Higher order genomic organization and epigenetic control maintain cellular identity and prevent breast cancer.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

Starvation and hypothyroidism exert an overlapping influence on rat hepatic messenger RNA activity profiles.

To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together with T3-independent mechanisms initiated in the starved animal may constitute redundant processes designed to conserve energy and substrate in the nutritionally deprived organism.

Hepatic messenger ribonucleic acid activity profiles in experimental azotemia in the rat. Relationship to food intake and thyroid function.

We have studied the hepatic messenger RNA (mRNA) activity profile in chronically azotemic rats and sought to determine whether the observed changes could be mediated either by reduced food intake or diminished thyroid function at the tissue level. mRNA activity profiles were produced by two-dimensional gel electrophoretic separation of radioactively labeled products of an in vitro reticulocyte lysate system which had been programmed by hepatic RNA. Of the approximately 240 translational products identified in this system, seven sequences were consistently altered in azotemia. In pair-fed animals six of these also decreased, but the alterations in three were depressed to a significantly lesser extent in the pair-fed group. Moreover, analysis of covariance suggested that food intake could account for the differences in only one sequence. The possibility that the mRNA activity profile in azotemia could represent the effects of diminished thyroid function was minimized by the finding that the reductions in plasma thyroxine (T4) and triiodothyronine (T3) levels observed were due largely to reduced plasma protein binding, with maintenance of the mean free T4 and free T3 concentrations within the normal range. The changes in only one mRNA sequence could be related to free T3 levels alone. Our findings, therefore, indicate that although diminished food intake and reduced thyroid function may contribute to some of the observed changes in the mRNA activity profiles, the bulk of alterations in azotemia appear to be mediated by other mechanisms. The striking overlap between the sequences affected by azotemia and pair-feeding raises the speculation that altered gene expression in azotemia may reflect an impaired hepatic response at the pretranslational level to metabolic signals associated with food intake.

Differential thyroid hormone-regulated rat thyrotropin beta gene expression detected by blot hybridization.

In the rat, there is a single TSH beta-subunit gene represented by three exons interrupted by two introns. This gene contains two promoters which determines the synthesis of two mRNAs with 5'-untranslated regions that differ by 43 base pairs. This study evaluates the steady state levels of these TSH beta mRNAs in various thyroidal states. Blot hybridization analyses of pituitary mRNA with synthetic probes designed to detect either one or both TSH beta mRNAs were performed. One probe corresponds to 24 bases in the 5'-untranslated region of mRNA1 and a second corresponds to 25 nucleotides in the coding region and detects both mRNA1 and mRNA2. These studies indicate the presence of TSH beta mRNA species of indistinguishable size consistent with the presence of two TSH beta mRNAs that contain slightly different 5'-untranslated regions. Comparison of pituitary RNA obtained from normal and hypothyroid rats reveals that the shorter mRNA (mRNA2) is increased approximately 6- to 8-fold with hypothyroidism while the abundance of the longer mRNA (mRNA1) is relatively unchanged. Treatment of either normal or hypothyroid animals with T3 decreases the abundance of mRNA2 while again mRNA1 is relatively unaffected. Thus, although both mRNAs are detected, only one mRNA is dramatically altered by thyroidal status. Therefore, the single rat TSH beta gene is transcribed into two mRNAs via the use of alternative promoters of which only one is markedly regulated by thyroid hormones.

Thyroid hormones regulate rat thyrotropin beta gene promoter activity expressed in GH3 cells.

