Visualization of X-chromosome inactivation mosaicism of Tfm gene in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) locus, which produces a deficiency in androgen receptors, is located on the X-chromosome. Steroid autoradiographic techniques were used to demonstrate the mosaicism of the X-chromosome inactivation in two androgen target tissues of XTfm/X+ heterozygous female mice. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), about half of the cells were receptor positive (Tfm gene inactive). Statistical analysis of coherent clone size was applied to the heterozygous mesenchyme of the urogenital sinus and the coherent clone size of receptor-positive cells was estimated to be two or three cells per clone. This small clone size suggests that considerable cell mixing occurred in the tissue during embryonic development. Androgen binding in the mammary gland rudiments was restricted to the mesenchymal cells only in close vicinity to the epithelial mammary bud. In the wild-type rudiments most of the mesenchymal cells beneath the epithelium were receptor positive, while in heterozygous rudiments, receptor-positive and -negative cells intermingled. This observation suggests that in the wild-type mammary gland rudiments the epithelial bud may induce the formation of androgen receptors in adjacent mesenchymal cells rather than attract pre-existing receptor-rich mesenchymal cells.     

Steroid hormone receptors and the sexual phenotype of the Harderian gland in hamsters

To investigate the participation of intracellular steroid hormone receptors in the sexual transformation process of the Harderian gland, a series of experiments were undertaken in adult golden hamsters. The invitro labelling of cytosolic steroid-binding sites with appropriate radioligands revealed the presence of androgen, oestrogen and glucocorticoid but not progestin receptors in the glands from animals of both sexes. The androgen receptor of the female gland was further characterized because it was found to be the predominant intracellular steroid receptor. Studies of binding kinetics using [3H]7 alpha,17 alpha-dimethyl-17 beta-hydroxy-4-oestren-3-one (DMNT) as ligand, demonstrated a high affinity androgen-binding site with an apparent dissociation constant (Kd) of 0.7 nmol/l and maximal saturation binding capacity of 84.0 +/- 3.0 (S.D.) fmol/mg protein. Specificity of the androgen receptor was assessed by displacement analysis; DMNT, 5 alpha-dihydrotestosterone, testosterone and 3 alpha-androstanediol were efficient competitors for the androgen-binding site, while oestradiol-17 beta, progesterone and dexamethasone exhibited very little, if any, competitive potency. The sedimentation coefficient of the androgen receptor in sucrose density gradients was 8-9 S. These data indicate that the physicochemical characteristics of the androgen receptor from the female gland are similar to those previously described in the male gland. The striking observation of a complete lack of oestrogen-inducible and oestrogen-insensitive progestin receptors in glands cytosol, even after stimulation with cholera toxin, adds further support to the concept that the androgen receptor is the key molecule mediating the hormone-induced sexual transformation of the Harderian gland in this species.     

Induction of androgen receptor formation by epithelium-mesenchyme interaction in embryonic mouse mammary gland.

The role of tissue interaction in the development of hormone responsiveness was studied in the embryonic mammary gland of the mouse, which becomes sensitive to testosterone on day 14. Previously, the mesenchyme had been identified as the sole target tissue for the hormone, although it was also demonstrated that its response to testosterone required the presence of mammary epithelium. Using autoradiography, we now show that [3H]testosterone or [3H]5 alpha-dihydrotestosterone is bound only by those mesenchymal cells closest to the epithelial mammary bud. When mammary epithelia were experimentally associated with mesenchyme of the mammary region and cultured together for 3 days in vitro, they also became surrounded by several layers of [3H]testosterone-binding mesenchymal cells. Correspondingly, this tissue association was accompanied by a substantial increase of androgen-binding sites in the explants. No hormone-building mesenchymal cells were seen in combinations with epidermis or pancreas epithelium; only salivary epithelium showed a weak positive effect. From these results we conclude that mammary epithelium induces the formation of androgen receptors in adjacent mesenchyme and thereby controls the development of androgen responsiveness in this tissue.     

