An insertion in the human thyrotropin receptor critical for high affinity hormone binding.

Thyrotropin (TSH), luteinizing hormone (LH), and chorionic gonadotropin (CG) are structurally related glycoprotein hormones, which bind to receptors that share a high degree of sequence similarity. However, comparison of the primary amino acid sequences of the TSH and LH-CG receptors reveals two unique insertions of 8 and 50 amino acids in the extracellular domain of the TSH receptor. The functional significance of these insertions were determined by site-directed mutagenesis. Deletion of the 50-amino acid tract (residues 317 to 366) had no effect on TSH binding or on TSH and thyroid-stimulating immunoglobulin (TSI) biological activities. In contrast, either deletion or substitution of the eight-amino acid region (residues 38 to 45) abolished these activities. This eight-amino acid tract near the amino terminus of the TSH receptor appears to be an important site of interaction for both TSH and TSI.

Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors.

We examined the relative effects of thyrotropin (TSH) and TSH receptor autoantibodies in the sera of patients with autoimmune thyroid disease on three TSH-lutropin/chorionic gonadotropin (LH/CG) receptor extracellular domain chimeras. Each chimera binds TSH with high affinity. Only the chimera with TSH receptor extracellular domains ABC (amino acids 1-260) had a functional (cAMP) response to thyroid stimulatory IgG. The chimeras with TSH receptor domains CD (amino acids 171-360) and DE (amino acids 261-418) were unresponsive. The lack of response of the chimera with TSH receptor domains DE was anticipated because it fails to transduce a signal with TSH stimulation, unlike the other two chimeras. A different spectrum of responses occurred when the TSH-LH/CG chimeras were examined in terms of autoantibody competition for TSH binding. IgG with TSH binding-inhibitory activity when tested with the wild-type TSH receptor also inhibited TSH binding to the chimera with TSH receptor domains DE. Dramatically, however, these IgG did not inhibit TSH binding to the chimera with TSH receptor domains CD, and had weak or absent activity with the chimera with TSH receptor domains ABC. Chimeras with TSH receptor domains ABC and DE were equally effective in affinity-purifying IgG with thyroid-stimulatory and TSH binding-inhibitory activities. Nonstimulatory IgG with TSH binding-inhibitory activity inhibited the action of stimulatory IgG on the wild-type TSH receptor, but not with the chimera containing TSH receptor domains ABC. In summary, TSH receptor autoantibodies and TSH bind to regions in both domains ABC and DE of the TSH receptor extracellular region. Stimulatory and inhibitory TSH receptor autoantibodies, as well as TSH, appear to bind to different sites in domains ABC, but similar sites in domains DE, of the receptor. Alternatively, TSH and the different TSH receptor antibodies bind with differing affinities to the same site in the ABC region.

Thyrotropin and adenosine 3',5'-monophosphate stimulate the activity of the ferritin-H promoter.

Fragments of the rat ferritin-H 5'-flanking region up to 1 kilobase in length were generated by the polymerase chain reaction using FRTL5 rat thyroid cell genomic DNA as template. Ferritin-H 5'-flanking region fragments of 219, 351, 666, and 1046 basepairs (bp), ligated up-stream to the reporter gene luciferase, were transiently transfected into FRTL5 thyroid cells and NIH-3T3 mouse fibroblasts. In both cell types, constitutive (nonstimulated) ferritin-H promoter activity increased progressively with constructs containing increasing lengths of 5'-flanking region. TSH or (Bu)2cAMP (dBcAMP) stimulation of FRTL5 cells transfected with the shorter (219 and 351 bp) ferritin-H 5'-flanking region fragments increased promoter activity 2- to 3-fold. However, with the longer DNA segments (666 and 1046 bp), the extent of TSH stimulation was less. Exposure of transfected NIH-3T3 cells to dBcAMP mimicked in all respects the effects of TSH and dBcAMP on ferritin-H promoter activity in FRTL5 cells. Transcription initiation sites in the luciferase reporter gene were unaffected by the length of the ferritin-H 5'-flanking region included in the construct or by dBcAMP stimulation. Plasmid constructs with 45 bp of the ferritin-H 5'-flanking region containing a potential cAMP response element did not reveal any promoter activity or dBcAMP responsiveness in this region. Gel shift mobility assays with the -219 bp ferritin-H 5'-flanking region fragment and NIH-3T3 nuclear proteins revealed specific protein-DNA interaction. Reduced DNA mobility was inhibited by excess unlabeled probe DNA, but not by DNA fragments corresponding to the recognition sites for a variety of known trans-activating factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the role of amino acids 38-45 in the expression of a functional thyrotropin receptor.

