Presence of TSH receptors in discrete areas of the hypothalamus and caudal brainstem with relevance for feeding controls-Support for functional significance.

AIMS: Previous studies have shown that brain-derived thyroid-stimulating hormone (TSH) and its receptor (TSHr) are present in hypothalamic extracts. No studies investigating both the anatomical location and functional significance of putative TSHr proteins in specific central nervous system (CNS) nuclei involved in feeding controls have yet been conducted. The aim was thus to determine whether TSHr are present in nuclei associated with feeding behavior, and if such receptors may be functional. METHODS: Brain tissue from adult rats was analyzed for gene expression and receptor protein expression was investigated with immunohistochemistry and western blotting. To investigate whether putative TSHr may be functional, we evaluated food intake of rats given intraparenchymal nanoinjections of TSH into the nucleus of the solitary tract (NTS). RESULTS: RT-qPCR confirmed previous reports that TSHr mRNA is expressed in CNS tissues of the adult rat. Immunohistochemistry showed TSHr-immunoreactivity in the arcuate, the ventromedial, the dorsomedial, and the paraventricular hypothalamic nuclei. We also found TSHr-ir in the dorsal hindbrain to be localized to the area postrema, NTS, dorsal motor nucleus of the vagus, and the hypoglossal motor nucleus. Further protein analysis with western blotting showed 120kDa TSHr-ir proteins present in the hypothalamus and brainstem. Injections of TSH into the NTS reduced food intake similar to the positive control, urocortin. CONCLUSIONS: These data suggest that functional TSHr are present in the caudal brainstem and hypothalamic nuclei of relevance for feeding control as a possibly uncleaved holoreceptor, and highlights a hindbrain component to central TSH inhibition of food intake.

Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource.

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.

Thyroid status co-regulates thyroid hormone receptor and co-modulator genes specifically in the hypothalamus.

Regulation of Thyrotropin Releasing Hormone (TRH) transcription in the hypothalamus represents the central control point of thyroid function. To examine the expression of potential TRH regulatory components, we simultaneously amplified, by semi-quantitative multiplex PCR system, nine key genes from < or = 100 ng total RNA from two brain areas (hypothalamus and cortex) under different thyroid states. Expression of TR1 and TR2 isoforms, key elements in TRH regulation, was modified by thyroid status in the hypothalamus but not in the cortex. Similarly, hypothyroidism increased specifically hypothalamic levels of three co-modulator genes. This study provides the first demonstration of tissue specific co-regulation of a set of genes by thyroid status within a defined brain area.

Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells.

The complementary DNA for the human TSH receptor (TSHR) translated region was amplified in the genome of stably transfected Chinese hamster ovary (CHO) cells using a dihydrofolate reductase minigene. Immunoprecipitation of TSHR in whole cells precursor-labeled with [35S]methionine and [35S]cysteine revealed an approximately 10-fold increase in TSHR expression in cells stabilized in 10,000 nM methotrexate (TSHR-10,000 cells) compared to cells with the same gene not subjected to amplification (TSHR-0 cells). Similarly, [125I]TSH cross-linking to the surface of intact CHO cells revealed a progressive increase in TSH-binding sites with dihydrofolate reductase minigene amplification, with a 12.8-fold increase in TSHR in TSHR-10,000 vs. TSHR-0 cells. Based on the known number of TSHR expressed by TSHR-0 cells, TSHR-10,000 express approximately 1.9 x 10(6) TSHR on their surface. Two ligand-TSHR complexes were evident under reducing conditions, representing the single chain holoreceptor of about 115 kDa and a dissociated A subunit of about 60 kDa. In the absence of TSH, basal cAMP levels in TSHR-10,000 cells were greater than those in TSHR-0 cells (6-fold in isotonic medium and 18.5-fold in hypotonic medium), indicating that the unliganded TSHR has significant constitutive activity. We assessed the kinetics of TSH binding to CHO cells overexpressing the TSHR using [125I]TSH in the presence of increasing concentrations of unlabeled TSH as well as by attempted saturation with labeled ligand. Surprisingly, in contrast to TSHR-0 cells (Kd = approximately 5 x 10(-10) M), we observed progressively lower affinities for TSH binding by TSHR-800 cells (Kd = approximately 10(-9) M) and TSHR-10,000 cells (Kd = approximately 2 x 10(-9) M). In summary, we report a high level of expression of TSHR in CHO cells and confirm the high constitutive activity of the TSHR in the absence of ligand as well as the binding of TSH to the single subunit, uncleaved TSHR. Moreover, we found that a high level of expression is associated with apparent negative cooperativity among the TSHR in terms of their affinity for ligand.

Recombinant thyrotropin receptor and the induction of autoimmune thyroid disease in BALB/c mice: a new animal model.

In a preliminary study, we observed the production of TSH binding-inhibiting (TBII) and thyroid-blocking (TBAb) antibodies accompanied by lymphocytic infiltration of the thyroid in a pool of male BALB/c mice immunized with the extracellular domain (ECD) of the human TSH receptor (TSHR) expressed as a maltose-binding protein (MBP) fusion in bacteria. In the present study we evaluated the humoral response to the same antigenic preparation in a new series of individual male and female BALB/c mice immunized ip on day 0 with 100 micrograms MBP-ECD and days 25, 39, and 53 with 50 micrograms MBP-ECD in an adjuvant composed of aluminum oxide, magnesium hydroxide, and Bordetella pertussis vaccine. Mice immunized with MBP served as control. Individual sera and immunoglobulins were tested for TBII, TBAb, and thyroid-stimulating antibodies (TSAb) on days 0, 32, 46, and 60, and total circulating T4 levels were measured by RIA. Animals were killed on day 120, their thyroids were examined histologically, the infiltrates were characterized using monoclonal antibodies specific for T-cells (total, activated, helper, and suppressor), B-cells, and macrophages. Sera and immunoglobulins G of the MBP-treated control group were all negative for TSAb, TBAb, and TBII activity. The receptor-immunized mice, despite having high titers of antibodies to the immunogen in an enzyme-linked immunosorbent assay, displayed a heterogeneous response in terms of biological activity, with 3 of 7 female and 4 of 8 male mice having TBAb/TBII activities that persisted and whose activity increased throughout the experiment. No significant TSAb antibody activity was observed. Total T4 levels were also heterogeneous even before immunization, but 9 of 15 MBP-ECD-treated mice had levels below the normal range after immunization, and 7 of these also had TBII/TBAb activities. At the end of the experiment, only 4 of the MBP-ECD-treated female mice survived, but all of them had a severe lymphocytic infiltration of their thyroid, composed mostly of activated T-cells, although B-cells and macrophages were also present. A similar infiltrate was seen in 4 of 8 male MBP-ECD-treated mice. No infiltrate was observed in male or female MBP-treated mice. The model described demonstrates the feasibility of using the TSHR as an immunogen to overcome tolerance and mimics some characteristics of human autoimmune disease of the thyroid.