Solubilization and properties of oxytocin receptors from rat mammary gland

Oxytocin (OT)-binding activity was extracted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate (CHAPSO) from rat involuted mammary glands with about 20% yield. The binding in detergent extracts was characterized and shown to be similar or identical to that of OT receptors on intact plasma membranes. Solubilized receptors had a high affinity site (Kd approximately 2 nM) and a lower affinity component, whereas the membrane receptor has only the high affinity site. Several synthetic OT analogs inhibited [3H]OT binding in the same rank order in both solubilized and intact membrane preparations. Both solubilized and membrane-associated receptor required Mn2+ for [3H]OT binding. The concentration of OT-binding sites in solubilized extracts of uterine myometrium from rats in late pregnancy was substantially greater in uteri from rats in labor than in that from rats 2 days before labor, as we have seen previously with receptors on intact membranes. The affinity of the solubilized myometrial receptor (Kd approximately 5 nM) was comparable to that of the membrane-associated receptor. Binding of [3H]OT to solubilized extracts of intestinal smooth muscle, which is not a target for OT, was negligible. Gel filtration analysis on columns of Sepharose 6B indicated that the solubilized [3H]OT-binding component from mammary gland was present in multiple mol wt forms, but the smallest major form eluted with an average apparent mol wt of about 40,000. These studies indicate that CHAPSO-solubilized binding sites for [3H]OT are the same as those in intact membranes and, therefore, are components of the OT receptor.     

Epidermal growth factor receptor levels in mouse mammary glands in various physiological states

Experiments were undertaken to demonstrate and characterize specific receptors for epidermal growth factor (EGF) in mammary glands of female BALB/c mice in various physiological states. The results of an in vitro desaturation technique are also presented which allow estimation of the total EGF-binding sites per mg membrane protein. Binding of the ligand [125I]iodo-EGF is both time and temperature dependent. Maximum binding to the membrane is achieved after 6 h of incubation with [125I]iodo-EGF at 23 C. Scatchard analysis of equilibrium binding using membrane preparations of mammary glands from virgin mice yields two classes of high affinity receptors with Kd values of 0.8 +/- 0.1 and 5.0 +/- 0.4 X 10(-10) M and receptor concentrations of 10 +/- 1.2 and 23.5 +/- 2 fmol/mg protein, respectively. Membrane preparations of mammary tissues from cycling, gestating, and lactating mice were used to correlate cellular receptor levels to the physiological state of the animal. Beginning at weaning, there is a constant decrease in high affinity receptor level with increasing age, as well as through the early stages of both gestation and lactation. On day 10 of gestation, receptor levels increase, reaching 15.2 +/- 1.6 fmol/mg protein, followed by a decrease to 3.8 +/- 0.9 fmol/mg protein on day 10 of lactation. We conclude that membrane preparations from the mouse mammary gland contain specific high affinity receptors for EGF, and that receptor levels are characteristic of the physiological state.     

Age-related changes in glucocorticoid and androgen receptors of cultured human pubic skin fibroblasts

In order to evaluate age-related changes in glucocorticoid receptor and androgen receptor of cultured human pubic skin fibroblasts in young and aged men, we determined both [3H]dexamethasone binding and [3H]methyltrienolone (R1881, potent androgenic steroid) binding, using dispersed whole cell assay. Scatchard analyses of specific [3H]dexamethasone binding to the fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean (+/- SD) binding site concentration (Bmax) of 12.69 +/- 2.36 X 10(4) and 12.87 +/- 12.21 X 10(4) sites/cell, respectively, and mean (+/- SD) dissociation constant (Kd) of 5.60 +/- 0.41 and 7.36 +/- 2.17 nM, respectively. Scatchard analyses of specific [3H]R1881 binding to the same cultured skin fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean Bmax of 5.77 +/- 1.02 X 10(3) and 2.82 +/- 0.97 X 10(3) sites/cell, respectively, and a mean Kd of 0.56 +/- 0.23 and 0.50 +/- 0.28 nM, respectively. These findings indicate that there were no significant age-related changes in binding site and affinity of glucocorticoid receptor in cultured human pubic skin fibroblasts, whereas binding sites of androgen receptor significantly decreased in those of aged men as compared to young men, without significant change in affinity.     

