Cloning of the LH/CG receptor implications for a unique G-protein coupled receptor.

The proteins encoded by the rat and porcine luteinizing hormone (LH)lchorionic gonadotropin (CG) receptor cDNAs appear to be unique members of the G -protein-coupled receptor family. Although they have the characteristic seven transmembrane domains, the LH/CG receptors have relatively low homology to other members o f this family, do not have a G-protein-coupling domain corresponding to that of the a-adrenergic receptor, and contain an unusual long extracellular domain. Experience in the molecular dissection of this receptor family will help guide investigation of the LHICG receptor's functional domains. Further studies are needed to clarify the origin and significance of the various mRNA species hybridizing on Northern blot analyses. Finally, the rat and porcine receptor cDNAs should permit cloning of the human LH/CG receptor, as well as cloning of the thyroid-stimulating hormone and follicle-stimulating hormone receptors.     

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.

To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction we constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. With respect to hormone binding, substitution of the entire extracellular domain of the LH/CG receptor for the corresponding region of the TSH receptor resulted in high-affinity human CG binding with complete loss of TSH binding. Surprisingly, however, there was at least one chimera with a substitution at each of the five domains that still retained high-affinity TSH binding. Substitution of residues 1-170 of the TSH receptor with the corresponding region of the LH/CG receptor was associated with the retention of high-affinity TSH binding but ligand specificity was lost in that TSH and human CG could interact functionally with the receptor. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites.     

Thyroid stimulatory autoantibodies in different patients with autoimmune thyroid disease do not all recognize the same components of the human thyrotropin receptor: selective role of receptor amino acids Ser25-Glu30.

To study the interaction between TSH, autoantibodies and the amino-terminal half of the TSH receptor extracellular region (amino acids 1-260; domains ABC), we constructed 20 LH/CG chimeric receptor cDNAs. The prototypic receptor for modification contained domains ABC of the TSH receptor and domains DE of the LH/CG receptor. Segments (6-13 amino acids) within the ABC domains were replaced with the corresponding amino acids of the rat LH/CG receptor. Fifteen of the 20 chimeric receptors could be expressed functionally in Chinese hamster ovary cells. Twelve retained both high affinity TSH binding and normal signal transduction. These 12 receptors were tested with a panel of 10 patients' immunoglobulin G (IgG) samples containing potent TSH receptor stimulatory activity. With 11 of the receptor variants, the cAMP responses were similar to those with the prototype receptor (TSH-LHR-6). However, the -A1 variant of TSH-LHR-6 (Ser25-Glu30) responded poorly to 6 of 10 IgGs. The same pattern was observed when the IgGs were tested for their ability to inhibit [125I] TSH binding to the receptor variants, suggesting qualitative differences between the different stimulatory TSH receptor autoantibodies. Therefore, we examined the dose-response relationship of 2 IgGs that were approximately equipotent when tested with TSH-LHR-6 and its -A1 variant and another 2 IgGs that displayed greatly diminished potency with respect to the -A1 variant. Despite dilution to nearly undetectable levels, the relative potencies of the 4 IgG samples for both types of receptors remained similar. These data demonstrate directly that stimulatory TSH receptor autoantibodies do not all recognize the same components of the TSH receptor. The segment of the TSH receptor discriminated by these autoantibodies is between amino acids Ser25-Glu30.     

Different binding of stimulatory-type and blocking-type TSH receptor antibody with guinea-pig testis membrane.

A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic alpha-subunit peptide (alpha 26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of alpha-subunit of TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor.     

Role of the carboxyl-terminal half of the extracellular domain of the human thyrotropin receptor in signal transduction.

We studied the role of the carboxyl-terminus of the extracellular region of the human TSH receptor in signal transduction (cAMP generation). For this purpose, we introduced homologous substitutions of smaller segments within amino acids 261-418 (domains D and E) of the TSH receptor with the corresponding amino acids of the rat LH/CG receptor. Amino acids 317-366 were not investigated in view of previous data indicating their noninvolvement. Mutant TSH receptor cDNAs, in a eukaryotic expression vector, were stably transfected into Chinese hamster ovary cells. Eight of nine plasmid constructs expressed TSH receptors that could be detected by radiolabeled TSH binding; six of these were of high affinity similar to the wild-type receptor and, therefore, provided informative data on signal transduction. Despite high affinity TSH binding, five of six TSH receptor mutants displayed a diminished cAMP response to TSH stimulation, suggesting the involvement of broad segments of domains DE in signal transduction. Amino acids 270-278 and 287-297 were particularly important in this respect. The conformation conferred by these segments of the TSH receptor, therefore, appears to be involved in transducing a signal from the extracellular to the intracellular region of the receptor.     

