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1.
Prolactin binding activity was studied in suspensions of cells which had been enzymatically dissociated from R3230AC mammary tumors, 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and lactating rat mammary glands. Prolactin bound specifically with high affinity (apparent binding affinity = 4.0 X 10(9) M-1) to R3230AC tumor cells. Hormone binding at room temperature was proportional to cell number and increased with time of incubation up to 120-180 min. Prolactin binding to R3230AC tumor cells from diabetic animals was reduced by about 50%. Specific prolactin binding activity was also demonstrated in preparations of cells from DMBA-induced tumors and lactating mammary gland. The levels of hormone binding in both dissociated cells and subcellular particles prepared from these tissues varied as follows: DMBA-induced tumors > lactating mammary gland > R3230AC mammary adenocarcinoma.  相似文献   

2.
3.
Characterization of prolactin receptors in pig mammary gland.   总被引:1,自引:1,他引:0       下载免费PDF全文
Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.  相似文献   

4.
Primary cultures of carcinogen-induced rat mammary tumors incubated at 37 degrees C with 125I-labeled ovine prolactin (5 ng/ml) accumulate intact prolactin. A steady state is reached at 24--48 h and loss of accumulated prolactin is slow t1/2 24 h). Accumulated prolactin is rapidly released when cryptic prolactin receptors are unmasked by energy depletion, suggesting that accumulated prolactin and cryptic receptors reside in the same cellular compartment. Under normal conditions, the accumulated prolactin is released slowly and is partially degraded. Subcellular fractionation on discontinuous sucrose gradients indicates that cryptic receptors reside in vesicle fractions (p less than or equal to 1.16). After energy depletion, the unmasked receptors are in cell surface membrane fractions (p = 1.18-1.20). Prolactin accumulation within receptor-containing vesicles in mammary tumor cells may account for their increased growth sensitivity (compared with normal mammary cells) to low physiologic levels of prolactin.  相似文献   

5.
Dwarf mice (DW/J;dw/dw) which exhibit a deficiency of prolactin and GH secretion were treated for 8 days with ovine prolactin and/or human GH (10 or 20 mug/day) and the effect on hepatic and testicular prolactin receptors was investigated. In both sexes there was a significant increase in body weight after all hormone treatments, but an increment in testicular weight was observed only after prolactin administration. Prolactin treatment increased the specific binding % of prolactin in liver membranes in females but not males, and in testicular homogenates (together with an increase in LH receptors). The results suggest that lack of prolactin but not of GH retards sexual development in these mice. Treatment with prolactin partly counteracts this deficiency, and the effect may be mediated by the induction of hepatic and testicular prolactin and LH receptors.  相似文献   

6.
Prolactin receptors were monitored by measuring 125I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (Kd) of 0.99 . 10(-9) M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydrocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (Kd) of the receptors.  相似文献   

7.
Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturable and specific, high affinity 125I-prolactin binding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes.  相似文献   

8.
B A Ponder  L V Crawford 《Cell》1977,11(1):35-49
Androgen-dependent and androgen-independent (autonomous), cloned, cultured cell lines of the androgen-dependent mouse mammary adenocarcinoma, Shionogi carcinoma 115, have been established. Growth of the dependent cells requires the presence of androgen, provided they are grown in suspension culture in medium containing dextran-charcoal-treated fetal calf serum. The growth rate of autonomous cells in the presence or absence of DHT is similar to that of dependent cells grown in its presence. An agar culture method has been developed that enables the proportion of dependent and autonomous cells in mixed populations to be determined. Autonomous cells appear in dependent clones, and their frequency increases with increasing time of subculture. Dependent cells from tumors preferentially in male animals, and dependent cell cytosols contain significant amounts (approximately 300 femtomoles per mg protein) of a specific androgen-binding macromolecule. Autonomous cells formed tumors equally well in both male and female mice, and autonomous cell cytosols contain very low levels (≤ 7 femtomoles per mg protein) of the specific androgen-binding macromolecule(s). These studies delineate a system which can be used to investigate the mechanism of steroid hormone-dependent and autonomous tumor growth, and the transitions between the hormone-dependent and autonomous states.  相似文献   