Evidence for an androgen receptor in porcine Leydig cells

Cytosol and nuclear androgen receptor concentrations were measured in freshly prepared and cultured Leydig cells of immature pig testis with exchange assays using [3H]methyltrienolone as labelled ligand. Androgen receptors in Leydig cells had high affinity for [3H]methyltrienolone and steroid binding specificity typical of an androgen receptor. The mean receptor concentrations were 76 fmol/mg protein and 210 fmol/mg DNA for cytosol and nuclei, respectively. In sucrose gradients, cytosol androgen receptors sedimented in the 4 S region. The cells maintained androgen receptors under culture conditions. Exposure of cultured cells to [3H]methyltrienolone (10 nmol/l) resulted in accumulation of androgen receptors in the nuclei with maximal uptake by 1 h. We conclude that methyltrienolone binding sites with characteristics of androgen receptors were identified in both cytosol and nuclei of porcine Leydig cells.     

Identification of serotonin 5HT2 receptors in bovine pineal gland

The concentration of serotonin in the pineal gland is extremely high, which prompted speculation that in addition to serving as a precursor of melatonin, serotonin may have an independent function of its own. By using [3H]-spiperone as a ligand, and ketanserine as a selective serotonin 5HT2 receptor antagonist, we have identified 5HT2 receptor in the bovine pineal gland, revealing a single population of binding sites with a dissociation equilibrium constant (Kd) value of 1.26 +/- 0.41 nM and a receptor density (Bmax) value of 193 +/- 38.85 fmol/mg protein. In displacement experiments, the concentrations of the drugs required to inhibit 50% of the specific binding of [3H]-spiperone in descending order of potency were methysergide greater than ritanserin greater than pirenperone greater than pipamperone greater than ketanserin greater than cyproheptadine greater than M-trifluoromethylphenyl-piperazine greater than prazosin greater than 5-methoxy-N-N-dimethyltryptamine hydrogen oxalate greater than 1-(3-chlorophenol) piperazine greater than serotonin. In the rat pineal gland, [3H]-spiperone revealed a low affinity serotonin binding site with a Kd value of 25.77 +/- 10.7 nM and a Bmax value of 1244 +/- 472 fmol/mg protein. The results of these studies are interpreted to indicate that the bovine pineal gland possess serotonin 5HT2 receptor. However, the rat pineal gland possess a serotoninergic binding site of unknown nature.     

Binding studies with rat myometrial and mammary gland membranes on effects of manganese on relative affinities of receptors for oxytocin analogs

Mg2+ increases the potency of oxytocin (OT) analogs in stimulating uterine contractions. Generally, the enhancing effects of Mg2+ are inversely related to the potency of the peptide. To determine the site of metal ion action, we measured the effects of Mn2+, another potentiating metal ion, on the ability of a series of peptides to inhibit the binding of [3H]OT to receptor sites on both uterine myometrial and mammary gland plasma membranes. The analogs used in this study were derivatives of 7-glycine oxytocin, which is about 10 times more active when the Mg2+ concentration in the uterine smooth muscle bath is increased from 0 to 0.5 mM. We found a generally good correlation between the ability of the analogs to inhibit [3H]OT binding to both receptor systems and their biological potencies. An increase in Mn2+ concentration from 1 to 10 mM enhanced the affinity of uterine membranes for the analogs, in inverse proportion to their potencies. This selective enhancement occurred regardless of the structural modification of peptide. These results suggest that the metal ion effect occurs at the receptor level and is not a property of the peptide per se. In contrast to the uterus, the affinities of mammary gland receptors for two low potency analogs were unaffected by increased Mn2+ concentration. The mechanisms of the metal ion effect are not entirely understood, but it appears that Mn2+ allows the conformation of the myometrial receptor to adapt to less well-fitting ligands. Although the metal ion effects on mammary gland receptors are more difficult to interpret, it is clear that uterine and mammary gland receptors are different with respect to the mechanisms of interaction with peptides.     

