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The retinoic acid receptor (RAR) requires coregulators to bind effectively to response elements in target genes. A strategy of sequential screening of expression libraries with a retinoic acid response element and RAR identified a cDNA encoding a coregulator highly related to RXR alpha. This protein, termed RXR beta, forms heterodimers with RAR, preferentially increasing its DNA binding and transcriptional activity on promoters containing retinoic acid, but not thyroid hormone or vitamin D, response elements. Remarkably, RXR beta also heterodimerizes with the thyroid hormone and vitamin D receptors, increasing both DNA binding and transcriptional function on their respective response elements. RXR alpha also forms heterodimers with these receptors. These observations suggest that retinoid X receptors meet the criteria for biochemically characterized cellular coregulators and serve to selectively target the high affinity binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate DNA response elements.

Characterization of several thyroid hormone (T3), retinoic acid, and estrogen response elements has led to the identification of conserved DNA half-sites (core binding motifs). We present evidence that differences in both the relative orientation and spacing of these motifs within hormone response elements determine the distinct transcriptional responses of three members of the nuclear receptor superfamily. When separated by 3 bp, direct repeat, palindromic, and inverted palindromic arrangements of these motifs impart selective transcriptional responses to retinoic acid, estrogen, and T3 receptors, respectively. Varying the spacing between core motifs alters the specificity. Without spacing, a direct repeat of the core motif paradoxically configures the T3 receptor to confer transactivation in the absence of T3 and repression in its presence. Such an element occurs naturally in the mouse beta-thyrotropin promoter, physiologically under negative regulation by T3. The orientation and spacing of core binding motifs may thus function in concert as a code that accounts for the selective patterns of transcriptional responses of hormonally regulated promoters.

The screening of a size-selected cDNA library from the ovary revealed the existence of a second form of PRL receptor in the rat. The polypeptide sequence deduced from cDNAs has a much longer cytoplasmic domain (357 amino acids) than the form previously identified in the liver (57 amino acids). Nucleotide sequence analysis and comparison with rabbit, mouse, and human PRL receptor cDNAs suggests that the two forms of rat PRL receptor result from alternative splicing of a primary transcript. Complementary DNAs encoding the long form of the receptor were also found in a library prepared from estradiol-treated rat liver, although they represent a minor fraction of total PRL receptor cDNAs obtained from this tissue. DNA polymerase chain reaction amplification of cDNA confirmed the presence of the two receptor forms in both the ovary and liver. Northern analysis, using probes that specifically hybridize with either form of mRNA, indicates a major transcript of 1.8 kilobases (kb) in estradiol-treated liver, which encodes the receptor with a short cytoplasmic domain, while the long form of the receptor is encoded by mRNAs of 2.5 and 3 kb. In the ovary, a complex pattern of hybridization to multiple mRNAs (1.8-5.5 kb) is obtained with the probe specific to the long form, and essentially only a 5.5-kb mRNA is obtained with the probe specific to the short form. The predicted size of the mature form of the long PRL receptor (PRL-R2) is 591 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin receptor (PRLR) and growth hormone receptor (GHR) are encoded by members of a gene family containing regions of identical sequences. To determine their chromosomal locations, cDNA probes for these genes were used. Analysis of hybridization to several somatic cell hybrids, together with hybridization in situ to metaphase chromosomes, resulted in the assignment of the loci for both receptors to human chromosome 5 in the region p13----p14. Thus, these proteins may be encoded by a cluster of related genes that evolved from a common ancestral gene, in a manner consonant with that of their ligands.

