Endocrinology Vol. 144, No. 4 1324-1330
Copyright © 2003 by The Endocrine Society
Targeted Restoration of Cleavage in a Noncleaving Thyrotropin Receptor Demonstrates that Cleavage Is Insufficient to Enhance Ligand-Independent Activity
Chun-Rong Chen,
Gregorio D. Chazenbalk,
Sandra M. McLachlan and
Basil Rapoport
Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Basil Rapoport, M.B., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: rapoportb{at}cshs.org.
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Abstract
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Two unusual features of the TSH receptor (TSHR) ectodomain are its intramolecular cleavage at the cell surface into disulfide-linked subunits and its constraint of ligand-independent (constitutive) activity inherent to the serpentine region. Whether ectodomain cleavage alters the level of TSHR constitutive activity is an important unanswered question. To address this issue, we used a TSHR engineered so as not to undergo spontaneous cleavage into subunits (deletion of amino acid residues 317–366 and GQE367–369NET substitution). Into this noncleaving TSHR (termed TSHR-D1-NET), we introduced thrombin recognition motifs (termed Thr 6 and Thr 18) at the site of spontaneous cleavage. Treatment of intact Chinese hamster ovary cells expressing TSHR-D1-NET-Thr 6 and -Thr 18 with thrombin induced cleavage into A and B subunits, as determined by 125I-TSH covalent cross-linking. Nevertheless, constitutive activity of the thrombin-cleaved TSHR was unaltered. The level of TSHR constitutive activity was, therefore, fully dissociated from intramolecular cleavage into subunits. Trypsin treatment of the same cells expressing the noncleaving TSHR also generated disulfide-linked A and B subunits but, in contrast to thrombin, enhanced TSHR constitutive activity. Therefore, the activating effect of trypsin appears to involve clipping at an additional, as-yet unidentified, site.
In summary, our data demonstrate that TSHR cleavage is, by itself, insufficient to reduce TSHR ectodomain constraint on ligand-independent constitutive activity. These data are consistent with other evidence that A subunit shedding consequent to TSHR cleavage is a critical factor in enhancing TSHR constitutive activity.
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Introduction
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TSH BINDING TO the ectodomain markedly activates the TSH receptor (TSHR), primarily via the cAMP pathway (1). However, even in the absence of ligand, the TSHR has clearly detectable activity, or "noise" (2). This high level of TSHR constitutive activity is not observed with the other glycoprotein hormone receptors and is of pathophysiological importance in explaining, at least in part, persistent low level thyroid function in central hypothyroidism. TSHR constitutive activity would be even higher were it not partially suppressed by the TSHR ectodomain (3, 4). For this reason, the TSHR ectodomain has recently been termed a tethered inverse agonist (4). Ligand binding reverses this suppressive effect by switching the TSHR ectodomain into a far more potent agonist.
Unlike the other glycoprotein hormone receptors, some mature TSHR on the cell surface undergo spontaneous intramolecular cleavage into two subunits (A and B) that remain linked by disulfide bonds (reviewed in Ref. 5). Cleavage occurs immediately upstream of a C peptide region corresponding approximately to a 50-amino-acid insertion in the TSHR relative to the noncleaving gonadotropin hormone receptors (Refs. 6 and 7 ; and Fig. 1
). Subsequent to cleavage at this initial upstream site, the C peptide region is removed through a process of progressive degradation (7). An intact C peptide cannot be recovered (7, 8), hence our use of the term "C peptide region." Remarkably, deletion by mutagenesis of the C peptide region does not, by itself, abrogate spontaneous TSHR cleavage into A and B subunits (9). An additional chimeric substitution with the noncleaving LH receptor (GQE367–369NET) is necessary to create a noncleaving TSHR (9).
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Figure 1. Schematic representation of the TSHR. A large, but variable, fraction of single polypeptide chain TSHR on the cell surface undergoes intramolecular cleavage within the ectodomain. Cleavage generates an extracellular A domain linked by disulfide bonds to a serpentine, membrane spanning B subunit. This process is associated with the loss of an intervening C peptide region that approximates a 50-amino-acid (50AA) insertion in the TSHR relative to the noncleaving gonadotropin receptors (reviewed in Ref. 5 ). Only the approximate locations of the cleavage sites are known. Nine leucine rich repeats are depicted as  -helices and ß-sheets. The disulfide bonds involved in A and B subunit linkage are not definitively established and those depicted represent deductions from available mutagenesis data (5 ).