We previously reported that deletion or substitution of a unique eight-amino acid tract (residues 38-45) in the extracellular domain of the human TSH receptor led to the loss of specific ligand binding to the surface of transfected cells. In the present study we analyzed this region in more detail. Using site-directed mutagenesis of the TSH receptor cDNA, we substituted amino acid residues 38-45, either in three overlapping groups of four amino acids each or individually. The resultant TSH receptor mutant cDNAs were stably transfected into Chinese hamster ovary cells, and the cells were tested for their TSH-binding ability. Our data demonstrate that amino acid residues 38-40 and 42-45 in this region of the human TSH receptor can be substituted without alteration in receptor function and are, therefore, not critical in forming or maintaining the TSH-binding site. However, substitution of Cys41, either alone or together with adjacent amino acids, leads to the loss of TSH binding to its receptor. These data suggest a central role for the amino acid in position 41 in preserving the biological function of the TSH receptor.

A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain.

The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis of the human thyrotropin receptor: role of asparagine-linked oligosaccharides in the expression of a functional receptor.

We studied the role of glycosylation in the expression of a functional human TSH receptor. Oligonucleotide-directed mutagenesis was used to replace, separately or together, the Asn codons with Gln in each of the six potential glycosylation sites in the receptor. Recombinant wild-type and mutated TSH receptors were stably expressed in Chinese hamster ovary cells. High affinity TSH binding and the cAMP response to TSH stimulation were abolished in the receptor mutated at Asn77 as well as in the receptor mutated at all six potential glycosylation sites. In the receptor mutated at Asn113, the affinity of TSH binding was markedly decreased (Kd, 2.6 x 10(-8) 3.3 x 10(-10) M in the wild-type receptor). This affinity was too low to permit the transduction of a signal, as measured by an increase in intracellular cAMP generation. Substitution of Asn at positions 99, 177, 198, and 302 did not appreciably affect the affinity of the TSH receptor for TSH binding or its ability to mediate an increase in intracellular cAMP levels. Therefore, either these four potential glycosylation sites are not glycolysated, or alternatively, oligosaccharide chains at these positions do not play a major role in the folding, intracellular trafficking, stability, or expression of a functional receptor on the cell surface. Conversely, our data suggest that N-linked glycosylation of Asn77 and Asn113 does play a role in the expression of a biologically active TSH receptor on the cell surface.

Thyrotropin can increase the steady state level of a messenger ribonucleic acid species (glyceraldehyde-3-phosphate dehydrogenase) in thyroid cells by decreasing its rate of disappearance.

A cDNA clone (G18) was selected from an FRTL5 rat thyroid cell cDNA library by a differential screening procedure designed to identify TSH-responsive genes in thyroid cells. Nucleotide sequence analysis indicated that G18 was rat alpha-glyceraldehyde-3-phosphate dehydrogenase (GAPD). On Northern blot analysis, TSH increased GAPD mRNA levels in FRTL5 cells, reaching a maximum (4- to 10-fold above basal in different experiments) after approximately 24 h of TSH stimulation. Run-on transcription assays with nuclei prepared from quiescent and TSH-stimulated FRTL5 cells indicated that the action of TSH to increase GAPD mRNA levels was not associated with an increase in the transcriptional activity of this gene. Examination of the rate of disappearance of GAPD mRNA levels after inhibition of mRNA transcription by actinomycin-D revealed that TSH stimulation of FRTL5 cells stabilized the GAPD mRNA (t 1/2 extended from 9 to 56 h). In summary, the present data provide the first demonstration that TSH can increase the level of a mRNA transcript in thyroid cells by reducing its rate of degradation. GAPD is a useful model for future studies on the mechanism by which TSH alters gene expression at a posttranscriptional level.

