Evidence that the thyroid-stimulating hormone (TSH) receptor transmembrane domain influences kinetics of TSH binding to the receptor ectodomain

Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (k(off)) of prebound (125)I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, (125)I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH K(d) values for these two receptor forms at 4 °C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.     

Dominant Negative Effect of Mutated Thyroid Stimulating Hormone Receptor (P556L) Causes Hypothyroidism in C.RF-Tshr(hyt/wild) Mice

C.RF-Tshr(hyt/hyt) mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshr(hyt/wild) heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshr(hyt/wild) mice are smaller than those from wild-type mice, and (125)I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained (125)I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshr(hyt/wild) mice.     

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.     

The hinge region of human thyroid-stimulating hormone (TSH) receptor operates as a tunable switch between hormone binding and receptor activation

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.     

中药固真方对促甲状腺素受体基因表达的调节作用

促甲状腺素（ＴＳＨ）通过甲状腺细胞膜上的ＴＳＨ受体（ＴＳＨＲ）,产生第二信使ｃＡＭＰ,从而激活ｃＡＭＰ反应性起动子,而使相应的基因获得表达．实验结果表明,在构建有ＴＳＨＲ和糖蛋白激素ｃＡＭＰ反应性起动子以及萤光素基因（Ｌｕｃ）的转染细胞中,经补肾益精中药固真方提取液（１×１０~（－４）稀释）处理３～５ｄ后,可下调ＴＳＨＲ基因的表达,并使ＴＳＨＲ数目减少．提示固真方可调整甲状腺细胞的功能,或许有利于调整甲状腺机能亢进．     

Quantitative high-throughput screening using a live-cell cAMP assay identifies small-molecule agonists of the TSH receptor

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.     

Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis

C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.     

Relationship between thyrotropin receptor hinge region proteolytic posttranslational modification and receptor physiological function

The glycoprotein hormone receptor hinge region is the least conserved component and the most variable in size; the TSH receptor (TSHR) being the longest (152 amino acids; residues 261-412). The TSHR is also unique among the glycoprotein hormone receptor in undergoing in vivo intramolecular cleavage into disulfide-linked A- and B-subunits with removal of an intervening 'C-peptide' region. Experimentally, hinge region amino acids 317-366 (50 residues) can be deleted without alteration in receptor function. However, in vivo, more than 50 amino acids are deleted during TSHR intramolecular cleavage; furthermore, the boundaries of this deleted region are ragged and poorly defined. Studies to determine the extent to which hinge region deletions can be tolerated without affecting receptor function ('minimal hinge') are lacking. Using as a template the functionally normal TSHR with residues 317-366 deleted, progressive downstream extension of deletions revealed residue 371 to be the limit compatible with normal TSH binding and coupling with cAMP signal transduction. Based on the foregoing downstream limit, upstream deletion from residue 307 (307-371 deletion) was also tolerated without functional alteration, as was deletion of residues 303-366. Addressing a related issue regarding the functional role of the TSHR hinge region, we observed that downstream hinge residues 377-384 contribute to coupling ligand binding with cAMP signal transduction. In summary, we report the first evaluation of TSHR function in relation to proteolytic posttranslational hinge region modifications. Deletion of TSHR hinge amino acids 303-366 (64 residues) or 307-371 (65 residues) are the maximum hinge region deletions compatible with normal TSHR function.     

Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor.

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317-319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.     

A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR)

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.     