Thyroid hormones suppress the synthesis of TSH in part by decreasing the rate of alpha and TSH beta gene transcription. Cis-acting DNA sequences present in the rat TSH beta subunit gene that are induced in transcriptional regulation by thyroid hormone have been identified by deletion-mutation and transient expression studies. Plasmid expression vectors were constructed including 2900, 900, 204, 77, 17 base pairs (bp) of 5'-flanking sequence and exon (5'-untranslated sequence, transcriptional start sites) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. The transfected chimaeric plasmids demonstrated expression (with TSH beta DNA sequences in the 5'- to -3'-but not 3'- to -5'-orientation) in both a clonal pituitary cell line, GH3, and primary pituitary cell cultures, both of which are responsive to thyroid hormones. T3 (10(-11) M to 10(-7) M) treatment of transfected cells produced a dose-dependent decrease in CAT expression with a maximal 70% decrease at 10(-8) M. While a decrease in the basal level of expression was noted with progressive removal of both 5'-flanking and intronic sequences adjacent to exon 1, the fold-decrease in response to T3 was equivalent even in the 57 bp construct. In contrast, T3 had no effect on CAT expression directed by the promoter of the herpes simplex virus thymidine kinase gene. Thus, the rat TSH beta gene 5'-flanking region can direct heterologous gene expression in GH3 cells and contains sequences which have properties of a putative cis-active T3 responsive regulatory element(s).2+he

Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells.

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the thyroid hormone-responsive messenger RNA spot 11 as apolipoprotein-A1 messenger RNA and effects of the hormone on the promoter.

The induction of rat hepatic mRNA S11 by L-T3 (T3) is a useful model for studying the mechanisms of thyroid hormone action. Although numerous reports have examined the response of mRNA S11 to various physiological and hormonal manipulations, the role of S11 protein in cellular metabolism remains unknown. In this study we show that mRNA S11 is abundantly expressed and regulated by T3 only in liver and small intestine. High levels of the mRNA are present at birth, but drop sharply between 30-60 days of age. These and other features of the S11 gene product were similar to those of rat apolipoprotein-A1 (Apo-A1). The sequence of S11 cDNA was identical to a portion of the Apo-A1 mRNA, thus confirming identity of the S11 mRNA. To examine whether DNA sequences immediately adjacent to the transcription start site mediate the effects of thyroid hormone, we measured the activity of an Apo-A1 gene fragment, U-1 (-474 to -7) using a transient transfection assay. The activity of the full-length U-1 DNA in HuH-7 hepatoma cells was 2- to 2.5-fold higher in the presence of thyroid hormone. This finding closely matched previous results using the in vitro nuclear run-on assay. Internal deletion of a motif that resembles a thyroid hormone response element from U-1 DNA not only abolished the induction by T3, but suppressed promoter activity by 3- to 4-fold in response to the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of detectable chorionic gonadotropin subunit messenger ribonucleic acids in the rat placenta throughout gestation.

We hypothesized that if a CG similar in structure to other glycoprotein hormones was present in the rat placenta, then mRNAs encoding its subunits would be detectable. To investigate the possible presence of a CG in the rat, we attempted to detect mRNAs encoding the alpha- and CG beta-like subunits in the placentae of timed pregnant rats by sensitive blot hybridization analyses using cloned cDNAs encoding alpha- and beta-subunits of LH derived from rat pituitary mRNA. No subunit-specific mRNAs in placentae of pregnant rats at 4-21 days gestation were detected at either high or low stringencies of hybridization. However, under similar conditions, subunit-specific mRNAs were readily observed in total pituitary RNA of normal as well as ovariectomized rats. Moreover, hybridization with a cDNA for alpha-tubulin, a major component of the cytoskeleton, yielded easily detectable bands in rat placental RNA. In addition, hybridization analysis, under low stringency conditions, of restriction enzyme digests of rat genomic DNA with a rat LH beta-cDNA, which would detect LH beta subunit-like genes, suggests the presence of a single gene that, in fact, encodes the rat LH beta-subunit. We conclude that mRNAs encoding for proteins structurally homologous to rat LH subunits are absent in the rat placenta and that only a single LH beta-like gene is present in the rat. The luteotropic activity associated with the rat placenta must be related to a gonadotropin-like hormone whose structure is dissimilar from that of CG.

A cryptic peptide from the preprothyrotropin-releasing hormone precursor stimulates thyrotropin gene expression.