Influence of histamine- and 5-hydroxytryptamine-containing thyroid mast cells on thyroid blood flow and permeability in the rat.

The possible significance of thyroid mast cells in the regulation of thyroid blood flow and capillary permeability was investigated in rats whose TSH secretion had been eliminated by exogenous T4. Mast cells were identified by their abundance of metachromatic granules, and their content of histamine and 5-hydroxytryptamine (5-HT) was examined by fluorescence histochemistry. Thyroid histamine levels were determined by fluorometry. The tissue uptake of 86Rb was used as an indicator of blood flow and permeability. Numerous histamine- and 5-HT-containing mast cells were found within the thyroid and in connective tissue adjacent to the thyroid, whereas juxtathyroidal muscle tissue was virtually devoid of mast cells. Administration of compound 48/80 evoked a prompt depletion of 5-HT, histamine and metachromatic granules from thyroid mast cells, and a concomitant increase in the thyroidal uptake of 86Rb. The 86Rb uptake by juxtathyroidal muscle tissue was unaffected. Exogenous 5-HT and histamine both induced prompt increments in thyroidal 86Rb uptake, and 5-HT also stimulated 86Rb uptake in juxtathyroidal muscle tissue. TSH, previously shown to induce a gradual amine release from mast cells within, but not outside, the thyroid, evoked a gradual increase in thyroidal, but not in muscular, uptake of 86Rb. The findings support the concept that, in the rat, histamine and/or 5-HT, released from intrathyroidal mast cells by TSH, stimulate thyroid blood flow and/or permeability.     

Low TSH requirement and goiter in transgenic mice overexpressing IGF-I and IGF-Ir receptor in the thyroid gland.

Through the cAMP signaling pathway, TSH stimulates thyroid follicular cell proliferation, differentiation, and function. Although the autocrine production of IGF-I in the thyroid gland suggests an important physiological function for this factor in these processes, the exact role of the IGF-I/IGF-I receptor system in vivo remains unclear. Although the mitogenic action of TSH requires the presence of IGF-I or insulin in primary culture of dog and human thyroid cells, IGF-I has an effect equal to and independent of the effect of TSH on cell proliferation in rat thyroid cell lines and may even be the main growth regulator in this case. To investigate the in vivo function of the IGF-I/IGF-I receptor system, transgenic mice overexpressing human IGF-I, IGF-I receptor, or both in the thyroid were generated. Adult transgenic mice did not present external signs of thyroid dysfunction, but mice overexpressing both transgenes had significantly increased gland weight and follicular lumen area. A decreased TSH level together with a slightly increased serum T(4) concentration and increased thyroidal iodine uptake were also observed, suggesting that IGF-I and IGF-I receptor stimulate thyroid function to some extent in vivo.     

Characterization of an antiserum to the rat luteal luteinizing hormone/chorionic gonadotropin receptor

The LH/CG receptor was purified from detergent extracts of rat luteal tissue using hCG affinity and wheat germ agglutinin chromatography. Analysis of the purified material by silver staining of sodium dodecyl sulfate-polyacrylamide gels (with or without reducing agents) revealed a prominent broad band corresponding to a 93K protein and several minor contaminants. That the 93K band represents the LH/CG receptor is supported by the following. 1) This band is absent in material purified from rat luteal tissue in which the LH/CG receptor had been down-regulated. 2) [125I]iodohCG binding to Western blots of both the initial detergent extract and the purified material resulted in binding to a 93K protein. The 93K protein representing the LH/CG receptor was excised after sodium dodecyl sulfate-gel electrophoresis of the purified material and was electroeluted. Half of the electroeluted receptor was incubated with reducing agents. After mixing with nonreduced receptor, this mixture was used to immunize one rabbit. Immune, but not preimmune, serum (or immunoglobulin G purified thereof) recognized a 93K protein on Western blots of partially purified (approximately 10% pure) rat luteal receptor. No other bands were seen. Using this approach it was determined that the antiserum recognized reduced, nonreduced, denatured, and native forms of the receptor. A polyclonal antibody to the LH/CG receptor will be useful for studies on the structure and regulation of this receptor.     