Gonadotropin receptor of a mouse luteoma: interactions with luteinizing hormone (LH) and its and subunits

A radioligand-receptor system for luteinizing hormone (LH), USING transplantable mouse luteoma, was used to investigate the interactions of LH, other peptide hormones, and LH subunits. Since tumor size decreased as did production of androgenic hormones following hypophysectomy, the luteoma is believed to have been dependent on pituitary tropic hormones; posthypophysectomy histologic changes supported this conclusion. An homogenate was prepared from 1-4 gm luteomas, which had been borne by mice for 4-10 months. Ovine LH, bovine LH, and human chorionic gonadotrophin reduced the binding of iodine-125 human luteinizing hormone (125-I-hLH). Growth hormone, adrenocorticotrophic hormone, and prolactin had no capacity to interfere with binding of 125-I-hLH. Though follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) reduced the binding somewhat, the reductions were consistent with the known presence of contaminating amounts of LH in the FSH and TSH. The accumulated results of a number of experiments suggest that binding to the luteoma LH receptor requires a particular polypeptide structural conformation, one found in the native hormone but found in neither alpha nor beta subunit alone.     

TSH is a negative regulator of skeletal remodeling

The established function of thyroid stimulating hormone (TSH) is to promote thyroid follicle development and hormone secretion. The osteoporosis associated with hyperthyroidism is traditionally viewed as a secondary consequence of altered thyroid function. We provide evidence for direct effects of TSH on both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption, mediated via the TSH receptor (TSHR) found on osteoblast and osteoclast precursors. Even a 50% reduction in TSHR expression produces profound osteoporosis (bone loss) together with focal osteosclerosis (localized bone formation). TSH inhibits osteoclast formation and survival by attenuating JNK/c-jun and NFkappaB signaling triggered in response to RANK-L and TNFalpha. TSH also inhibits osteoblast differentiation and type 1 collagen expression in a Runx-2- and osterix-independent manner by downregulating Wnt (LRP-5) and VEGF (Flk) signaling. These studies define a role for TSH as a single molecular switch in the independent control of both bone formation and resorption.     

Biochemical roles of the oligosaccharide chains in thyrostimulin, a heterodimeric hormone of glycoprotein hormone subunits alpha 2 (GPA2) and beta 5 (GPB5)

Thyrostimulin is a heterodimeric hormone composed of GPA2 and GPB5, and shares the thyroid-stimulating hormone receptor (TSHR). Thyrostimulin has three N-linked oligosaccharide chains, two in GPA2 and one in GPB5. The roles of these N-linked oligosaccharides in secretion, heterodimer formation and signal transduction were analyzed. Recombinant GPA2s lacking either of the two oligosaccharides were obtained from conditioned medium, whereas dual site-disrupted GPA2 and the GPB5 mutant were not expressed in either the conditioned medium or cell lysate. The binding between GPA2 and GPB5 was weaker than that between TSH subunits GPA1 and TSH beta. Neither of the oligosaccharides in GPA2 had significant effects on heterodimerization. Disruption of either of the oligosaccharides in GPA2 significantly decreased receptor activation, suggesting their critical role in receptor activation.     

The thyrotropin receptor hinge region is not simply a scaffold for the leucine-rich domain but contributes to ligand binding and signal transduction

The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Delta) in the TSH receptor (TSHR) hinge region (residues 277-418). TSHRDelta287-366, Delta287-371, Delta287-376, and Delta287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRDelta287-384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371-384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRDelta287-371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371-384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.     

Expression of functional TSH receptor in white adipose tissues of hyt/hyt mice induces lipolysis in vivo

To determine the relative importance of TSH in white adipose tissue, we compared the adipose phenotypes of two distinct mouse models of hypothyroidism. These models differed in that the normal reciprocal relationship between thyroid hormone and TSH was intact in one and disrupted in the other. One model, thyroidectomized (THYx) mice, had a 100-fold increase in TSH and a normal TSH receptor (TSHR); in contrast, the other model, hyt/hyt mice, had a 120-fold elevation of TSH but a nonfunctional TSHR. Although both THYx and hyt/hyt mice were in a severe hypothyroid state, the epididymal fat (mg)/body wt (g) (F/B) ratio of THYx mice was much smaller than that of hyt/hyt mice (8.2 ± 0.43 vs. 14.4 ± 0.40, respectively, P < 0.001). The fat cell diameter in THYx mice was also smaller than that in hyt/hyt mice (79 ± 2.8 vs. 105 ± 2.2 μm, respectively, P < 0.001), suggesting that TSH induced lipolysis in adipose tissues. When we transferred a functional mouse TSHR gene and a control plasmid into opposite sides of epididymal fat of hyt/hyt mice by plasmid injection combined with electroporation, fat weight of the TSHR side was decreased to 60% of that of the control side. Messenger RNA levels of hormone-sensitive lipase in epididymal fat containing the transferred TSHR gene were twofold higher than those in tissue from the control side. These results indicated that TSH worked as a lipolytic factor in white adipose tissues, especially in mice in a hypothyroid state.     

Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL (ID50 = 0.44 nM) was comparable to that of 125I-oPRL by unlabeled oPRL (ID50 = 0.35 nM), while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82. These findings indicate that monoclonal antibodies can be readily prepared from partially purified PRL receptors from rabbit mammary gland; two antibodies (M110 and A82) are hormone binding site specific while the other (A917) binds a domain partially but not entirely distinct from the hormone binding site, and that all three antibodies have strong species specificity.     

Parathyroid hormone binding to cultured avian osteoclasts.

Parathyroid hormone (PTH) increases serum calcium concentration via a controversial cellular mechanism. We investigated whether PTH binds avian osteoclasts. Isolated hypocalcaemic hen osteoclasts were incubated with [125I]--bovine PTH (1-84). Specific binding of the hormone to the cells, which reached the equilibrium within 60 min, was observed. Half maximal binding was reached by 10 min. Binding was competitively inhibited by increasing doses of unlabeled PTH, and was about 55% displaced by adding, at the equilibrium, 10(-6) M unlabeled PTH. Autoradiography demonstrated specific label on the osteoclast. The cellular mechanism activated by the hormone remains to be elucidated.     

TSH receptor interaction with the extracellular matrix: role on constitutive activity and sensitivity to hormonal stimulation

Using immunocytochemistry, we have observed that the TSH receptor (TSHR) is concentrated at the leading edge of lamellipodia in both cultured human thyroid cells and in various transfected cells. This segregation of the receptor is due to its interaction with extracellular matrix (ECM) and specially with fibronectin. The TSHR, which interacts with the ECM, is known to undergo cleavage by a matrix metalloprotease. The homologous LH receptor, which does not interact with ECM, is not cleaved. The attachment to the ECM modifies the functional properties of the receptor: it increases adenylate cyclase stimulation by hormone, whereas PLC stimulation is not modified. Furthermore, the constitutive activity of the TSHR is only observed in attached cells, suggesting that it is dependent on TSHR interaction with the ECM. Thus, aside from its classical properties of hormone binding and signalization through G proteins, the TSHR is also involved in cell-matrix interactions, which modulate its functional properties.     

Regulation of the phosphatidylinositol 3-kinase,Akt/protein kinase B,FRAP/mammalian target of rapamycin,and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.     

Existence of parathyroid hormone binding sites on murine hemopoietic blast cells

We demonstrated that 125I-labeled human parathyroid hormone (1-34;8,18-Nle,34-Tyr)[[125I]hPTH(1-34)] bound specifically to hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Half-maximal inhibition of binding was achieved at concentrations of unlabeled hPTH(1-34) of about 5 x 10(-9)M. Insulin and hPTH(39-68) did not compete for PTH binding sites. Specific binding of hPTH(1-34) was detected in neither macrophages nor multinucleated cells (MNC's). Furthermore, treatment of hemopoietic blast cells with hPTH(1-34) stimulated MNC formation, and the range of concentrations (10(-10)-10(-8)M) over which hPTH(1-34) caused these effects was similar to that which inhibited the binding of [125I]hPTH(1-34). These findings suggest the presence of a PTH receptor on osteoclast precursors and the direct effect of PTH on them, resulting in osteoclast-mediated bone resorption.