The nature of the thyroid disorder presented by patients with normal T4 and T3 but blunted TSH response to TRH has not been clarified. In this study, we compared thyroid function tests in 16 such patients with those of 14 controls and 10 hyperthyroid patients. Basal total T4, free T4, total T3, iodine uptake and cholesterol of the study group were similar to controls but significantly (P less than 0.001) lower than in hyperthyroid patients, except for cholesterol which was higher. In contrast, the basal TSH, increase in TSH after TRH stimulation, and decrease of T4 during T3 suppression tests were similar to data obtained in hyperthyroid patients but significantly (P less than 0.001) lower than in controls. Pulse rate was mid-way between the control and the hyperthyroid group. Thyroid stimulating antibody (TSAb) was measured with human thyroid cells in culture; the assay was positive in four subjects in the 16-patient group and in all hyperthyroid patients tested. TSH stimulation test showed a hyporesponse in iodine uptake in the four patients with positive TSAb (26 +/- 29%), as well as in hyperthyroid patients (6 + 5%). However, there was a hyper-response to TSH (213 +/- 52%) in the remaining 12 patients in the group, none of whom had TSAb. Thus TSAb is not seen as responsible for the thyroid disorder in the majority of patients with normal T3 and T4 and absent or blunted TSH response to TRH; surprisingly, most of these patients have thyroid hypersensitivity to TSH. These two characteristics, absence of TSAb and hypersensitivity to TSH, delineate a thyroid disorder clearly different from Graves' disease.

In order to better understand the difference between familial hypocalciuric hypercalcemia (FHH) and primary hyperparathyroidism (PHP), 4 adults with this disease (FHH(+], from the same kindred, and spanning 2 generations, were compared with 6 patients with PHP, 10 normal controls (N) and 3 unaffected members of the same kindred (FHH(-]. Clinically speaking, the FHH(+) patients were asymptomatic while those with PHP had recurring renal stones. Various phosphocalcic balance elements were measured in blood and urine, in a fasting state, following oral loading of 1 g calcium, as well as in 24-hour urine specimens. Serum calcium levels were slightly higher (11.90 +/- .14 vs 11.37 +/- .24 mg/dl; p less than .05), in patients with FHH(+), but serum phosphorus (2.47 +/- .38 vs 2.88 +/- .40 mg/dl), serum parathyroid hormone (301 +/- 147 vs 291 +/- 224 pg/ml) and serum 1,25(OH)2D (52.8 +/- 6.7 vs 55.0 +/- 12.0 pg/ml) values were similar to those observed in patients with PHP. Urinary calcium present in FHH(+), while fasting (.064 +/- .043 mg/100 ml G.F.), after the calcium loading (.150 +/- .071) and in the 24-hour specimen (.089 +/- .016), was below the 95% confidence limit of values obtained in patients with PHP (.225 +/- .026, p less than .001; .413 +/- .066, p less than .001; 314 +/- .080, p less than .001), but similar to those obtained in N and FHH(-).(ABSTRACT TRUNCATED AT 250 WORDS)

The primary structure of the rat liver prolactin receptor has been deduced from a single complementary DNA clone. The sequence begins with a putative 19 amino acid signal peptide followed by the 291 amino acid receptor that includes a single 24 amino acid transmembrane segment. In spite of the fact that the prolactin receptor has a much shorter cytoplasmic region than the growth hormone receptor, there is strong localized sequence identity between these two receptors in both the extracellular and cytoplasmic domains, suggesting that the two receptors originated from a common ancestor.

The rat liver prolactin receptor has been purified to homogeneity, and partial amino acid sequences have been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 and 598 amino acids, respectively). Both the short and long forms of the prolactin receptor show regions of strong sequence identity with the human and rabbit growth hormone receptors, suggesting that the prolactin and growth hormone receptors originate from a common ancestor.

Human PRL receptor cDNA clones from hepatoma (Hep G2) and breast cancer (T-47D) libraries were isolated by using a rat PRL receptor cDNA probe. The nucleotide sequence predicts a mature protein of 598 amino acids with a much longer cytoplasmic domain than the rat liver PRL receptor. Although this extended region has additional segments of localized sequence identity with the human GH receptor, there is no identity with any consensus sequences known to be involved in hormonal signal transduction. This cDNA will be a valuable tool to better understand the role of PRL in the development and growth of human breast cancer.

Two lambda gt11 clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.