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Considerable evidence supports the concept that these two unusual properties of the TSHR (high constitutive activity and intramolecular cleavage) are linked. Thus, tryptic clipping of the TSHR ectodomain on the surface of intact cells enhances constitutive activity (10) and, under identical conditions, converts residual single chain TSHR on the cell surface into two subunit forms of the receptor without loss of TSH binding (9). The same trypsin treatment also deletes a monoclonal antibody (mAb) epitope (residues 354–359; Ref. 10). This epitope is now known to be within the C peptide region that is lost consequent to physiological TSHR cleavage into A and B subunits. However, functional studies on a TSHR mutant engineered so as not to cleave into subunits (see above) did not support the concept of a relationship between cleavage and constitutive activity (11).
We performed the present study to resolve the question of the relationship between TSHR intramolecular cleavage and constraint of constitutive activity. By introducing thrombin recognition motifs in the noncleaving TSHR mentioned above, we restored intramolecular cleavage at the physiological spontaneous cleavage site. We now show that TSHR cleavage is, by itself, insufficient to reduce TSHR ectodomain constraint. These data are consistent with other evidence (3, 4) suggesting that A subunit shedding consequent to TSHR cleavage is a critical factor in enhancing TSHR constitutive activity.
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Materials and Methods
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Construction and expression of TSHR mutants
The following TSHR mutants were created previously but required further modification to introduce a H601Y substitution, and to remove most (1.3 kb) of the 3'-untranslated region that reduces the level of TSHR expression (12): 1) deletion of amino acid residues 317–366 (13); 2) deletion of residues 317–366 and substitution of residues 367–369 (GQE367–369NET), in brief "D1-NET" (9, 14). The reason for the H601Y substitution (QuikChange Site-Directed Mutagenesis Kit, Stratagene, San Diego, CA) is described below (see Discussion). H601 in our TSHR cDNA is not a cloning artifact because it was present in multiple TSHR cDNA clones that we isolated and because the identical substitution has been observed in another patient (15).
Thrombin recognition sites were introduced into TSHR-D1-NET (see above) by the PCR using overlapping oligonucleotide primers and Pfu polymerase (Stratagene) with the TSHR-D1-NET cDNA as template. Two different thrombin recognition sites were used: 1) the original sequence LDPRS (16), modified to LVPRGS to facilitate cleavage (17); and 2) LDPRSFLLRNPNDKYEPF, which contains both thrombin binding sites (16). Each thrombin recognition site was inserted directly after TSHR amino acid residue 316 and was followed by TSHR residue 367. The PCR products were restricted with AflII and SpeI and substituted for the same fragment in TSHR-D1-NET. Nucleotide sequences of all mutations were confirmed by dideoxynucleotide sequencing.
Plasmids were expressed in Cos-7L cells cultured in DMEM supplemented with 10% fetal calf serum, penicillin (100 U/ml), gentamicin (50 µg/ml), and fungizone (2.5 µg/ml). Cells in 12-well plates were transiently transfected with 1 µg plasmid DNA using FuGENE6 (Roche, Indianapolis, IN) according to the protocol of the manufacturer and were tested 48 h after transfection. For ligand cross-linking studies, plasmids were stably transfected into Chinese hamster ovary (CHO)-K1 cells with Superfect (QIAGEN, Santa Clarita, CA). Selection was with 400 µg/ml G418 (Life Technologies, Inc., Gaithersburg, MD). Surviving clones (>100 per 100-mm-diameter culture dish) were pooled and propagated for further study. Cells were cultured in Ham’s F-12 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), gentamicin (50 µg/ml), and fungizone (2.5 µg/ml).
Flow cytometry
Cells were detached from cell culture dishes by treatment with 1 mM EDTA and EGTA in PBS, 10 mM HEPES (pH 7.4). Flow cytometric analysis was according to the protocol described previously (18). We used 1) a mouse monoclonal antibody to the TSHR, 2C11 (Serotec Ltd., Oxford, UK; 1:100 dilution) whose epitope is reported to contain amino acid residues 354–359 (10) and, 2) serum from a mouse containing polyclonal antibodies to the TSHR ectodomain (1:50 dilution). As a second antibody we used fluorescein isothiocyanate-conjugated, affinity-purified goat antimouse IgG (1:100 dilution; Caltag Laboratories, Inc., Burlingame, CA).