Role of amino acids 261-418 in proteolytic cleavage of the extracellular region of the human thyrotropin receptor.

Previous in vivo cross-linking studies of TSH to the recombinant TSH receptor revealed that the receptor exists at least in part as a single chain glycoprotein of approximately about 100 kilodaltons (kDa), with intramolecular disulfide bonds. TSH also binds to a 54-kDa amino-terminal fragment of the TSH receptor (cleaved up-stream of amino acid residue 317). In the present study in order to better understand the structure of the TSH receptor, we covalently cross-linked radiolabeled TSH to six TSH-LH receptor extracellular region chimeras and the wild-type TSH receptor expressed on Chinese hamster ovary cells in vivo. In these chimeras, different regions of the TSH receptor were substituted with the homologous regions of the LH receptor. When analyzed under nonreducing conditions by polyacrylamide gel electrophoresis, the TSH-cross-linked products were similar to all TSH-LH receptor chimeras and the wild-type receptor. In contrast, differences among the receptors were noted when the TSH-cross-linked products were examined under reducing conditions. With the exception of two chimeras, as noted previously with the wild-type receptor, two TSH-cross-linked products were observed, representing TSH cross-linked to a holoreceptor of about approximately 100 kDa and a fragment of the receptor of about approximately 54 kDa. However, in the two chimeras in which both domains D and E (amino acids 261-418) of the TSH receptor were substituted, only the holoreceptor and not the smaller fragment was detected. Substitution of domains ABC (amino acids 1-260) did not prevent proteolytic cleavage of the TSH receptor. In conclusion, amino acids 261-418 are necessary for proteolytic cleavage of the extracellular region of the human TSH receptor.

Thyrotropin-induced expression of a gene for a ribosomal protein related to the trk oncogene.

By differential screening of an FRTL5 rat thyroid cell cDNA library, we isolated a clone (G7) corresponding to an mRNA transcript whose steady-state level is increased by thyrotropin (TSH) stimulation by a non-transcriptional mechanism. The nucleotide sequence of the G7 cDNA (0.85 kb) revealed homology with two other genes. First, there was 89% homology with the cDNA for a protein whose amino-terminal end forms the amino terminus of the chimeric tyrosine kinase human oncogene, trk-2h. Second, TSH-responsive G7 is 95% homologous with the 'surf-3' gene within the mouse surfeit locus which codes for the mouse L7a ribosomal protein. These findings are of interest in view of the frequent occurrence in thyroid cancers of an oncogene (PTC) that consists of an unidentified amino terminus linked to a downstream tyrosine kinase moiety.

Transcriptional regulation of ferritin H messenger RNA levels in FRTL5 rat thyroid cells by thyrotropin.

We examined the effect of thyrotropin (TSH) stimulation of FRTL5 rat thyroid cells on ferritin H mRNA levels. On Northern blot analysis, TSH (in the presence of serum and insulin) increased ferritin H mRNA levels, with an initial response evident after 1 h of stimulation. Ferritin H mRNA levels increased approximately 4-fold over basal levels after 4 h of TSH stimulation and showed little increase thereafter, maintaining a plateau for up to 48 h of TSH stimulation. Inducers of cAMP also increased ferritin H mRNA levels in FRTL5 cells to about the same extent, but the rate of response was not as rapid as with TSH stimulation. In serum-poor medium without insulin, the TSH effect was considerably weaker, increasing only about 2-fold after 24 h of stimulation. Also in serum-poor medium, insulin-like growth factor-I alone had a weak stimulatory effect on ferritin H mRNA levels. TSH and insulin-like growth factor-I had additive effects under these conditions. Nuclear run-on transcription assays were performed using nuclei prepared from FRTL5 cells. In serum-containing medium, TSH increased the transcriptional activity of ferritin H mRNA 3-4-fold without an increase in beta-actin transcriptional activity. The kinetics of TSH stimulation of ferritin H transcriptional activity were similar to the cellular ferritin H mRNA response to TSH stimulation. Dibutyryl-cAMP (dB-cAMP) increased ferritin H transcriptional activity about 4-fold, but not as rapidly as did TSH stimulation. In summary, our data indicate that ferritin H mRNA levels in FRTL5 thyroid cells are transcriptionally regulated by both TSH and dBcAMP stimulation. These data contrast with the predominantly nontranscriptional regulation of ferritin H mRNA levels observed in other tissues. The difference in the kinetics of the response to TSH and dBcAMP is consistent with the concept that not all effects induced by TSH stimulation of thyroid cells are mediated by cAMP as a second messenger.