The precursor peptide of TRH (prepro-TRH) contains five copes of pro-TRH linked by other peptide sequences. These peptides are coprocessed with TRH in the median eminence of the hypothalamus and released into the portal circulation, rendering this family of peptides available to act at the level of the anterior pituitary. Therefore, we tested the potential bioactivity of one cryptic peptide, prepro-TRH amino acids 160-169 [prepro-TRH-(160-169)], in a TRH-responsive pituitary cell line (GH3). In a heterologous TSH expression assay, we found that prepro-TRH-(160-169) stimulated TSH beta gene promoter activity in a time- and dose-dependent manner; moreover, the effect of prepro-TRH-(160-169) was more rapid and of greater magnitude than that of TRH on TSH beta-directed chloramphenicol acetyltransferase synthesis. In the same cells, we found that prepro-TRH-(160-169) stimulated PRL synthesis and secretion, but the effect was similar in magnitude and duration to that of TRH. The effect of prepro-TRH-(160-169) appears to be additive to that of TRH, suggesting that prepro-TRH-(160-169) may act through a mechanism separate from that of TRH. Thus, prepro-TRH-(160-169) has potent endocrinological effects at the level of the genome.

Rapid simultaneous measurement of rat alpha- and thyrotropin (TSH) beta-subunit messenger ribonucleic acids (mRNAs) by solution hybridization: regulation of TSH subunit mRNAs by thyroid hormones.

Using a sensitive method for the simultaneous measurement of TSH subunit mRNAs, we have investigated their hormonal regulation in the pituitary glands of normal and hypothyroid rats. Oligodeoxyribonucleotides (probes) complementary to coding regions of rat alpha- and TSH beta-subunit mRNAs were synthesized. These probes were 5'-end labeled with gamma-[32P] ATP and hybridized with total pituitary RNA obtained from T3-treated and untreated normal and hypothyroid rats. The samples were then exposed to S1 nuclease to digest single stranded nucleic acids. Specific hybridization of probes to the TSH subunit mRNAs would yield double stranded structures resistant to this enzyme. Measurement of the amount of undigested probes by denaturing polyacrylamide gel electrophoresis, autoradiography, and densitometry permits quantification of these mRNAs. Both rat alpha and TSH beta mRNAs were detected with as little as 0.1 microgram total pituitary RNA, representing a more than 10-fold increase in sensitivity compared to a standard RNA blot hybridization assay. Thyroidectomy resulted in a 3- to 5-fold increase, whereas T3 treatment caused a significant decrease in the subunit mRNAs in both normal and hypothyroid animals. However, in all treatment groups, the TSH beta mRNA was affected to a greater extent than the alpha mRNA by the changes in thyroid status. The ratio of alpha- to beta-subunit mRNAs was decreased with hypothyroidism and increased with T3 treatment. This assay allows simultaneous quantification of multiple mRNAs from a single pituitary gland within 48 h and should facilitate studies of the regulation of mRNAs encoding TSH subunits specifically and other pituitary proteins in general.

Distribution of thyroid hormone-responsive translated products in rat liver polysome and postribosomal ribonucleoprotein populations.

We investigated the possibility that hypothyroidism, like starvation, leads to a major disaggregation of polyribosomes and redistribution of mRNA to the nonpolysomal mRNA particle (mRNP) pool. The analysis involved the use of a translational assay of hepatic RNA obtained from bound and free polysomes and the subribosomal fractions (mRNP) of euthyroid and hypothyroid rats. Two-dimensional gel electrophoretograms of the in vitro synthesized 35S-labeled proteins revealed distinct protein patterns associated with each subcellular compartment. Of the 18 quantified T3-responsive sequences, the activities of 7 were preferentially associated with bound polysomes, 9 with free polysomes, and 2 with subribosomal mRNP in euthyroid rat livers. No major redistribution occurred in the transition from the euthyroid to the hypothyroid state. The distribution of mRNA activity of 1 T3-responsive sequence, that coding for Spot 14 (mRNAS14), was compared with the distribution of hybridizable mRNA, as determined with the use of a specific cDNA probe. In the euthyroid state, both techniques showed that approximately 70% of the total hepatic mRNAS14 was associated with free polysomes and 30% was associated with subribosomal complexes. A similar distribution was characteristic of the hypothyroid state. These findings demonstrate that T3-responsive sequences are found in all three mRNA compartments and that thyroid hormone action is not typically associated with a redistribution of mRNA among subcellular pools.

A cryptic peptide (160-169) of thyrotropin-releasing hormone prohormone demonstrates biological activity in vivo and in vitro.