Progesterone receptors in mammary gland of the pregnant rat

The characteristics of pregnant rat mammary gland progesteronee receptors have been studied by the use of two synthetic probes, [3H]R5020 (3H-labeled 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) amd [3H]ORG 2058 (3H-labeled 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione). [3H] ORG 2058 bound with high affinity (Kd at 4 C, approximately 0.5 nM) to an apparent single class of displaceable sites, with a hierarchy of competition as follows: ORG 2058 greater than R5020 greater than or equal to progesterone greater than dexamethasone. In contrast, [3H]R5020 showed binding in mammary gland cytosols which was nonlinear with protein concentration, of intermediate affinity (Kd at 4 C, approximately 5 nM), and poorly displaced by nonradioactive R5020; the hierarchy of competitors was R5020 greater than progesterone greater than dexamethasone = ORG 2058. Parallel studies in rat uterine cytosols showed both [3H]ORG 2058 and [3H]R5020 to bind with high affinity to an apparently identical class of sites; the hierarchy of competition for both was ORG 2058 greater than or equal to R5020 greater than progesteron greater than dexamethasone. We conclude that in pregnant rat mammary gland, as opposed to rat uterus and other tissues, R5020 is unsuitable as a progesterone receptor probe; however, the affinity, capacity, ad specificity characteristics of progesterone receptors in this tissue can be described by the use of ORG 2058.     

Androgens control androgen-binding sites in rat epididymis

Previous results suggested that the number of androgen-binding sites in rat epididymis was controlled by androgens. Using an exchange technique for receptor determination, we now report that cytoplasmic receptor number decreased to about half the control value after 4 days of castration and reached its lowest level (30% of the control value) after 12 days of castration. The Kd for [3H]methyltrienolone for cytoplasmic receptor varied from 2.8 X 10(-9) M in controls to 0.6 X (10-9) M (P less than 0.001) after 6 or more days of castration. Nuclear binding sites were reduced to 17% of the control value from the second day of castration on, but no change in the affinity for methyltrienolone was noted. The administration of testosterone propionate (400 micrograms/day) to rats castrated for 20 days significantly increased the number of nuclear and cytoplasmic binding sites from the second and third days of treatment, respectively. Labeled thymidine incorporation into DNA rose significantly after a 4-day latency period. The proportion of total binding sites capable of translocation into the nucleus was elevated during the early phase (2-4 days) of androgen treatment. These results also suggest the heterogneity of the cytoplasmic binding site population. Control epididymides were composed of 61.5% receptor-containing epithelial cells, and this proportion was significantly reduced to 53.2% after 20 days of castration. Androgen administration elevated this percentage after an 8-day latency period. Proteolytic activity in cytosol was increased over control values after castration (for 20 days) and until the fourth day after the onset of androgen treatment. However, this activity does not seem to be an important factor in the decrease in binding sites caused by orchidectomy.     

3H]cortivazol: a unique high affinity ligand for the glucocorticoid receptor

Cortivazol (CVZ) and deacylcortivazol (DAC) are pyrazolosteroids with potent glucocorticoid activity. In previous work we showed that DAC is 40-fold more potent than dexamethasone (DEX) in lysing leukemic lymphoblasts. To assess the interaction between these atypical steroids and the glucocorticoid receptor, we examined the binding of [3H]CVZ to cytosol from glucocorticoid-sensitive and -resistant variants of the human leukemic cell line CEM C7. In glucocorticoid-sensitive cells [3H]CVZ causes a 2-fold induction of glutamine synthetase and binds to a protein in the 4.6 S region of high salt sucrose gradients. On DEAE-cellulose chromatography, [3H]CVZ-receptor complexes show a shift from high (0.25 M KP) to low salt (0.09 M KP) eluting forms upon activation. CVZ competes for a 97,000-dalton protein labeled by [3H]dexamethasone mesylate. Scatchard analysis of the binding of [3H]CVZ in glucocorticoid-sensitive cells revealed a curvilinear plot which resolved into high (0.4 nM) and low (11 nM) affinity components. The receptor concentration of the low affinity site (0.30 pmol/mg protein) was approximately twice that of the high affinity site (0.14 pmol/mg protein). Dissociation experiments with dilution and/or excess unlabeled CVZ supported the presence of independent sites. In contrast, the binding of [3H]DEX to C7 cytosol revealed a single class of binding sites (Kd = 1.9 nM; receptor concentration, 0.46 pmol/mg protein). Examination of the binding of [3H]CVZ using 10(-5) M DEX as the competing ligand showed that DEX binds only to the low affinity site detected by [3H]CVZ. In cytosol from a glucocorticoid-resistant cell line with virtually no [3H]DEX binding, [3H]CVZ detected a single high affinity binding site that was similar in dissociation constant (0.8 nM) and receptor concentration (0.13 pmol/mg protein) to the high affinity site detected in the glucocorticoid-sensitive cell line C7.     