Trypsin treatment of TSHR on the cell surface
To prevent TSHR trafficking or recycling to the cell surface, cells in 12-well plates were preincubated for 30 min at 37 C in growth medium supplemented with 20 µg/ml Brefeldin A and 50 µM Monensin (both from Sigma, St. Louis, MO). The cells were then treated for 2 min with Krebs-Ringer HEPES buffer containing 0.01% trypsin (Sigma), with or without 0.01% trypsin inhibitor (Sigma) exactly as described by Van Sande et al. (10). After rinsing with Krebs-Ringer HEPES buffer containing 0.1% BSA and 0.01% trypsin inhibitor, the cells were incubated for 1 h at 37 C in the same buffer supplemented with 25 µM Rolipram (Sigma), Brefeldin A, and Monensin. The buffer or medium was aspirated, intracellular cAMP was extracted with 95% ethanol, evaporated to dryness, and resuspended in 0.5 ml of 50 mM sodium acetate, pH 6.2. After acetylation, cAMP was measured by RIA using cAMP, 2 O-succinyl-[125I]iodotyrosine methyl ester and a rabbit anti-cAMP antibody (Fitzgerald, Concord, MA). cAMP levels were normalized for the concentration of cellular protein measured by the Bradford assay, and the data were expressed as pmol cAMP/mg of protein per well.
Covalent cross-linking of radiolabeled TSH
Highly purified bovine TSH (National Hormone and Distribution Program) was radiolabeled with 125I to a specific activity of approximately 60 µCi/µg using Bolton-Hunter reagent (4400 Ci/mmol; NEN Life Science Products, Boston, MA). Confluent 100-mm-diameter dishes of TSHR-expressing cells were incubated for 2.5 h at 37 C with approximately 5 µCi 125I-TSH followed by cross-linking with 1 mM disuccinimidyl suberate (Sigma) and processing as described previously in detail (6). After addition of Laemmli sample buffer (19) containing 0.7 M ß-mercaptoethanol (30 min at 50 C) the samples were electrophoresed on 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad Laboratories, Inc., Hercules, CA). Radiolabeled proteins were visualized by autoradiography on Biomax MS x-ray film (Eastman Kodak Co., Rochester, NY).
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Results
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To determine the relationship between TSHR cleavage and the degree of ectodomain constraint on constitutive functional activity, we used a TSHR with the minimal modification necessary to prevent spontaneous intramolecular cleavage into A and B subunits. Based on the noncleaving LH receptor, this TSHR has the 50-amino-acid insertion (amino acid residues 317–366) deleted and a chimeric three residue substitution (GQE 367–369 NET; Ref. 9). TSHR cleavage occurs on the cell surface, so we first used flow cytometry on intact cells to compare the level of expression of this noncleaving receptor, termed TSHR-D1-NET, vs. the wild-type TSHR. Using a mouse polyclonal antibody to the TSHR ectodomain, both the wild-type TSHR and TSHR-D1-NET expressed to comparable degrees on the surface of transiently transfected CHO cells (Fig. 2, middle panels). Mouse mAb 2C11 (20) recognizes an epitope (containing amino acids 354–359; Ref. 10) within the C peptide region. With this antibody, detection of the wild-type TSHR, but not TSHR-D1-NET, on the cell surface confirmed the absence of the C peptide region in the latter receptor (Fig. 2, lower panels).
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Figure 2. Flow cytometry to assess the level of expression on the cell surface of noncleaving TSHR-D1-NET and the spontaneously cleaving wild-type TSHR. COS-7L cells transfected with the cDNA for the wild-type TSHR or TSHR-D1-NET were tested using a mouse monoclonal antibody (2C11) to the C peptide region (lower panels) or a polyclonal antibody raised to the TSHR ectodomain (middle panels). Normal mouse serum was used as a negative control (upper panels).
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Using noncleaving TSHR-D1-NET as a template, we attempted to restore cleavage by introducing thrombin cleavage motifs at the initial spontaneous TSHR cleavage site, deduced to be very close to amino acid residue 316 (Fig. 3A; and Refs. 6, 7 and 9). One thrombin motif involved 6 amino acids (D1-NET-Thr 6) and the other 18 amino acids (D1-NET-Thr 18). To determine whether thrombin had the intended proteolytic effect, we cross-linked 125I-TSH to intact cells before and after treatment with thrombin. Receptor cleavage was determined by electrophoresis of the cross-linked products. Under reducing conditions, the TSH-binding A subunit (if present) will separate from the disulfide-linked B subunit. In the absence of thrombin treatment (control), TSHR-D1-NET was uncleaved, with TSH cross-linking only to the single polypeptide chain form of the receptor on the cell surface (Fig. 3B). The D1-NET-Thr 6 receptor was also uncleaved, whereas D1-NET-Thr 18 revealed a minor degree of spontaneous intramolecular cleavage. Treatment of intact cells with thrombin largely cleaved D1-NET-Thr 6 and D1-NET-Thr 18 into receptors with two subunits. TSHR-D1-NET, which lacked a thrombin cleavage motif was unaffected by thrombin and remained as a single polypeptide chain. In parallel, we treated the same cells with trypsin rather than with thrombin. Trypsin cleaved TSHR-D1-NET whether or not the thrombin recognition motifs were present (Fig. 3C). The sizes of the 125I-TSH/A subunit adducts were similar (
75 kDa) whether the single chain receptors were cleaved with thrombin or with trypsin. This size is also the same as reported previously after spontaneous cleavage of the wild-type TSHR (reviewed in Ref. 5).