TRH is synthesized as a precursor peptide containing five copies of the sequence Gln-His-Pro-Gly, QHPG, flanked by paired basic amino acids, and linked by other peptides. We tested one cryptic peptide, PPT (160-169, SFPWMESDVT), as a possible physiological regulator of pituitary activity in vivo. Male rats were cannulated (jugular) and received a single dose of either PPT or TRH (10(-8)-10(-6) M). PPT caused no consistent effects on either TSH or PRL secretion, while TRH stimulated the secretion of both hormones. However, PPT stimulated a dose-dependent increase in both pituitary TSH beta and PRL mRNA content at 240 min similar to TRH. In primary cultures of rat pituitaries, PPT stimulated a maximum 4-fold increase in TSH beta mRNA and a 2-fold increase in PRL mRNA in 4 h, while TRH increased both TSH beta and PRL mRNA approximately 3-fold. Again, PPT had no significant effect on TSH or PRL secretion into the medium. Thus, PPT appears to be a physiological regulator of both TSH and PRL synthesis, but, unlike TRH, does not act as a secretagogue.

Effects of triiodothyronine and glucose on cultured rat hepatocyte gene expression.

Both thyroid hormone and carbohydrate feeding are known to influence hepatic gene expression in vivo. In order to determine whether these compounds act directly on the liver, we have utilized an isolated adult rat hepatocyte culture to examine the in vitro influence of either T3 or glucose on a wide range of mRNA activities. RNA was extracted from hepatocyte cultures, translated in the rabbit reticulocyte system, and the translated products separated by two-dimensional gel electrophoresis. We have found a strong similarity between the hepatic mRNA response to T3 administered either to the animal or added to the culture medium and between the response to high carbohydrate feeding and an increase in the medium glucose concentration. Our studies suggest that the isolated hepatocyte culture is a useful model for the study of the influence of hormones and metabolites on the expression of specific mRNA's.

Hepatic messenger ribonucleic acid activity profile of rats subjected to alterations in thyroidal and adrenocortical states: evidence for significant interaction.

We have previously established the value of 2-dimensional electrophoretic mRNA activity profiles for investigating the hepatic genomic response to several metabolic perturbations, such as thyroid hormone or GH treatment, diabetes, high carbohydrate diet, starvation, and uremia. We now report the effects of adrenalectomy and dexamethasone treatment, and compare these with alterations due to thyroidectomy and T3 treatment. Total rat hepatic RNA was isolated and translated in a reticulocyte lysate system. The [35S]methionine-labeled translated products were separated by 2-dimensional gel electrophoresis and quantified with computerized videodensitometry. Of 200 consistently quantifiable products, 14 (7%) were altered by adrenalectomy and dexamethasone, including 4 products (46, 47, 56, and 57) which have not been observed to change in previous studies from this laboratory. Adrenalectomy increased 5 and decreased 2 products, whereas dexamethasone increased 1 and decreased 8 products. Two products maintained the same directional shift in the transitions form adrenalectomy to control and from control to the dexamethasone-treated state. Thyroidectomy and T3 altered 13 products. Thyroidectomy increased 2 and decreased 7 products, whereas T3 treatment increased 6 and decreased 3 products. Four products maintained the same directional shift in the transitions from thyroidectomy to control and from control to the T3-treated state. In all of the manipulations performed (adrenalectomy, thyroidectomy, dexamethasone treatment, and T3 treatment), a total of 20 separate products changed. One third were affected by alterations of both the steroidal and thyroidal states. However, when adrenalectomy and thyroidectomy were compared, only 7% of the shifts were concordant, whereas 30% of the shifts were concordant when treatment with dexamethasone and T3 were compared. These results demonstrate that the mRNA activity response is highly specific for each hormonal manipulation. In addition, unanticipated interrelationships between steroidal and thyroidal states were observed. In some, the presence of T3 appears necessary for the suppressive effect of dexamethasone. Others show that T3 appears to inhibit a stimulatory effect of dexamethasone. Specificity of response to dexamethasone is emphasized by the lack of response to vitamin D, deoxycorticosterone, and dihydrotestosterone and by a different response to estradiol from dexamethasone.