9.
Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.  相似文献   

10.
Prolactin receptors have been identified for the first time on human peripheral blood lymphocytes. These receptors are present on T- and B-cells as well as monocytes. The specific binding of [125I]prolactin to these cells can be selectively enhanced at certain concentrations and blocked by higher concentrations of cyclosporine , a known immunosuppressive agent which inhibits the mitogenesis of T-cells. Prolactin also induces ornithine decarboxylase, a key growth regulatory enzyme, in lymphocytes. Therefore, we suggest that the lymphocyte prolactin receptor may be involved in regulating lymphocyte function, and that one of the actions of cyclosporine is to block this rather ubiquitously occurring receptor.  相似文献   

11.
Purification and protein sequence analysis of rat liver prolactin receptor   总被引:4,自引:0,他引:4  
Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

12.
The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated, by the use, of labelled ovine prolactin) was increased 2–3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.  相似文献   

13.
Prolactin receptors were monitored by measuring 125I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (Kd) of 0.99 · 10?9 M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (Kd) of the receptors.  相似文献   

14.
Rat mammary tumors contain a unique class of cryptic cell-surface prolactin receptors that can be unmasked by depleting the cells of energy. These cryptic receptors, which are found in mammary tumors and nonlactating normal mammary cells but not in differentiated mammary tissue, are continuously inserted and rapidly removed from the cell surface. In this report we demonstrate that prolactin regulates the level of cryptic receptors. Treatment of primary cultures of rat mammary tumor cells with prolactin at concentrations between 0.1 and 0.5 ng/ml caused cryptic receptor levels to increase within 24 h, and this increase was maintained for up to 6 days. At prolactin concentrations of 10-50 ng/ml, receptor levels were the same as in cells incubated without hormone, while a decrease in the steady-state level of cryptic receptors was induced within 24 h by 100-500 ng prolactin/ml. Concentrations of 1,000-5,000 ng prolactin/ml caused a rapid, dose-dependent down regulation of cryptic receptor sites. Down regulation at 5,000 ng prolactin/ml was (1) complete (84 +/- 5% reduction) in 1 h; (2) specific for lactogenic hormones; (3) completely reversed within 10 h after prolactin removal; (4) energy dependent; and (5) not blocked by the cytoskeleton active agents cytochalasin B and colchicine or by NH4Cl, which inhibits hormone degradation. We conclude that rat mammary tumor cells have the capacity to auto-regulate cryptic prolactin receptors, a property that supports our notion that such receptors play a role in regulating prolactin responsiveness. The observed pattern of cryptic receptor autoregulation in response to prolactin concentration and time of exposure suggests that a pool of cryptic sites provides these cells with the capacity to respond to prolactin concentrations from pg to microgram/ml, a range well beyond the Kd for the receptor itself. Since prolactin receptors in mammary tumors are not down regulated unless prolactin concentrations are well beyond the saturation point, these cells may have a selective growth advantage over cells in normal mammary tissue.  相似文献   

15.
Prolactin regulates a variety of physiological processes, including mammary gland growth and differentiation, and recent findings support an important role in breast cancer development and progression. However, little is known about the trafficking of its receptor, a member of the cytokine receptor superfamily. In the present study, we examined the effect of ligand on the endogenous "long" isoform of the prolactin receptor in breast cancer cells. We found that prolactin caused rapid and prolonged down-regulation of this receptor. The prolactin-induced increase in degradation was blocked by inhibitors of both proteasomes and lysosomes. However, the ubiquitin-conjugating system was not required for internalization. Prolactin also resulted in the concomitant appearance of a cell-associated prolactin receptor fragment containing the extracellular domain. This latter process required proteasomal, but not metalloprotease, activity, distinguishing it from ectodomain "shedding" of other membrane receptors, which are secreted as binding proteins. The prolactin receptor fragment was labeled by surface biotinylation and independent of protein synthesis. Together, these data indicated that prolactin binding initiates limited proteasomal cleavage of its receptor, generating a cell-associated fragment containing the extracellular domain. Our findings described a new potential mediator of prolactin action and a novel mechanism whereby proteasomes modulate cellular processes.  相似文献   