Receptor-binding activity of highly purified bovine luteinizing hormone and thyrotropin, and their subunits

Highly purified preparations of bovine TSH (bTSH) and LH (bLH) and their subunits have been obtained by affinity chromatography using immobilized antibodies directed against counterpart subunits. The purified preparations were assessed for biological activity in radioligand-receptor assays for TSH and LH. After affinity purification against bLH beta, a TSH preparation whose initial potency in the LH assay had been 0.15% that of LH, failed to compete with [125I]LH in amounts up to 100 microgram. Thus, it appears that bTSH does not bind to LH receptors in the rat testis and that interaction of less purified TSH with gonadotropin receptors is attributable to LH contamination. In contrast, LH, whose initial potency in the TSH receptor assay was 0.6% that of TSH, retained a potency of 0.004% of TSH (equivalent to 3.6 mU/mg) after immunoadsorption by anti-bTSH beta. The retention of TSH receptor-binding activity by affinity-purified LH indicates that the LH molecule (like hCG) has a low intrinsic thyroid-stimulating activity. Affinity-purified LH subunits have little or no demonstrable affinity for the LH receptor in vitro. Affinity-purified TSH subunits and affinity-purified LH, however, exhibit very weak receptor-binding activity in the TSH radioligand receptor assay. An evaluation of the capacity of the immunoadsorbents to remove TSH from artificial mixtures suggests that the residual binding does not result entirely from contamination, and therefore, that alpha-subunits as well as LH have some intrinsic TSH-binding activity.     

The cloned equine thyrotropin receptor is hypersensitive to human chorionic gonadotropin; identification of three residues in the extracellular domain involved in ligand specificity

The receptors for TSH, LH/chorionic gonadotropin (CG), and FSH belong to the same subfamily of G protein-coupled receptors. The specificity of recognition of their cognate hormone involves a limited number of residues in the leucine-rich repeats present in the N-terminal ectodomain of the receptor. It is admitted that receptors of this subfamily coevoluted with their respective ligands. The secretion of CG is restricted to gestation of primates and Equidae. We hypothesized that, facing the challenge of a new hormone, the glycoprotein hormone receptors would have evolved differently in Equidae and human so that distinct residues are involved in hormone specificity. In particular, it is known that equine CG has a dual (FSH and LH) activity when administered to other species. In the present work, we cloned and characterized functionally the equine TSH receptor (TSHR), which shares 89% homology with the human TSHR. The equine TSHR is not responsive to equine CG but is more sensitive to human CG than the human TSHR. Three residues, at positions 60, 229, and 235 of the ectodomain, are responsible for this difference in sensitivity as shown by modelization and targeted mutagenesis, followed by in vitro functional characterization. The phylogenetic approach is a suitable approach to identify determinants of specificity of receptors.     

Production and characterization of polyclonal antiserum to rat luteinizing hormone/chorionic gonadotropin receptor and its immunohistochemical application for studying receptor location and down-regulation

A polyclonal antiserum against the affinity-purified nondenatured rat 90K LH/CG receptor polypeptide was raised in rabbits, characterized, and used to study the location of the LH/CG receptor in pseudopregnant rat luteal cells and the fate of the receptor-hCG complex together with the specific anti-hCG serum during hCG-induced down-regulation by immunochemical techniques. Even at a 1:3000 dilution, the antiserum recognized a single 90K polypeptide on Western blots of both the affinity-purified receptor and the initial detergent extract of the pseudopregnant rat ovarian membranes. It recognized sodium dodecyl sulfate-denatured and reduced, sodium dodecyl sulfate-denatured, and native forms of the receptor on dot blots; the immunoreaction was the most intense with the native receptor. The antiserum also contained antibodies that recognized the hormone-binding site, or a region near to it, and the occupied receptor. The majority of the LH/CG receptors were located on the luteal cells in pseudopregnant rat ovaries before the induction of down-regulation. The receptor content seemed to vary among the luteal cells, however, and on single cells, suggesting both functional heterogeneity and a functional polarization of the luteal cells. Upon induction of down-regulation with hCG both the receptors and the bound hormone disappeared from the luteal cell surfaces at a very slow rate, without any simultaneous appearance of receptor- or hCG-specific immunostaining in the luteal cell interior. No accumulation of receptor degradation products capable of [125I]iodo-hCG or antibody binding could be detected on Western blots of the tissue. The polyclonal LH/CG receptor antiserum described here is useful for studying the structure and function of this receptor, particularly for immunohistochemical investigations into receptor location and regulation.