Thyrotropin-releasing hormone stimulates c-jun and c-fos messenger ribonucleic acid levels: implications for calcium mobilization and protein kinase-C activation.

The hypothalamic neuropeptide TRH, through G-protein-coupled transmembrane pituitary receptors, rapidly stimulates intracellular signaling events that, in turn, stimulate gene transcription. Our previous studies in transfected pituitary tumor cells indicated that TRH stimulation of thyrotropin beta-subunit (TSH beta) gene expression involves both calcium mobilization and protein kinase-C activation. To characterize the gene-proximal elements of the intracellular signaling pathways involved, we examined the effects of TRH, ionomycin, and phorbol ester (TPA) on cellular protooncogenes (c-jun and c-fos) known to be responsive to calcium mobilization and protein kinase-C activation. TRH stimulated a 3-fold increase in both c-jun and c-fos mRNA levels within 1 h, followed by a rapid decline in steady state mRNA levels. A secondary response to the single administration was noted, culminating in a 5-fold stimulation at 20 h. The increase in c-jun and c-fos mRNA levels occurred before the increased steady state mRNA levels of both PRL and TSH beta chimera in transfected pituitary GH3 cells. Furthermore, we examined the role of calcium in these effects using the ionophore ionomycin to elevate and TMB-8 to decrease intracellular calcium. We used the phorbol ester TPA to investigate the effects of increased protein kinase-C activity and H7 or pretreatment with TPA to monitor the decreased kinase activity. Our data indicate that calcium mobilization and protein kinase activation represent distinct components of the signaling events initiated by TRH resulting in increased c-jun and c-fos mRNA levels. Only c-fos mRNA is increased by all three factors, suggesting that c-fos may be a key element in mediating the intracellular processes reflecting TRH action.

Screening of candidate oncogenes in human thyrotroph tumors: absence of activating mutations of the G alpha q, G alpha 11, G alpha s, or thyrotropin-releasing hormone receptor genes.

Activating mutations encoding substitutions at positions Arg201 and Gln227 of the alpha-subunit of the stimulatory G protein. G10 have been found in about 40% of pituitary somatotroph tumors. Although the etiology of thyrotroph adenomas is unknown, their autonomous behavior and blunted response to stimulatory hypothalamic hormone superficially resemble those of somatotroph tumors. We hypothesized that a subset of thyrotroph tumors might be caused by dominant somatic mutations that lead to inappropriate activation of the Gq/phospholipase C beta/Ca2+/protein kinase C. pathway normally triggered by occupancy of the TRH receptor (TRHR). We, therefore, screened samples from nine thyrotroph tumors for the presence of activating mutations of the alpha q, alpha 11, and TRHR genes. Fragments of alpha q and alpha 11 complementary DNA encompassing residues (Arg183 and Gln209) that correspond to Arg201 and Gln227 of alpha q were amplified and sequenced. Temperature gradient gel electrophoresis was used to screen for heterozygous mutations in the TRHR coding sequence as well as for known alpha s mutations. No mutations were detected. We conclude that mutations in these regions of the alpha q, alpha 11, alpha s, and TRHR genes occur infrequently, if at all, in human thyrotroph tumors. Alternative mechanisms underlying thyrotroph tumorigenesis, including changes in the expression levels of G protein alpha-subunits or TRHR, dysregulation of downstream components, inappropriate activation of other stimulatory pathways, or loss of inhibitory inputs, remain to be explored.

Expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen. Application to the human thyrotropin receptor.

The clinical applicability of a newly described polymerase chain reaction directed protein expression system was assessed for the in vitro synthesis and partial epitope mapping of large radiolabeled human thyrotropin receptor (hTSH-R) protein segments. PCR amplification of targeted regions within the hTSH-R cDNA followed by in vitro transcription and translation permitted rapid synthesis of protein segments ranging in size from 18 to 62 kDa. Initial epitope mapping was directed at a 52 amino acid segment unique to the hTSH-R compared to otherwise homologous glycoprotein hormone receptors. Sera from Graves' disease patients known to have autoantibodies against the hTSH-R were used to immunoprecipitate two protein fragments differing only by the presence of the unique region in the larger fragment (E5) but not in the smaller fragment (E4). Dense precipitation bands were obtained using Graves' sera to immunoprecipitate E5 whereas little or no specific immunoprecipitation of E4 occurred. Normal sera gave only weak immunoprecipitation bands of E5. The technique provides significant advantages over conventional cloning methods and should have general applicability in the study of other protein targets of autoimmune disease.