Prostaglandin F2 alpha receptors on enzyme-dissociated pig luteal cells throughout the estrous cycle

A deficiency of luteal cell prostaglandin F2 alpha (PGF2 alpha) receptors might help explain the well documented refractoriness of pig corpora lutea to the luteolytic effects of PGF2 alpha administered in vivo before day 12 of the estrous cycle. Accordingly, experiments were conducted to measure the levels of [3H] PGF2 alpha-binding sites/receptors on collagenase-dispersed pig luteal cells taken at different stages of the estrous cycle. Pig corpora lutea were obtained surgically at various stages of the estrous cycle and dissociated with collagenase in medium 199. Dissociated mixed luteal cells (approximately 5-15 x 10(4) large luteal cells/tube) were assayed for [3H]PGF2 alpha-binding activity by saturation (Scatchard) analysis. In preliminary experiments it was determined that PGF2 alpha binding was maximal after incubation for 45 min at 30 C in assay buffer of pH 5.75. Additionally, it was determined that [3H]PGF2 alpha binding was displaceable by PGF2 alpha = PGD2 greater than PGE2 greater than 13,14-dihydro-15-keto-PGF2 alpha. Other eicosanoids did not inhibit [3H]PGF2 alpha binding. Two distinct classes of binding sites (high affinity Kd = 19-64 nM; low affinity Kd = 262-3103 nM) were observed at all stages of the estrous cycle. From studies using enriched (by elutriation) large (greater than 30 microns) and small (10-20 microns) luteal cells it appeared that the high affinity binding site was largely confined to large luteal cells, whereas the low affinity binding site was found on both large and small luteal cells. The concentrations (number per large luteal cell) of high affinity PGF2 alpha-binding sites of mixed (unelutriated) luteal cell preparations was low on days 6-8 (0.6 x 10(6) sites/cell) and increased gradually up to 1.4 x 10(6) sites/cell on day 12. The concentrations of binding sites were increased approximately 3-fold on day 13 (4.6 x 10(6) sites/cell; P less than 0.05 vs days 6-12) and remained elevated on days 14 and 16-17 (approximately 3 x 10(6) sites/cell). In summary, these results indicate the existence of a high affinity PGF2 alpha-binding site in pig (large) luteal cells, which is probably the luteal PGF2 alpha receptor. The numbers of these putative PGF2 alpha receptors are low during the early luteal phase (before day 12), but increase thereafter (days 13, 14, and 16-17). This may provide one explanation for the observed refractoriness in vivo of pig corpora lutea to PGF2 alpha before, and increased sensitivity to PGF2 alpha after, day 12 of the estrous cycle.     

Distribution of specific androgen binding sites within the ovine ovarian follicle

Two specific androgen binding sites were characterized in the ovine follicle with [3H]DHT, [3H]T and [3H]R-1881 as ligands, different incubation times and a charcoal separation step: the first, with characteristics very similar to testicular ABP in terms of its capacity, affinity, association and dissociation rates and specificity for natural and synthetic androgens, was found in serum, follicular fluid and the 27000 X g particulate and cytosol fractions of granulosa cells; the second, classic androgen receptors, were found in the cytosol with high affinity and low capacity for the synthetic androgen R-1881 and a very slow steroid-protein rate of dissociation. Saturation analysis on purified nuclei showed only the presence of the androgen receptor binding R-1881 with capacity similar to cytosol receptor. Isolated follicles showed a direct correlation between the total concentrations of androgen ([3H]-[3H]R-1881) binding sites and the follicular diameter. The complex actions which androgens exert on granulosa cell function may be mediated by interactions in vivo between these extra- and intracellular specific androgen binding proteins.     