Despite total thyroidectomy, a patient with metastatic follicular carcinoma of the thyroid remained biologically euthyroid three months after stopping thyroxine (T4) therapy. Thyroid hormone production was investigated by means of a modified tri-iodothyronine (T3) suppression test, in which serum T4 was used as a suppression marker. After three weeks of oral T3 (Cytomel) therapy (50 micrograms/day), the serum T4 decreased from normal (108 nmol/L) to undetectable values. However, even though suppressive therapy was effective in preventing TSH dependent hormone secretion by the tumor, it did not prevent tumor growth and the eventual death of the patient.

Rat liver prolactin receptor has been partially characterized and purified to homogeneity using monoclonal antibodies. Pure receptor was digested with trypsin and amino acid sequence of receptor fragments determined. This allowed us to clone the prolactin receptor cDNA. Our approach to clone the receptor cDNA consisted of synthesizing oligonucleotides corresponding to the amino acid sequence of receptor fragments, and to screen a cDNA library. Sequencing reveals that prolactin receptor is a 291 amino acid protein, containing an extracellular domain of 210 residues, a single transmembrane segment of 24 amino acids and a cytoplasmic domain of 57 amino acids. Introduction of the prolactin receptor cDNA into various cell types demonstrates that the single protein is sufficient to bind prolactin with the same affinity and specificity reported for the prolactin receptor.

Sex steroids are major regulators of PRL receptor expression in rat liver. Using a probe encoding the rat PRL receptor we have studied receptor mRNA levels in female rat liver during ontogeny and in response to estrogen treatment. Steady state mRNA levels were determined by Northern blot and densitometric analysis. Messenger RNA levels have been compared to the number of binding sites, which was assessed by Scatchard analysis of [125I]ovine PRL binding in membrane preparations. Our results show that steady state mRNA and binding levels of PRL receptors are both regulated by development and estrogens, but that binding does not exactly parallel mRNA levels. From the developmental stages of prepuberty to adult, receptor numbers increase 8-fold, whereas mRNA levels increase 3-fold. Estrogen treatment stimulates receptor levels 6-fold, but mRNA levels are only increased 3-fold. These results suggest that PRL receptor gene expression in rat liver is regulated at the transcriptional or posttranscriptional level as well as at the translational level.

This study was designed to follow the evolution of serum 1,25(OH)2D after surgery for primary hyperparathyroidism. Ten patients were studied before and for up to 85 d after removal of a single parathyroid adenoma. Blood and 24 h urine were obtained at various time points, for the measurement of serum or urinary phosphate and calcium indices. Before surgery, serum calcium (2.91 +/- 0.06 mmol/l; mean +/- SEM), parathyroid hormone (354 +/- 47 pg/ml) and 1,25(OH)2D (61.2 +/- 7.8 pg/ml) were elevated while serum phosphate (1.01 +/- 0.07 mmol/l) tended to be low. Relative hypoparathyroidism was evident for up to 5 d after surgery with the lowest value for serum parathyroid hormone (41 +/- 16 pg/ml) on day 1, serum calcium (2.12 +/- 0.06 mmol/l) on day 3 and highest value for serum phosphate (1.41 +/- 0.13 mmol/l) on day 5. As expected, serum 1,25(OH)2D levels decreased to 35.9 +/- 4.2 pg/ml 24 h after surgery. Stabilization of serum and urinary parameters to normal values was seen between day 5 and day 27; the only exception was serum 1,25(OH)2D, which increased again at day 27 to 57.6 +/- 5.0 pg/ml, a value as high as that before surgery. It was still elevated at day 50 (58.3 +/- 4.3 pg/ml), but returned towards normal values in three out of four patients (44 +/- 3.9 pg/ml) by day 80. No variation in 25(OH)D or 24,25(OH)2D was seen throughout the study. 1,25(OH)2D values could be related to serum parathyroid hormone values before surgery (r = 0.659, P less than 0.05) but not after. The secondary increase in serum 1,25(OH)2D could not be related to variations in serum calcium, phosphate, parathyroid hormone or diet. Further studies will be required to explain this phenomenon.