Prokaryotic expression of the thyrotropin receptor and identification of an immunogenic region of the protein using synthetic peptides

Graves' disease is characterized by hypersecretion of thyroid hormones due to binding of autoantibodies to the thyrotropin receptor (TSHR). In order to study immunological aspects of the TSHR we expressed the extracellular domain of the rat TSHR (ETSHR) as a fusion protein with beta-galactosidase in a prokaryotic system. The identity of this ETSHR-fusion protein was confirmed by Western blot, using antibodies to synthetic peptides derived from TSHR. Patients' sera reacted to a significantly greater extent with the affinity purified ETSHR relative to control sera. Similarly, sera from patients with Graves' disease displayed significant reactivity with only one of five peptides, RH2 (residues 352-366), when compared with normal sera. These data, together with the predicted hydrophilicity of the peptide RH2, suggest that amino acids 352-366 which lie within one of the unique regions of the extracellular domain of the TSHR may be important for antibody binding.     

Subunit structure of thyrotropin receptors expressed on the cell surface

We studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305-316) would generate a B subunit of 51-52 kDa. However, only smaller B subunits (40-46 kDa) were detected, corresponding to N termini from residues approximately 370 (site 2) extending downstream to the region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45-52 kDa) than the majority of B subunits recovered with a C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding.     

Defining structural and functional dimensions of the extracellular thyrotropin receptor region

The extracellular region of the thyrotropin receptor (TSHR) can be subdivided into the leucine-rich repeat domain (LRRD) and the hinge region. Both the LRRD and the hinge region interact with thyrotropin (TSH) or autoantibodies. Structural data for the TSHR LRRD were previously determined by crystallization (amino acids Glu(30)-Thr(257), 10 repeats), but the structure of the hinge region is still undefined. Of note, the amino acid sequence (Trp(258)-Tyr(279)) following the crystallized LRRD comprises a pattern typical for leucine-rich repeats with conserved hydrophobic side chains stabilizing the repeat fold. Moreover, functional data for amino acids between the LRRD and the transmembrane domain were fragmentary. We therefore investigated systematically these TSHR regions by mutagenesis to reveal insights into their functional contribution and potential structural features. We found that mutations of conserved hydrophobic residues between Thr(257) and Tyr(279) cause TSHR misfold, which supports a structural fold of this peptide, probably as an additional leucine-rich repeat. Furthermore, we identified several new mutations of hydrophilic amino acids in the entire hinge region leading to partial TSHR inactivation, indicating that these positions are important for intramolecular signal transduction. In summary, we provide new information regarding the structural features and functionalities of extracellular TSHR regions. Based on these insights and in context with previous results, we suggest an extracellular activation mechanism that supports an intramolecular agonistic unit as a central switch for activating effects at the extracellular region toward the serpentine domain.     

Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin

The thyrotropin receptor (TSHR) has a unique 50 residue (317-366) ectodomain insertion that sets it apart from other glycoprotein hormone receptors (GPHRs). Other ancient members of the leucine-rich repeat G protein-coupled receptor (GPCR) (LGR) family do exhibit ectodomain insertions of variable lengths and sequences. The TSHR-specific insert is digested, apparently spontaneously, to release the ectodomain (A-subunit) leaving the balance of the ectodomain attached to the serpentine (B-subunit). Despite concerted efforts for the last 12 years by many laboratories, the enzyme involved in TSHR cleavage has not been identified and a physiologic role for this process remains unclear. Several lines of evidence had suggested that the TSHR protease is likely a member of the a disintegrin and metalloprotease (ADAM) family of metalloproteases. We show here that the expression of ADAM10 was specific to the thyroid by specially designed DNA microarrays. We also show that TSH increases TSHR cleavage in a dose-dependent manner. To prove that ADAM10 is indeed the TSHR cleavage enzyme, we investigated the effect of TSH-induced cleavage by a peptide based on a motif (TSHR residues 334-349), shared with known ADAM10 substrates. TSH increased dose dependently TSHR ectodomain cleavage in the presence of wild-type peptide but not a scrambled control peptide. Interestingly, TSH increased the abundance of non-cleaved single chain receptor, as well higher molecular forms of the A-subunit, despite their enhancement of the appearance of the fully digested A-subunit. This TSH-related increase in TSHR digested forms was further increased by wild-type peptide. We have identified for the first time ADAM10 as the TSHR cleavage enzyme and shown that TSH regulates its activation.     