Prolactin and tadpole metamorphosis. Evidence of prolactin receptors in premetamorphic Rana catesbeiana liver and tail fin.

The binding characteristics of ovine prolactin (OPRL) to a particulate fraction from liver and tail fin of Rana catesbeiana tadpoles were studied. The specific binding of [125I]oPRL to both tissues was found to be a saturable process with a single class of binding sites in each tissue. Although the dissociation constants were similar for each tissue, the tail fin demonstrated a 10-fold higher binding capacity than the liver tissue. Pretreatment of the liver and tail fin particulate fractions with degradative enzymes revealed that trypsin and phospholipase C reduced the subsequent specific [125I]oPRL binding in both tissues. However, neuraminidase treatment decreased the prolactin binding in the liver while having no effect on the tail fin. The binding of prolactin to the amphibian tissues was found to be specific for prolactin and growth hormones. [125I]oPRL binding to both tissues was a reversible process although the dissociation rate was faster for the tail fin than for the liver. Therefore, prolactin receptors are associated with both a prolactin responsive tissue, the tail, and an unresponsive tissue, the liver, in the tadpole.

Characterization of solubilized prolactin receptors from Rana catesbeiana tadpole tissues.

Prolactin (PRL) receptors were solubilized from Rana catesbeiana tadpole liver and tail fin and female Sprague-Dawley rat-liver membranes by treatment with 1% Triton X-100. Binding of [125I]oPRL to tadpole-liver and tail-fin solubilized extracts reached equilibrium by 12 h at 19 degrees C. The binding was linear up to 50 micrograms of tadpole liver and tail-fin protein and 30-40 micrograms of rat-liver protein. Solubilization did not affect the dissociation constant for [125I]oPRL binding but did result in a greater number of sites. The binding was specific for oPRL and hGH, which has PRL-like activity. Neuraminidase pretreatment of membranes altered the binding affinity of oPRL to tadpole-liver membranes and solubilized extracts but not to tadpole-tail fin membranes or extracts. Pretreatment of membranes with neuraminidase did not affect the binding capacity of tadpole-liver or tail-fin membranes or solubilized extracts.

Changes in specific prolactin binding in Rana catesbeiana Tadpole tissues during metamorphosis and following prolactin and thyroid-hormone treatment.

The prolactin-binding affinity (KD) and number of binding sites *N) in Rana catesbeiana tadpole liver, tail fin and kidney tissues were studied during metamorphosis and following administration of oPRL and L-T3 to premetamorphic tadpoles. With increasing developmental stage there was an increase in N; a maximum was found at stage XVIII followed by a gradual decrease in N through metamorphic climax for all 3 tissues. No change in KD was noted. L-T3 treatment of premetamorphic tadpoles for 7 days caused a significant decrease in tail length and height and body length and an increase in hindlimb length with a concurrent increase in N of approximately 3-fold while treatment for 1 or 3 days was without effect on tadpole morphology or oPRL binding. OPRL treatment for 7 days caused a significant increase in tail length and height and body length with no significant changes in hindlimb length and a 3-5-fold increase in N. Treatment with both L-T3 and oPRL for 7 days resulted in an inhibition of the T3-induced decrease in tail length and height and body length and no inhibition of the hindlimb length increase. N increased in all tissues similar to that found with eight treatment alone. No change in KD was noted in any of these studies. Therefore, oPRL and L-T3 are able to regulate the numbers of specific oPRL-binding sites in amphibian tissues. The change in N with development parallels the reported change in tadpole pituitary capacity to stimulate growth but occurs prior to the reported surge of endogenous T3 during metamorphosis. Thus, the variation in the number of oPRL-binding sites may be due to the changes in endogenous PRL levels during development.