16.
Two types of prolactin receptors were identified in sow mammary gland. When light membranes were prepared on a discontinuous sucrose gradient (0.3 and 1.7 M) and then diluted and washed with 0.3 M sucrose solution, a large amount (about 50%) of receptors were released from membranes and appeared in the supernatant fraction. These two forms (hydrophobic and water-soluble) of receptors were characterized as having the same binding specificity for lactogenic hormones and a similar affinity constant for ovine prolactin (K alpha approximately 10-12 X 10(9) M-1). Polyclonal antibodies and one monoclonal (mAb M110) antibody, obtained against partially purified prolactin receptors from rabbit mammary gland, cross-reacted effectively with sow mammary receptors. They completely inhibited the specific binding of [125I]oPRL to membrane and water-soluble receptors. The present studies indicate that the two types of sow prolactin receptors could represent the same molecular entity and confirm that prolactin receptors from rabbit and sow mammary gland exhibit numerous antigenic similarities.  相似文献   

17.
We have examined the binding capacity and properties (affinity, specificity) of LH and prolactin (Prl) receptors in a transplantable rat Leydig cell tumor (H-540) grown in intact, castrated and hypophysectomized rats. LH receptors in adult rat testis and Prl receptors in the rat ventral prostate were examined simultaneously for comparison. The results can be summarized as follows: The qualitative properties (affinity, specificity) of LH and Prl receptors in tumor Leydig cells appear to be identical to those of corresponding receptors in non-tumor tissues. The levels of LH receptors in tumor Leydig cells are only some 1% of that present in normal Leydig cells from adult rats. Tumor Leydig cells grown in hypophysectomized rats had even lower levels of LH receptors; ca. 1/3 of that found in tumors from intact rats. The levels of Prl receptors in the tumor Leydig cells are almost as high as in normal Leydig cells from adult rats. In tumors grown in hypophysectomized rats, the levels of Prl receptors were much lower (ca. 20%) than in tumors from intact or castrated rats. There were great variations in the number of LH and Prl receptors in individual tumors, and there was a positive correlation (r = 0.88; P less than 0.01) between LH and Prl receptors in individual tumors. No differentiation toward a "LH receptor tumor" or "Prl receptor tumor" was observed. Thus, receptors for LH and Prl in tumor cells are qualitatively normal, but the number is greatly (LH) or moderately (Prl) reduced. These receptors in the tumor Leydig cells are stimulated by pituitary hormones.  相似文献   

18.
Optimal conditions for the quantitation of free prolactin binding components of human prostatic tissue obtained by TURP were studied by applying γ receptor assay. The radioligand used was 125I-prolactin. Significantly greater heat stability of the prostate membrane prolactin binding sites, when compared to that of androgen cytoplasmic receptors, was confirmed. The saturability and specificity of the prolactin binding components was demonstrated by the results of both Scatchard plot analysis and displacement studies. Free prolactin receptors were found in none of the poorly differentiated (G3) prostatic tumors examined, and only in 62.5% of medium differentiated (G2) prostatic malignancies. The majority of tissue specimens coming from patients with either BPH or well differentiated prostatic tumor (G1) contain measurable amounts of free prolactin membrane binding components. In the present study we report also the case in which the change in tumor differentiation toward a higher grade (G2 to G1, provoked by the successful chemohormonal treatment) is accompanied with the appearance of previously absent free prolactin binding components. In histologically proven BPH tissue specimens free prolactin receptor negative status has been found in most patients with a slight increase in serum PAP values, while receptor rich status was detected in the majority of those with elevated PSA concentrations. We believe therefore that the prolactin receptor values, when used as part of the multivariable analysis, may participate in further delineation of the role of prolactin in the development of prostate cancer, but may also play a role in a subclinical prediction related to the conversion of either an adenoma or a latent adenocarcinoma to the clinically manifest prostatic malignancy.  相似文献   

19.
The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of 125l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased 125l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.  相似文献   

20.
The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low pH than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pKa value of a histidine side chain is ~6.8 making histidine residues obvious candidates for examination. From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone–receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology. Copyright © 2010 John Wiley & Sons, Ltd.  相似文献   

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