observed in a time frame known to cause VSM cell relaxation. The level of myosin light chain phosphorylation is a major determinant of smooth muscle contraction. Thus, treatment of VSM cells with isoproterenol, a vasodilator, caused a significant decrease in radiolabeled phosphate incorporation into the myosin light chains, whereas T3 had no effect on phosphorylation of these proteins. Primary cultures of vascular endothelial cells exposed to T3 showed no nitric oxide production as measured by cellular cGMP content and nitrite release, suggesting that T3 acted directly on the VSM cell to cause vascular relaxation.

Immunohistochemical localization and biochemical analysis of tyrosine kinases in the central nervous system

The regulation of cell activity, growth, and metabolism by a number of growth factor receptors and protooncogene products involves tyrosine kinase activity that results in the phosphorylation of tyrosine on protein substrates, the possible autophosphorylation of the kinase itself, and its direct interaction with “docking” proteins. The availability of specific antibodies against receptor kinases and phosphotyrosine (PY) provides a panel of immunochemically based methods that allow the identification, isolation, and precise localization of receptor kinases and PY protein substrates in defined brain areas. Immunochemistry coupled with ultrastructural analysis is able to define the location of receptor kinases and the protein substrates within a cell. Additional Information can be gathered by biochemical analysis, i.e., receptor isolation and hormone binding studies, measurement of the tyrosine kinase activity, Western blotting of PY proteins, and evaluation of the direct interactions of the kinases with other proteins potentially involved in signal transduction. The combination of morphological and biochemical methods provides the first step in understanding the functional role of these kinases in the central nervous system and related areas.

Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor. Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation.

A previous report from this laboratory (Reese, J.C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperature-sensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

Developmental regulation of ecdysteroid receptors in the nervous system of Manduca sexta.

The technique of steroid hormone autoradiography has been used to study the cellular distribution of ecdysteroid binding sites in the ventral nervous system of the tobacco hornworm moth, Manduca sexta. The ligand was 26-[125I]iodoponasterone. Tissue was examined from the subesophageal ganglia, thoracic ganglia, and abdominal ganglia of larvae at two times during the larval-pupal transient: the 2nd day of wandering and the prepupal stage. The patterns of neuronal binding seen were compared with those found in earlier autoradiographic studies of hormone binding in tissue sampled on the 1st day of wandering, in the pharate adult, and in the 4-day-old moth (Fahrbach and Truman, '89). The pattern of binding was reproducible but dependent upon developmental stage: whereas only a subset of neurons exhibited nuclear accumulation of radiolabeled ecdysteroids on the 1st day of wandering, less than 24 hours later nearly every neuron in the ventral nervous system was labeled. A limited pattern of binding, however, was seen again in the prepupal nervous system. Thus, the insect nervous system is able to use a single hormone both as a general cue for metamorphic development and as a single targeted to stage-specific subsets of neurons by alternating periods of ubiquitous expression of receptor with periods during which the capacity to bind the steroid hormone is highly restricted.

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.

Electronic determination of size and number in isolated unfixed adipocyte populations

Adipose tissue cellularity and metabolism are traditionally expressed in terms of mean cell size and number. The need for a simple method allowing rapid determination of cell size and number of freshly isolated, unfixed adipocyte preparations led us to compare estimates of cell size determined by the established method of optical sizing to a proposed method of electronic cell sizing and counting. In collagenase-isolated, unfixed adipocytes whose mean diameters ranged from approximately 40 to 65 microns (obtained from healthy rats weighing 100-360 g) the electronic method provided estimates of the mean cell diameter and size distribution that did not differ from the optical sizing technique. Estimates of mean cell diameter and cell number by the electronic method were rapid and reproducible (coefficients of variation 0.5 and 3.8%, respectively) and a less than 20 sec delay until sample analysis, after mixing of the adipocyte suspension, did not alter these estimates. Electronic determination of cell size and number, using freshly isolated, unfixed rat adipocyte populations (mean cell diameter less than or equal to 60 microns), is rapid and reliable. It will be particularly useful for studies of hormone binding and transport processes where it may be necessary to tightly control cell density.