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Figure 3. Conversion of a noncleaving TSHR variant into a cleaving receptor by insertion of thrombin recognition sites. A, Schematic representation of a TSHR-D1-NET, a receptor that does not undergo spontaneous intramolecular cleavage into A and B subunits (9 ). This receptor lacks the 50-amino-acid insertion (amino acid residues 317–366; corresponding approximately to the C peptide region) and has TSHR amino acids 367–369 substituted with the corresponding residues in the noncleaving LH receptor. During spontaneous intramolecular cleavage of the wild-type TSHR, the C peptide region is deleted with a proximal cleavage site occurring closely upstream of residue 317 (6 7 9 ). At this position in noncleaving TSHR-D1-NET, we inserted thrombin recognition sites (bold letters, underlined) of 6 residues (D1-NET-Thr 6) or 18 residues (D1-NET-Thr 18; see Materials and Methods). The vertical line indicates the sites of thrombin cleavage. B and C, Covalent cross-linking of radiolabeled TSH to noncleaving TSHR with or without thrombin recognition sites. TSHR-D1-NET, D1-NET-Thr 6, and D1-NET-Thr 18 are shown schematically in panel A. Monolayer cultures of intact CHO cells stably expressing the indicated receptors were pre-treated for 1 h with thrombin (panel B) or for 2 min with trypsin (panel C), as described in Materials and Methods. Control represents preincubation in the same medium without additive. The medium was then removed and 125I-TSH added for 2.5 h followed by covalent cross-linking (Materials and Methods). Cell membranes were solubilized and resolved on 7.5% polyacrylamide gels under reducing conditions. The gels were then dried and autoradiography performed. The data shown are representative of three experiments. D, Covalent cross-linking of radiolabeled TSH to the wild-type (WT) TSHR shown to indicate its degree of spontaneous intramolecular cleavage compared with noncleaving TSHR-D1-NET. This experiment is representative of scores performed in our laboratory in which a proportion (typically 60–70%) of TSHR expressed on the cell surface undergo cleavage into A and B subunits.
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Having established that thrombin could restore cleavage at a targeted site of a noncleaving TSHR, the critical question was whether this proteolytic effect of thrombin would result in an increase in TSH-independent constitutive activity. Constitutive cAMP generation by cells expressing TSHR-D1-NET, as well as TSHR-D1-NET with either short or long thrombin recognition site, was clearly detectable relative to cells transfected with vector alone (Fig. 4). The same cells in monolayer were also treated with thrombin under conditions identical to those used in the TSH cross-linking experiments. As expected, because of the absence of a thrombin cleavage motif in TSHR-D1-NET, thrombin treatment had no functional effect on constitutive cAMP generation by cells expressing TSHR-D1-NET. Most important, despite its ability to cleave TSHR-D1-NET-Thr 6 and D1-NET-Thr 18 into A and B subunits (Fig. 3B
), thrombin had no effect on the level of constitutive activity of these receptors (Fig. 4). In contrast, trypsin treatment of parallel cultures of the same cells increased receptor constitutive activity, as is well established for the wild-type TSHR (10).
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Figure 4. Restoration of TSHR cleavage by thrombin does not alter constitutive activity. COS-7L cells were transfected with the cDNAs for the receptors indicated in Fig. 3A. As controls, cells were also transfected with the empty vector. Cells were treated with medium containing no additive (control), thrombin for 1 h or trypsin for 2 min (Materials and Methods). Cells were then incubated for a further 1 h with phosphodiesterase inhibitor before assay of intracellular cAMP levels. Data represent the mean + SD of values obtained in duplicate wells, each measured in duplicate. The data shown are representative of three experiments.
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Finally, we addressed the question of the relationship between TSHR amino acid residues 317–366, corresponding approximately to the C peptide region (6), and TSHR constitutive activity. As shown above, trypsin activates TSHR-D1-NET despite the absence of these amino acids in this receptor (Figs. 3A and 4). However, in addition to this deletion, TSHR-D1-NET has a small chimeric substitution with the noncleaving LH receptor (GQE367–369NET). We therefore examined a spontaneously cleaving TSHR in which only amino acids 317–366 are deleted (13). Tryptic treatment of intact cell monolayers enhanced the basal activity of this receptor in a manner similar to the wild-type TSHR (Fig. 5).