Immunochemical mapping of antigenic regions on the human thyrotropin beta-subunit by antipeptide antibodies

To study antigenic sites present in the beta-subunit of human thyrotropin (hTSH), we produced site-specific antibodies directed against synthetic peptides analogous to the 1-18, 44-59, and 85-112 regions of the thyrotropin beta-subunit. The hTSH beta(1-18) peptide-carrier conjugate elicited antisera capable of binding to both radiolabeled hTSH and its beta-subunit whereas antibodies elicited against the hTSH beta(44-59) peptide-carrier conjugate bound only to the peptide. Thus, the NH2-terminal region of hTSH beta appears to be accessible at the surface of the hormone whereas the hTSH beta(44-59) region may be poorly accessible. Two monoclonal antipeptide antibodies that bound to 125I-hTSH beta, designated as TS01 and TS02, were selected after immunization with the hTSH beta(85-112) peptide-carrier conjugate. The antigenic site recognized by TS01 was located on the eight COOH-terminal(105-112) amino acid residues. TS02 antibody bound to an antigenic region included within Cys95 and Cys105. Both antigenic sites appeared to be more accessible on the free hTSH beta than on the hormone. Immunoblots performed on various preparations containing TSH revealed that TS02 antibody detected the beta-subunit from both the human and bovine species but not the rat TSH beta. Under reducing conditions, a low molecular weight material was identified in hTSH beta, likely caused by intrachain nicking.     

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.     

G13-dependent activation of MAPK by thyrotropin

Stimulation of the thyrotropin receptor (TSHR) activates G proteins of all four subfamilies (G(s), G(i/o), G(q/11), and G(12/13)). Whereas G(s)/cAMP-dependent cellular responses upon TSHR stimulation are well established, other signaling pathways are less characterized. We evaluated TSH-elicited cellular responses in human follicular thyroid carcinoma cells stably expressing the TSHR and in primary, nonneoplastic human thyrocytes. In these cellular models, stimulation with TSH caused activation of p44/42 MAPK and subsequent induction of c-Fos. MAPK stimulation occurred independently of G(s), G(i/o), and G(q/11) signaling. Dominant negative constructs of G(12) or G(13) as well as shRNA-mediated suppression of Galpha(12) or Galpha(13) revealed that MAPK activation was dependent on G(13) but not on G(12) signaling. Furthermore, G(13)-dependent transactivation of the epidermal growth factor receptor was necessary for MAPK activation in follicular carcinoma cells, whereas EGFR was not involved in MAPK activation in nonneoplastic primary thyrocytes. The use of bacterial inhibitors of monomeric GTPases revealed that MAPK activation proceeded independently of Rho proteins but was clostridial toxin B-sensitive, suggesting involvement of Cdc42 or Rac. Thus, our data shed new light on cAMP-independent TSHR signaling and identify the first G(13)-dependent TSHR signaling pathway in human thyrocytes.     

Identification of immunogenic regions in human thyrotropin receptor for immunoglobulin G of patients with Graves' disease.

To identify immunogenic regions in human thyrotropin (TSH) receptor for immunoglobulin G (IgG) of patients with Graves' disease, seven different peptides (each consisting of 14-29 residues long) corresponding to segments of the extracellular domain of the receptor were synthesized. Graves' sera and IgG significantly bound to two out of seven peptides (the amino acid sequence of peptide #1, HQEEDFRVTCKDIQRIPSLPPSTQT; that of peptide #5, LRQRKSVNALNSPLHQEYEENLGDSIVGY). The present data indicate the characteristic existence of immunogenic regions in human TSH receptor for IgG of patients with Graves' disease.     

Inhibition of thyrotropin binding to receptor by synthetic human thyrotropin beta peptides

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.     

Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.     

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.     

A full biological response to autoantibodies in Graves' disease requires a disulfide-bonded loop in the thyrotropin receptor N terminus homologous to a laminin epidermal growth factor-like domain

We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin gamma1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. Removal in various permutations of the four cysteines at TSHR positions 24, 29, 31, and 41 (signal peptide residues are 1-21) revealed Cys(41) to be the key residue for receptor expression. Forced pairing of Cys(41) with any one of the three upstream Cys residues was necessary for trafficking to the cell surface of a TSHR with high affinity TSH binding similar to the wild-type receptor. However, for a full biological response to TSAb, forced pairing of Cys(41) with Cys(29) or with Cys(31), but not with Cys(24), retained functional activity comparable with the wild-type TSHR. These data suggest that an N-terminal disulfide-bonded loop between Cys(41) and Cys(29) or its close neighbor Cys(31) comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin gamma1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease.     

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.     

The cysteine bonding in TSH receptor and it function in autoantibodies recognition

The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.     