Structural requirements for signal transduction of the insulin receptor

Structural requirements for signal processing by human placental insulin receptors have been examined. Insulin binding has been found to change the physico-chemical properties of (alpha beta)2 receptors solubilized with Triton X-100, indicating a marked alteration of the form, i.e. size and shape, of the molecular complex. (a) The Stokes radius decreases from about 9.5 nm to 7.9 nm, as determined by PAGE with Triton X-100 in the buffer (Triton X-100/PAGE), and from 9.1 nm to 8.7 nm, as assessed by gel filtration. (b) The sedimentation coefficient s20,w rises from 10.1 S to 11.4 S. Upon dissociation of the receptor-hormone complex, the alterations are reversed. After autophosphorylation of hormone-bound (alpha beta)2-insulin receptors, phosphate incorporation was found for 7.9-nm receptor forms when receptor-insulin complexes were crosslinked with disuccinimide suberate prior to Triton X-100/PAGE. However, phosphate incorporation was demonstrated for the 9.5-nm receptor forms when receptor-insulin complexes were not prevented from dissociation. This strongly indicates that the (alpha beta)2 receptor is autophosphorylated after assuming its 7.9-nm form upon insulin binding. Moreover, the insulin-dependent structural alterations are not affected by autophosphorylation. In contrast to (alpha beta)2 receptors, the diffusion and the sedimentation behaviour of alpha beta receptors, which carry a dormant tyrosine kinase even in the hormone-laden state, has been found to be insensitive to insulin binding. Different molecular properties of alpha beta and (alpha beta)2 receptors have also been detected by hormone binding studies. Insulin binding to (alpha beta)2 and alpha beta receptors differs markedly with respect to pH, ionic strength, and temperature. This might indicate that the structure of the hormone binding domain of alpha beta receptor changes on association into the (alpha beta)2 species. Alternatively, distinct hormone-induced conformational alterations at the molecular level of alpha beta and (alpha beta)2 receptor species may lead to the different binding properties. Our data demonstrate that the (alpha beta)2-insulin receptor undergoes extended conformational alterations upon insulin binding. This capacity for structural changes coincides with the hormone-inducable enhancement of tyrosine autophosphorylation of the 7.9-nm insulin-bound receptor form. In contrast, alpha beta receptors appear to be locked in an inactive nonconvertable state. Thus, interaction between two alpha beta receptor units is required to allow extended conformational alterations, which are assumed to be the triggering event for augmented auto-phosphorylation.

Do insulin and the insulin like growth factors (IGFs) stimulate growth of the exocrine pancreas?

Previous in vivo studies have suggested a long term regulatory role for insulin in the exocrine pancreas. Furthermore, we reported that pancreatic acini have specific receptors for IGF I and II, and using different techniques (acid washing, trypsinisation, electron microscope autoradiography), that CCK8 reduces the internalisation of IGF II. To now directly study the long term role for IGF and insulin in the exocrine pancreas we used AR42J cells, a rat cell line that is derived from a transplantable tumour of the acinar pancreas. Hormone binding studies with 125I-labelled hormones indicated that those cells have insulin receptors, relatively fewer receptors for IGF II but in contrast with normal acini no detectable IGF I receptors. Insulin at concentrations as low as 1 nm stimulated the growth of AR42J cells, as measured by an increase in cell number, DNA and protein content. At 100 nM insulin had maximal effects stimulating the growth by about 50%. IGF I and II had only very weak growth promoting effects probably due to their interaction with the insulin receptor. Additionally insulin increased amylase synthesis over the same concentration range that it stimulated growth. But immunoprecipitation studies revealed that insulin induced a selective increase of amylase synthesis over general protein synthesis. These studies indicate, therefore, that insulin is a growth promoting hormone for AR42J cells and that additionally it seems to specifically regulate amylase synthesis. The role for the IGFs in the exocrine pancreas, however, still remains to be determined.

The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition.

The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross-linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification.

Monoclonal antibodies to the rabbit liver growth hormone receptor: production and characterization.