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Figure 5. Deletion of the C peptide region in the spontaneously cleaving wild-type TSHR does not abrogate the trypsin-induced increase in ligand-independent constitutive activity. COS-7L cells were transiently transfected with empty vector (vector), vector containing the cDNA for the wild-type TSHR, or with the TSHR with the C peptide region deleted (amino acid residues 317–366). Unlike TSHR-D1-NET, the latter receptor spontaneously cleaves into subunits. Following pretreatment for 2 min with 0.01% trypsin, either alone or together with 0.01% trypsin inhibitor, cells were incubated for a further 1 h in the presence of phosphodiesterase inhibitor before assay of intracellular cAMP levels (Materials and Methods). Data represent the mean + SD of values obtained in duplicate wells, each measured in duplicate. The data shown are representative of three experiments.
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Discussion
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TSHR cleavage into disulfide-linked subunits is an enigmatic process. Cleavage does not alter ligand binding affinity (21) and is not necessary for TSH action (22). Recently, it has been suggested that only the two subunit, and not the single chain, TSHR forms dimers or multimers (23). TSHR cleavage is also followed by shedding of some A subunits from the cell surface (24, 25), a process of potential importance in the initiation or amplification of the autoimmune response to the TSHR in Graves’ disease (26). Finally, as mentioned above, cleavage of the TSHR into subunits may contribute to its high ligand-independent constitutive activity, also of pathophysiological importance.
In the present study, we investigated the relationship between high TSHR constitutive activity and intramolecular cleavage into A and B subunits. Study of this relationship is complicated by interassay and intertechnique variability in assessing the extent of intramolecular cleavage of the wild-type TSHR on the cell surface (for example, see Refs. 21 and 27). We overcame this difficulty by using, as a substrate, a TSHR (TSHR-D1-NET) modified so as not to cleave into A and B subunits on the cell surface (9). Previous experiments with cells expressing this modified TSHR had suggested that its constitutive activity was similar to that of the wild-type TSHR (11). However, subsequent information revealed that a polymorphism (H601Y; Ref. 28) in the receptor used in this study was not silent but, instead, significantly reduced constitutive activity (29, 30). This polymorphism explained our previous inability (data not shown) to confirm the report of tryptic enhancement of TSHR constitutive activity (10) and required us to reevaluate our conclusions regarding TSHR cleavage and constitutive activity. In the present study, we mutated TSHR-D1-NET to the common phenotype (Y601).
By inserting thrombin recognition sites at, or very close to, the site of spontaneous TSHR cleavage (7, 9, 21), thrombin cleaved TSHR-D1-NET into A and B subunits. Under identical conditions, thrombin treatment of intact cells in monolayer culture did not alter the level of receptor constitutive activity. In contrast, tryptic treatment of the same cells produced a similar structural change to thrombin but did increase basal functional activity. There was, therefore, a dissociation in the functional effects of the two enzymes. These data provide clear and unequivocal evidence that TSHR cleavage into subunits is, in itself, insufficient to enhance constitutive activity.
The present data are also instructive in terms of the mechanism by which the ectodomain constrains TSHR constitutive activity. Tryptic enhancement of TSHR constitutive activity is associated with removal of TSHR amino acid residues 354–359, comprising, at least in part, the epitope of mouse mAb 2C11 (10). These data are the basis for the present concept that deletion of a short segment in this region of the TSHR ectodomain triggers a subtle conformational change responsible for activation of basal activity (10). However, in our study trypsin activated, as effectively as the wild-type, receptors in which amino acid residues 317–366 (encompassing residues 354–359) were deleted. These data conclusively demonstrate that the tryptic deletion of TSHR amino acid residues 354–359 is insufficient, or is not directly related to, altered constitutive activity. The TSHR activating effect of trypsin must, therefore, involve clipping at an additional site that remains to be determined.
In summary, by restoring cleavage at a targeted site in a noncleaving TSHR, our study provides a conclusive answer to the question of whether TSHR ectodomain cleavage alters the level of TSHR constitutive activity. TSHR cleavage is, by itself, insufficient to reduce TSHR ectodomain constraint on ligand-independent constitutive activity. A subunit shedding that follows TSHR cleavage is likely to be more important in enhancing TSHR constitutive activity.
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Footnotes
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Abbreviations: CHO, Chinese hamster ovary; mAB, monoclonal antibody; Thr 6 and Thr 18, thrombin recognition motifs; TSHR, TSH receptor.
Received November 1, 2002.
Accepted for publication December 20, 2002.
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Top
Abstract
Introduction
