Growth hormone action in rat insulinoma cells expressing truncated growth hormone receptors

Transfection of the insulin-producing rat islet tumor cell line RIN-5AH with a full length cDNA of the rat hepatic growth hormone (GH) receptor (GH-R1-638) augments the GH-responsive insulin synthesis in these cells. Using this functional system we analyzed the effect of COOH-terminal truncation of the GH receptor. Two mutated cDNAs encoding truncated GH receptors, GH-R1-294 and GH-R1-454, respectively, were generated by site-directed mutagenesis and transfected into the RIN cells. Both receptor mutants were expressed on the cell surface and displayed normal GH binding affinity. Whereas GH-R1-638 had a molecular mass of about 110 kDa, GH-R1-294 and GH-R1-454 showed molecular masses of 49 and 80 kDa, respectively. Cells expressing GH-R1-454 internalized GH to a similar extent as cells transfected with the full length receptor and the parent cell line, but GH-R1-294-expressing cells showed a markedly reduced capability of GH internalization. In contrast to cells transfected with GH-R1-638, none of the cell lines expressing truncated GH receptors exhibited any increase of the GH-stimulated insulin production. We conclude that domains within the COOH-terminal half of the cytoplasmic part of the GH receptor are required for transduction of the signal for GH-stimulated insulin synthesis, whereas cytoplasmic domains proximal to the transmembrane region are involved in receptor-mediated GH internalization.     

Localization of thyroid hormone in subpopulations of rat alveolar macrophages

Summary Radioimmunoassay and immunocytochemical staining methods were used to study the distribution of thyroid hormone in rat alveolar macrophages. The cells were fractionated into six subpopulations by Percoll density gradient. Positive immunoreactive tri-iodothyronine (T₃) was observed in all subpopulations of macrophages, especially in low-density (1.040 and 1.050 g cm^–3) groups, by avidin-biotin-peroxidase immunostaining techniques. The macrophages also showed various patterns of cellular T₃ stainability. Results from radioimmunoassay of macrophage extracts also demonstrated that macrophages of low density had a higher level of total T₃ than those of higher densities (1.060 g cm^–3).     

Localization of hormone receptors inTetrahymena

Summary It can be shown by light and electron microscopic autoradiography that¹²⁵I triiodothyronine and¹²⁵I thyroxine bind to the cell membrane ofTetrahymena. For thyroxine the finer localization is at the cilia as well as in pinocytotic and food vacuoles. Some grains also appear above the nucleus. On the other hand, insulin only binds to the membrane ofTetrahymena, and binding can be inhibited by unlabelled insulin.     

Ontogenesis of hepatic growth hormone receptors in the rat

Detailed ontogenic studies of the binding of human (hGH) and bovine growth hormone (bGH) have been performed in liver preparations from male and female rats during the neonatal, weanling, pre- and post-pubertal periods. Specific binding of both hormones was readily detected at all ages, with no apparent interference due to occupancy by endogenous hormones. No sex difference in binding was observed prior to weaning (22 days) for hGH, which binds to both somatotrophic and lactogenic sites. However, after weaning a marked sex-related dissociation in the pattern of binding did occur, with female rats binding 3-4 times more hGH than in the pre-weaning period and male rats binding hGH to only half their pre-weaning levels. A very similar pattern was seen for binding of bGH (which binds only to somatotrophic sites) except that in male rats, the post-weaning levels did not fall. Binding patterns for either hGH or bGH prior to weaning did not mirror the known age-related pattern of circulating rat GH levels, suggesting the absence of a definitive auto-regulation system for the GH-GH receptor system under normal circumstances in vivo. The possible role of the weaning process per se in the post-weaning changes of GH binding seen in male and female rats still requires elucidation.     

Increased number of beta-adrenergic receptors on cells of the rat adenohypophysis after thyroid hormone administration

The authors studied the effect of administration of thyroid hormones on the beta-adrenergic receptors of rat adenohypophyseal cells. The administration of triiodothyronine and thyroxine was followed by an increase in specific binding for 3H-dihydroalprenolol. No significant differences were found in cyclic adenosine monophosphate levels before and after isoprenaline stimulation. The significance of changes in these receptors for the hyperplastic reaction after oestrogens is discussed with reference to the inhibitory effect of the thyroid hormones on hyperplasia of the adenohypophyseal cells after the administration of oestradiol.     

Characterization of a third human thyroid hormone receptor coexpressed with other thyroid hormone receptors in several tissues

A cDNA that encodes a third type of human thyroid hormone receptor (hTR alpha 1) has been isolated from a skeletal muscle library. The cDNA encodes a 410 amino acid protein, Mr = 46,820. When expressed and translated in vitro, hTR alpha 1 binds T3 with an association constant (ka) of 1.8 x 10(9) M-1. Comparison of the DNA sequence of hTR alpha 1 and a previously identified alpha type thyroid hormone receptor (hTR alpha 2) suggests that they could be transcribed from the same gene, and that alternative RNA splicing results in the synthesis of either hTR alpha 1 or hTR alpha 2. Two mRNA (3.2 kilobases and 6 kilobases) of hTR alpha 1 have been detected in several tissues. At least three types of thyroid hormone receptors (hTR alpha 1, alpha 2, beta), which possess similar affinities for hormone ligands, can be expressed in the same tissue.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Ovarian hormone receptors in human mammary stromal cells

Mesenchymal cells of the rodent breast express both estrogen and progesterone receptors. Searches for these molecules in the human breast have yielded conflicting results. Following immunohistochemical staining of samples of normal human breast tissue, the authors detected estrogen receptor alpha protein and progesterone receptor protein in extralobular (non-specialized) fibroblasts and estrogen receptor alpha protein in adipocytes. Tissues from young teenage girls and pregnant women contained the greatest number of receptor positive fibroblasts. These observations confirm prior reports of the presence of ovarian hormone receptors in mammary fibroblasts. The findings also illustrate similarities in the organization of the rodent and human breasts and thereby suggest that regulation of the gland by ovarian hormones involves similar mechanisms in both species.     

Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat granulosa cells

We have investigated the effects of bFGF on both the FSH-induced LH receptor expression and cAMP production in cultured rat granulosa cells. Concentrations of pure FGF, from 10(-12) M to 10(-10) M, progressively inhibit the stimulatory actions of FSH with an ED50 of approximately 4 x 10(-12) M for both parameters. Higher FGF concentrations, from 4 x 10(-10) M to 10(-8) M, lead to a gradual reduction of the growth factor inhibitory effect. The effects of FGF are more prominent on the modulation of LH receptors than on the FSH-induced cAMP production. Moreover, FGF impairs the LH receptor formation induced by cholera toxin or 8-Bromo-cAMP, indicating that the growth factor also acts at a step distal to cAMP formation. The inhibitory effect of FGF on LH receptor expression increases during the entire course of granulosa cell differentiation, from 24 to 96 h, and is not due to variations in cell number or viability, but rather to a change in the content of LH receptors with no significant modification of binding affinity (KD congruent to 0.8 x 10(-10) M). These results suggest that bFGF may acutely regulate the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the ovarian follicular maturation.     

Light microscopic immunocytochemical demonstration of thyroid-stimulating hormone (TSH) receptors on normal rat thyroid cells

We present a method for immunohistochemical demonstration of surface-associated thyroid-stimulating hormone (TSH) receptors on direct imprints of rat thyroid follicular cells at the light microscopic level, by use of dinitrophenyl (DNP)-labeled bovine TSH as the primary probe in a DNP-hapten sandwich staining (DHSS) procedure. The light microscopically invisible diaminobenzidine (DAB) product of the enzyme reaction was amplified with a DAB enhancement technique (silver amplification), which yielded a reliable and distinct light microscopically visible cell surface staining. The consistently negative control results after inhibition with TSH receptor-specific reagents provide evidence in support of immunohistochemical localization of surface membrane-associated TSH receptors.     

Hormone selectivity in thyroid hormone receptors

Separate genes encode thyroid hormone receptor subtypes TRalpha (NR1A1) and TRbeta (NR1A2). Products from each of these contribute to hormone action, but the subtypes differ in tissue distribution and physiological response. Compounds that discriminate between these subtypes in vivo may be useful in treating important medical problems such as obesity and hypercholesterolemia. We previously determined the crystal structure of the rat (r) TRalpha ligand-binding domain (LBD). In the present study, we determined the crystal structure of the rTRalpha LBD in a complex with an additional ligand, Triac (3,5, 3'-triiodothyroacetic acid), and two crystal structures of the human (h) TRbeta receptor LBD in a complex with either Triac or a TRbeta-selective compound, GC-1 [3,5-dimethyl-4-(4'-hydroy-3'-isopropylbenzyl)-phenoxy acetic acid]. The rTRalpha and hTRbeta LBDs show close structural similarity. However, the hTRbeta structures extend into the DNA-binding domain and allow definition of a structural "hinge" region of only three amino acids. The two TR subtypes differ in the loop between helices 1 and 3, which could affect both ligand recognition and the effects of ligand in binding coactivators and corepressors. The two subtypes also differ in a single amino acid residue in the hormone-binding pocket, Asn (TRbeta) for Ser (TRalpha). Studies here with TRs in which the subtype-specific residue is exchanged suggest that most of the selectivity in binding derives from this amino acid difference. The flexibility of the polar region in the TRbeta receptor, combined with differential recognition of the chemical group at the 1-carbon position, seems to stabilize the complex with GC-1 and contribute to its beta-selectivity. These results suggest a strategy for development of subtype-specific compounds involving modifications of the ligand at the 1-position.     

Localization of transferrin receptors (TFRs) in human non functioning thyroid nodules and in extranodular thyroid tissue

In this research the TFR localization in non functioning human thyroid nodules and in the extranodular thyroid tissue, using an immunohistochemical technique, has been studied. For this study a monoclonal antibody (B3/25) against TFR and the peroxidase technique have been utilized. Moreover a morphometric comparative analysis was carried out based on the following parameters: 1) mean immunoreactive area for microscopic field, 2) mean value of immunoreactive follicular perimeter, 3) integrated optical density, 4) % of immunoreactive area on total examined area in nodular and extranodular tissue. The immunoreactivity was detected in some follicular cells in a number of follicles randomly distributed in the extra nodular tissue. As concern the non functioning thyroid nodules, the positivity was localized in the generality of the follicles both in the flattened epithelial cells of the larger follicles and in the cuboidal cells of the smaller ones. The morphometric parameters confirm a statistically significant difference of immunoreactivity between extranodular and nodular tissue. These results suggest that TF might play a role in the cellular proliferation of thyroid gland.