Monoclonal antibodies to the GH receptor (GHR) have been produced by the application of hybridoma technology to splenic lymphocytes from BALB/C mice immunized with a human (hGH) affinity purified preparation of rabbit liver GHR. Primary screening of 384 wells yielded 4 antibodies able to immunoprecipitate [125I]iodo-hGH complexes with purified GHR and one able to inhibit binding of [125I]iodoovine GH ([125I]iodo-oGH) to rabbit liver microsomes. These cells were cloned and grown as ascitic tumors with loss of 1 of the 4 precipitators. Ascitic fluids contained monoclonal antibodies of high titer (inhibitor and 2 precipitators, 1:2.0 X 10(5); one precipitator, 1:2.0 X 10(4)) and high affinity (precipitators, 2.5-6.0 X 10(9) M-1; inhibitor, high affinity component, 6.4 X 10(10) M-1), which were isotyped as IgG1K and IgG2aK (1 precipitator). These antibodies did not cross-react with rabbit insulin or PRL receptors in the appropriate receptor assays and did not possess antihormone activity. Binding of [125I]iodo-MAb7, the inhibitory antibody, was totally blocked by the addition of excess unlabeled oGH or hGH, although these hormones had no effect on binding of the 125I-labeled precipitators. Scatchard analysis of [125I]iodo-oGH binding in the presence of MAb7 showed decreased binding by loss of sites rather than affinity. Antibody dilution curves and Scatchard plots for MAb7 binding provided evidence for two types of GHR in the rabbit liver, in accord with previously published data based on hormone binding studies. All precipitating antibodies gave an enhancement of [125I] iodo-oGH binding with purified receptor (up to 360% of polyethylene glycol-precipitated control), but only minimal enhancement with solubilized microsomal membranes. This enhancement was shown to be due to an increase in receptor number rather than affinity. After examining a number of hypotheses, we concluded that the enhancement was an artifact resulting from a nonpolyethylene glycol-precipitable species of GHR which could be totally precipitated by the monoclonal antibodies. We have produced and characterized four monoclonal antibodies to the GHR which will be of value in characterizing the structure and function of this receptor.

Corticosteroids and control of nasal salt gland function in the lizard Tiliqua rugosa

The scincid lizard Tiliqua rugosa possesses a well-developed nasal gland composed of both mucoserous and salt-secreting cells. Confusion over its secretory capacities (see H. Saint-Girons, M. Lemire, and S. D. Bradshaw, 1977, Zoomorphologie 88, 277–288) has been resolved and NaCl- and KCl-injected animals can secrete a hyperosmotic fluid with an F P ratio of about 3.6. The concentration of Na + in the secretion varied from a mean of 434 mmol/liter when sodium loaded to 167 mmol/liter when potassium loaded. Potassium concentrations varied from 226 to 433 mmol/liter, respectively. Na:K ratios thus vary from 1.98 with NaCl lcading to 0.42 with KCl loading, demonstrating the gland's capacity to vary the nature of the secretion. Rates of fluid production did not differ significantly between NaCl- and KCl-loaded individuals and varied from 13.3 to 19.5 μl (100 · g hr) −1. Adjacent studies on the north African agamid lizard Uromastix acanthinurus suggested that aldosterone may influence the rate and composition of the nasal gland secretions and this possibility was investigated in Tiliqua by hormone binding studies. High affinity binding for both corticosterone and aldosterone was demonstrated during the breeding season, with a K d of 5.2 × 10 −9 and 12.9 × 10 −9 M, respectively. Binding of aldosterone to nasal gland receptors was not evident in nonbreeding animals and the binding of corticosterone was primarily nonspecific in these individuals. These data suggest that hormone receptor concentrations and affinity vary on a seasonal basis and in concert with reproductive activity.

Changes in specific dexamethasone binding during aging in WI-38 cells.

Normal human diploid cell line WI-38, which shows enhanced proliferation in the presence of glucocorticoids, was found to contain high affinity binding sites for dexamethasone (DEX) and hydrocortisone. Hormone binding studies carried out in intact cell monolayers showed rapid uptake and binding of the [3H]DEX, which was independent of cell density at 37 C. The number of binding sites per cell decreased with increased age in vitro. Competition studies with several unlabeled steroids demonstrated high molecular specificity and a strong correlation between the ability of a steroid to compete for specific DEX binding sites and its ability to stimulate cell proliferation. Our observation that the number of specific glucocorticoid binding sites per cell decreased 40% without any significant change in affinity is similar to observations made in target tissues in aged animals. This finding may explain the previously reported decrease in responsiveness to these glucocorticoids in aging WI-38 cells.