Relationships between mammary estrogen receptor and estrogenic sensitivity. II. Binding of cytoplasmic receptor to chromatin

Mammary glands from nulliparous mice are responsive to estradiol, whereas mammary glands from lactating mice are unresponsive, despite the presence of high concentrations of estrogen receptors. This study examined the relation between mammary estrogenic sensitivity and ability of mammary estrogen receptors to bind to intact chromatin as well as to partially deproteinized chromatin. Mammary chromatin was prepared from nulliparous and lactating mice, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine chloride (Gdn X HCl). The binding of receptors to these various chromatin preparations was determined using partially purified [3H]estradiol-receptor complexes ([3H]ER) obtained by fractionation on diethylaminoethyl cellulose columns. The binding pattern of [3H]ER from nulliparous mice to chromatin fractions from either nulliparous or lactating mice revealed maximal binding activity with chromatin previously extracted with 4-6 M Gdn X HCl. Binding was of high affinity [dissociation constant (Kd) 3.6 X 10(-10) M], saturable and steroid receptor and species specific. However, mammary [3H]ER preparations from lactating mice bound poorly to intact chromatin as well as to the Gdn X HCl extracted chromatin fractions isolated from either mammary gland of nulliparous or lactating mice. In mixing experiments the estrogen receptor preparation from lactating mice decreased substantially the binding activity of [3H]ER from nulliparous mice to chromatin suggesting the presence of an inhibiting factor. Thus, these studies reveal that the unresponsiveness of lactating mammary glands to estradiol coexists with the inability of estrogen receptors from lactating mice to interact with specific high affinity sites on mammary chromatin and also that this impeded interaction of estrogen receptors with chromatin may be due to some inhibitor(s) present in the cytosol of lactating mammary glands.     

Purification of the muscarinic acetylcholine receptor from porcine atria.

The muscarinic acetylcholine receptor from porcine atria has been purified 100,000-fold to homogeneity by solubilization in digitonin/cholate and sequential chromatography on wheat germ agglutinin-agarose, diethylaminoethylagarose, hydroxylapatite, and 3-(2'-aminobenzhydryloxy)tropane-agarose. The yield of purified receptor was 4.3% of that found in the membrane fraction, and the purified receptor bound 11.1-12.8 nmol of L-[3H]quinuclidinyl benzilate per mg of protein, corresponding to a binding component Mr of 78,400-90,000. The purified receptor preparation consisted of two polypeptides in approximately equimolar amounts when examined on silver-stained sodium dodecyl sulfate/polyacrylamide gels. The larger polypeptide (Mr 78,000 on 8% polyacrylamide gels) was specifically alkylated with [3H]propylbenzilylcholine mustard, whereas the smaller polypeptide (Mr 14,800) was not labeled. The possibility that the small polypeptide is a contaminant fortuitously appearing in equimolar amounts with the large polypeptide cannot be ruled out at this time. The purified preparation was highly stable, with no measurable change in the number of ligand binding sites or the gel pattern after 1 month's storage on ice. Scatchard analysis showed a single class of binding sites for the antagonist L-[3H]quinuclidinyl benzilate with a dissociation constant of 61 +/- 4 pM. Equilibrium titration experiments demonstrated that the antagonist L-hyoscyamine displaced L-[3H]quinuclidinyl benzilate from a single class of sites (Kd = 475 +/- 30 pM), whereas the agonist carbamoylcholine interacted at two populations of sites (53% +/- 3% high affinity, Kd = 1.1 +/- 0.3 microM; 47% +/- 3% low affinity, Kd = 67 +/- 14 microM). The ligand binding data were very similar to that for the membrane-bound receptor, suggesting that the receptor has not been altered radically during purification.     

Demonstration and affinity labeling of a stereoselective binding site for a benzomorphan opiate on acetylcholine receptor-rich membranes from Torpedo electroplaque.