Eleven amino acids (Lys-201 to Lys-211) and 9 amino acids (Gly-222 to Leu-230) in the human thyrotropin receptor are involved in ligand binding.

Our previous studies involving chimeric thyrotropin-lutropin/choriogonadotropin (TSH-LH/CG) receptors suggest that multiple segments spanning the entire extracellular domain of the human TSH receptor contribute to the TSH binding site. Nevertheless, the mid-region (segment C, amino acid residues 171-260) of the receptor extracellular domain is particularly important in TSH binding. In the present studies, we constructed seven new chimeric receptors in order to analyze segment C in further detail. Seven small segments spanning segment C of the TSH receptor were replaced with the counterpart of the rat LH/CG receptor. These mutant receptors were stably introduced into Chinese hamster ovary cells and were tested for hormone binding and cAMP responsiveness to hormone stimulation. The results indicate that 11 amino acids of the TSH receptor (Lys-201 to Lys-211) and the corresponding region of the LH/CG receptor (Thr-202 to Ile-212) are important for specific TSH and human CG binding, respectively. In addition, nine amino acids of the TSH receptor (Gly-222 to Leu-230) are also involved in TSH binding. A further conclusion from these data is that TSH and human CG bind to partially overlapping sites on their respective receptor molecules.

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.

Extracellular domain chimeras of the TSH and LH/CG receptors reveal the mid-region (amino acids 171-260) to play a vital role in high affinity TSH binding.

We constructed a series of TSH-LH/CG receptor chimeras by homologous substitution of relatively small regions of the TSH receptor extracellular domain for the corresponding region of the extracellular domain of the LH/CG receptor. Constructs were stably expressed in Chinese hamster ovary cells. Of the five chimeric receptors, only TSH-LHR-14, which contains mid-region domain C (amino acid residues 171-260) of the extracellular component of the TSH receptor, exhibited TSH binding of relatively high affinity. Consistent with this TSH binding, chimera TSH-LHR-14 was the only one that demonstrated a functional response to TSH stimulation in terms of intracellular cAMP generation. These data indicate that domain C plays a vital role in TSH receptor function.

Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis.

The thyrotropin (TSH) receptor belongs to a family of guanine nucleotide protein-coupled receptors with seven transmembrane-spanning regions joined regulatory together by extracellular and intracellular loops. The cytoplasmic domain comprises three cytoplasmic loops and a cytoplasmic tail that are likely to be important in coupling of the receptor to the guanine nucleotide proteins. To address the question of which portions of the cytoplasmic domain of the TSH receptor are important in this process, we have altered groups of amino acids in the region of the TSH receptor by site-directed mutagenesis. Because of the low affinity of TSH binding to the TSH receptor mutated in the amino terminus of the second cytoplasmic loop and the amino terminus of the cytoplasmic tail, definitive conclusions cannot be made regarding the roles of these regions in signal transduction. However, our data indicate that the first cytoplasmic loop (residues 441-450), the carboxyl-terminal region of the second cytoplasmic loop (residues 528-537), and the carboxyl-terminal (but not the amino-terminal) region of the third cytoplasmic loop (residues 617-625) are important in the ability of the TSH receptor to mediate an increase in intracellular cAMP production. Furthermore, two-thirds of the carboxyl-terminal end of the cytoplasmic tail (residues 709-764; corresponding to the region not conserved between the TSH and lutropin/chorionic gonadotropin receptors) can be removed without functional impairment of the TSH receptor.