THE PRESENCE OF LACTOGEN BUT NOT GROWTH HORMONE BINDING SITES IN THE ISOLATED RAT HEPATOCYTE

The binding of 125I-labelled human growth hormone (hGH) and bovine growth hormone (bGH) has been studied in hepatocytes isolated from female rats by perfusion with collagenase in situ. The cells appeared to retain normal membrane function, in that amino acid ([14C]alpha-aminoisobutyric acid) transport was both saturable and temperature-dependent. Amino acid ([14C]leucine) incorporation into protein was also linear over 3 h and was inhibited by cycloheximide. Binding of 125I-labelled hGH was dependent on time, temperature, hepatocyte concentration and hGH concentration. At 22 degrees C, binding reached a steady-state after 2-5 h and had a half-life of dissociation of 2-3 h. Hormone specificity studies indicated that binding was specific for hormones with prolactin-like activity (hGH, prolactins) and not for growth hormones themselves (bGH). Scatchard analysis revealed a single class of binding site with a binding capacity of 26-74+/-3-73 fmol/10(6) cells and a binding affinity of 1-24 X 10(9)+/-0-17 X 10(9) (S.E.M.) 1/mol (n=10). There was a significant sex difference in binding (female greater than male) and binding was subject to marked regulation by oestrogens (stimulation of binding) and by androgens (inhibition). The lactogen-binding sites, therefore, were comparable in many respects to those previously reported in rat liver membranes. No distinct GH binding sites were demonstrable as shown by the lack of specific binding by 125I-labelled bGH, purified either by Sephadex chromatography or by binding to and elution from GH receptors in rabbit liver membranes. The value of receptor purification of tracer for use in hormone binding studies was indicated by a substantial lowering of non-specific binding.

A comparative study of prostaglandin E 1 binding and activation of adenylate cyclase in normal, malignant and hybrid mammalian cells

Summary Partially purified plasma membranes have been prepared from a number of normal, malignant and hybrid cell lines which exhibit a wide variation in basal and prostaglandin E1-stimulated adenylate cyclase activity. The two lymphoma cell lines, EB-2 and P388F-36, are both characterized by low basal and hormonally stimulated levels of cAMP in intact cells. In broken cell preparations, P388F-36 exhibits a striking increase in hormonal response whilst the poor responsiveness of EB-2 cells is maintained. Hormone binding studies of P388F-36 correlate well with hormone activation. No specific binding of prostaglandin E1 can be observed in plasma membrane preparations of EB-2 indicating that low levels of the receptor may be the cause of the poor hormone responsiveness. The data thus suggests that distinct biochemical alterations have resulted in abnormally low levels of cAMP which are observed in the malignant cell lines. Comparison of hormone binding and activation in PCMI, a hybrid cell line produced by fusion of P388F-36 and CH23 cell lines suggests that the receptors of both parental lines are expressed in the hybrid but that the large variation in the affinities of the two types of receptor for the hormone results in the functional expression of only the high affinity form.

Insulin and Glucagon-Receptor Interactions in the Genetically Obese Zucker Rat: Studies of Hormone Binding and Glucagon-Stimulated Cyclic AMP Levels in Isolated Hepatocytes1

Insulin and glucagon binding and glucagon- stimulated cAMP accumulation were studied in liver cells isolated from 12–15 week-old female Zucker rats (lean: Fa/-; obese: fafa). The specific binding of [125I]iodoinsulin and [I25I]iodoglucagon was measured in cells isolated from 24 h-fasted rats and from fed rats that received 3 g glucose/kg BW ip 90 min prior to sacrifice. In all situations an apparent steady-state of binding was achieved after 60 min at 30 C for both [125I]iodoinsulin and [125I]iodoglucagon. Studies of binding, over a wide range of hormone concentrations showed that the specific binding of insulin was lower in the obese than in the lean rats but only in 24 h-fasted rats and at low insulin concentrations. In fed rats, no difference was observed between the fafa and the Fa/-. The specific binding of glucagon was similar in obese and in lean animals, but in both groups the binding was significantly higher in fed than in 24 h-fasted rats; this was observed over a wide range of glucagon concentr...