The interaction of an optically pure benzomorphan opiate, (-)-N-allyl-N-normetazocine [(-)-ANMC], with the nicotinic acetylcholine receptor from Torpedo electroplaque was studied by using radioligand binding and affinity labeling. The binding was complex with at least two specific components having equilibrium dissociation constants of 0.3 microM and 2 microM. The affinity of the higher affinity component was decreased by carbamoylcholine but not by alpha-bungarotoxin. The effect of carbamoylcholine was not blocked by alpha-bungarotoxin. In comparison, the affinity of [3H]phencyclidine, a well-characterized ligand for a high-affinity site for noncompetitive blockers on the acetylcholine receptor, is increased by carbamoylcholine and the increase is blocked by alpha-bungarotoxin. The binding of (-)-[3H]ANMC was inhibited by a number of other benzomorphans, with (-) isomers being 4- to 5-fold more potent than (+) isomers. Phencyclidine inhibits the binding of (-)-[3H]ANMC to its high-affinity site by a mechanism that is not competitive. UV-catalyzed affinity labeling indicated that the high-affinity-binding site for (-)-[3H]ANMC is at least partially associated with the delta subunit. Tryptic degradation of the Torpedo marmorata delta chain suggested that (-)-ANMC labeled a 16,000-dalton COOH-terminal portion of the subunit. In contrast, 5-azido-[3H]trimethisoquin, a photoaffinity label of the high-affinity site for noncompetitive blockers, labels a 47,000-dalton NH2-terminal fragment of the delta subunit. These results suggest that (-)-[3H]ANMC binds to sites completely distinct from the binding sites for acetylcholine. The high-affinity-binding site for (-)-ANMC and that for phencyclidine and 5-azidotrimethisoquin are allosterically coupled but are regulated differently and are probably physically distinct.     

Chimeric and point-mutated receptors reveal that a single glycine residue in transmembrane domain 6 is critical for high affinity melatonin binding

To delineate domains of high affinity melatonin receptors that are essential for melatonin binding, we generated chimeras between the human Mel1a melatonin receptor and the melatonin-related orphan H9 receptor. The latter receptor displays no high affinity melatonin binding. The chimeric receptors were transiently expressed in COS-7 cells and analyzed by radioligand binding using 2-[126I]iodomelatonin ([125I]Mel). Replacement of individual transmembrane domains (TMs) of the Mel1a receptor by the corresponding H9 helixes revealed that TM6 plays a critical role in ligand binding. Substitution of H9-TM6 into the Mel1a receptor abolished any detectable [125I]Mel binding, whereas the remaining TMs could be readily exchanged without affecting ligand binding. Subsequent site-directed mutagenesis showed that glycine 20 in TM6 of the Mel1a receptor occupies an important position in the binding site. Thus, the mutation of glycine 20 to threonine, the corresponding H9 residue, severely reduced the receptor's affinity for melatonin. Furthermore, the double mutation of alanine 14 to cysteine and of glycine 20 to threonine in TM6 completely eliminated high affinity [125I]Mel binding. This strongly suggests that molecular modifications in TM6 that involve glycine 20 lead to steric incompatibilities in the binding pocket that prohibit high affinity melatonin binding.     

Hormone regulation of the rodent Harderian gland: binding properties of the androgen receptor in the male golden hamster

Studies were conducted in castrated golden hamsters to assess whether sexual dimorphism and sensitivity to sex steroid hormones in the rodent Harderian gland are mediated by an interaction of androgens with specific intracellular receptors. Physical properties, binding kinetics and stereospecificity of the androgen receptor were analysed using [3H]mibolerone as the radioligand. The presence of [3H]mibolerone-androgen receptor complexes with a sedimentation coefficient of 7-8S was demonstrated in Harderian gland cytosol by a linear sucrose gradient ultracentrifugation technique using a vertical rotor. Kinetic analysis revealed an androgen-binding site with an apparent dissociation constant of 0.3 +/- 0.07 (S.D.) nmol/l and a saturation binding capacity of 113 +/- 15 fmol/mg protein. Displacement studies indicated that unlabelled mibolerone, methyltrienolone, 5 alpha-dihydrotestosterone and testosterone were efficient competitors for the androgen-binding sites, while progesterone, 17 beta-oestradiol, dexamethasone, dehydroepiandrosterone, ethiocholanolone and 5 alpha-16-androsten-3-one were not. Experiments in longterm castrated animals revealed that the Harderian gland androgen receptor concentration and sedimentation coefficient remained unmodified. The results of these studies were interpreted as demonstrating the presence of a specific high-affinity intracellular androgen receptor in the male hamster Harderian gland.