Rabbit antibodies toward extracellular loops of the membrane spanning region of human thyrotropin receptor possess thyroid stimulating activities.

We have synthesized three different peptides, E1 (amino acid residues 478-497), E2 (amino acid residues 561-580) and E3 (amino acid residues 649-652), corresponding to the first, the second and the third extracellular loops of the membrane spanning region of human thyrotropin receptor (TSH-R), respectively. We have produced rabbit antibodies toward these peptides and evaluated their thyroid stimulating antibody (TSAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. Although only slight TSAb activity was observed in E1 antibodies, E2 and E3 antibodies possessed strong TSAb activities, the values of which were 1118% and 910%, respectively. None of these antibody had TBII activities. These results suggest that antibodies against the extracellular loops of the TSH-R can stimulate cAMP formation in thyroid cells and that these regions may be one of the candidates for the epitope against autoantibodies from patients with Graves' disease.     

Autoantibodies interacting with purified native thyrotropin receptor.

Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti-TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (alpha-subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves' disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25-1 x 10-10 M) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) than in control subjects (1.76 +/- 0.21) (mean +/- SEM), there was an overlap between the groups. Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves' disease. The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects. Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves' disease. What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation.     

Targeted expression of the human thyrotropin receptor A-subunit to the mouse thyroid: insight into overcoming the lack of response to A-subunit adenovirus immunization

The thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves' hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, "non-self" regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.     

Fish gonadotropin and thyrotropin receptors: the evolution of glycoprotein hormone receptors in vertebrates

We have cloned and characterized, for the first time in fish, two different gonadotropin receptors (GTHR) and a single thyrotropin receptor (TSHR) from amago salmon (Oncorhynchus rhodurus) and Nile tilapia (Oreochromis niloticus). Phylogenetic analyses and intron/exon structure suggest that the two GTHRs in fish are comparable to tetrapod follicle stimulating hormone and luteinizing hormone receptors. Temporal and spatial expression patterns, examined by Northern blot analysis and in situ hybridization, paralleled those seen in mammals and birds. Consequently, genetic and functional divergence of two GTHRs and TSHR probably occurred before the teleost and tetrapod split.     

Rabbit antibodies against two different extracellular domains of human thyrotropin receptor possess thyroid stimulating activities

We have produced rabbit antibodies against synthetic peptides corresponding to the mid-region (amino acid residues 172-202, C peptide) and to the unique segment near the transmembrane region (amino acid residues 341-370, P peptide) in the extracellular component of the human thyrotropin (TSH) receptor and evaluated their biological activities. Both anti-C peptide antibodies raised in two rabbits showed strong thyroid stimulating activities (TSAb) (4127% and 2548%). Anti-P peptide antibodies raised in two rabbits were also strongly positive for TSAb activities (359% and 3468%). However, none of these antibodies had TSH-binding inhibitor immunoglobulin (TBII) activities. These results suggest that the domains responsible for TSAb are likely to span the entire extracellular component of the TSH receptor.     

Growth hormone, thyrotropin and prolactin responses to simultaneous administration of human growth hormone-releasing factor and thyrotropin releasing hormone in the bovine

The effects of intravenous injection of synthetic human pancreatic growth hormone-releasing factor-44-NH2 (hpGRF-44) and synthetic thyrotropin releasing hormone (TRH), or hpGRF-44 in combination with TRH on growth hormone (GH), thyrotropin (TSH), and prolactin (PRL) release in dairy female calves (6- and 12-month-old) were studied. When 0.25 microgram of hpGRF-44 per kg of body weight (bw) was injected in combination with TRH (1.0 microgram per kg of bw), the mean plasma GH concentration of the 12-month-old calves rose to a maximum level of 191.5 ng/ml (P less than 0.001) at 15 min from the value of 6.8 ng/ml before injection at 0 min. The maximum level was 3.1 and 6.1 times as high as the peak values obtained after injection of hpGRF-44 (0.25 microgram per kg of bw) and TRH (1.0 microgram per kg of bw), respectively (P less than 0.001). The area under the GH response curve for the 12-month-old calves for 3 hr after injection of hpGRF-44 in combination with TRH was 2.5 times as large as the sum of the areas obtained by hpGRF-44 and TRH injections. In contrast, the mean plasma GH level was unchanged in saline injected calves. The magnitudes of the first and the second plasma GH responses in the 6-month-old calves to two consecutive injections of hpGRF-44 in combination with TRH at a 3-hr interval were very similar. The peak values of plasma GH in the calves after hpGRF-44 injection were 2-4 times as high as those after TRH injection.(ABSTRACT TRUNCATED AT 250 WORDS)