Endocrinology, doi:10.1210/en.2003-0430
Endocrinology Vol. 144, No. 9 3821-3827
Copyright © 2003 by The Endocrine Society
Evidence that the C Terminus of the A Subunit Suppresses Thyrotropin Receptor Constitutive 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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The TSH receptor (TSHR), unlike the LH receptor (LHR), has considerable ligand-independent adenylyl cyclase activity, a feature of pathophysiological importance. The TSHR ectodomain partially suppresses constitutive activity, an effect reversed by trypsin treatment of intact cells. Localizing the functional site of trypsin action would provide insight into how the TSHR ectodomain exerts its constraint. For this purpose, we examined the effect of trypsin on intact cells expressing a series of modified TSHR. Trypsin did not increase cAMP production by a chimeric TSH-LH receptor involving substitution of TSHR residues 261–418 (the ectodomain C terminus). In contrast, with the wild-type TSHR, trypsin enhanced constitutive activity despite mutation of the following potential tryptic cleavage sites [arginine (R) and lysine (K) residues]: 1) K565, K651, K660 in the extracellular loops of the serpentine region; 2) B subunit juxtamembrane residues K371, K401, K415; 3) A subunit residues R310, R312, K313. We previously excluded K337 and K339 from being implicated in TSHR tryptic activation. By exclusion, only one R/K cluster remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293. Mutation of this cluster is incompatible with TSHR cell surface expression. However, tryptic clipping at this locus would reproduce a previously demonstrated structural effect of trypsin on the TSHR, removal of about a 2-kDa polypeptide fragment extending downstream from the locus to the C terminus of the A subunit. Taken together, these data suggest that the C terminus of the A subunit functions as a suppressor of TSHR constitutive activity.
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Introduction
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THE TSH RECEPTOR (TSHR) differs from the other glycoprotein hormone receptors in having ligand-independent (or constitutive) TSHR activity (1, 2, 3). This phenomenon is clinically important. It is well known that hypothyroidism is not as severe with TSH deficiency (secondary or tertiary hypothyroidism) as in athyreosis (whether iatrogenic or disease associated). Moreover, in thyroid cancer therapy, even total TSH suppression cannot be expected to abolish the thyrocyte growth stimulatory effect of the TSHR. Therefore, understanding the mechanism for TSHR ectodomain constraint has the potential for developing novel therapeutic options.
There is an apparent paradox that complicates understanding the mechanism of TSHR ectodomain constraint. On the one hand, it is clearly established that mutation of the TSHR to delete most or all of the ectodomain increases constitutive activity (4, 5). On the other hand, tryptic clipping of the wild-type TSHR ectodomain increases constitutive activity to a quantitatively similar extent but does not remove detectable quantities of ectodomain (6). Therefore, ectodomain constraint can be released even without removal of the ectodomain. Clearly, localizing the site(s) at which trypsin clipping mimics removal of the ectodomain would provide insight into how the ectodomain exerts its suppressive effect.
A number of structural effects of trypsin on the TSHR ectodomain have previously been determined (Fig. 1
): 1) loss of a monoclonal antibody (mAb) epitope that includes amino acid residues 354–359 (6), 2) removal of a polypeptide fragment encompassing residues approximately 287–310 (7), and 3) completion of the conversion of residual single-chain TSHR on the cell surface into the two-subunit form of the receptor (7). All of these alterations are interrelated. The mAb epitope lies within a C peptide region (residues 
317–366) that is removed during spontaneous intramolecular cleavage of the TSHR on the cell surface into A and B subunits that remain linked by disulfide bonds (Fig. 1) (8, 9). As described above, trypsin completes this process and also deletes the adjacent C terminus of the A subunit (residues 287–310).
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FIG. 1. Schematic representation of the TSHR focusing on potential tryptic cleavage sites. The ectodomain of the TSH holoreceptor (418 amino acid residues including the signal peptide) comprises the A subunit and the N-terminal region of the B subunit. A variable proportion of TSHR on the cell surface (typically 50–70%) undergoes intramolecular cleavage with loss of an intervening C peptide region, approximating a 50-amino-acid insertion in the TSHR relative to the noncleaving gonadotropin receptors (reviewed in Ref.18 ). Only the approximate locations of the cleavage sites are known (jagged arrows). After cleavage and removal of the C peptide region, the A and B subunits remain tethered by disulfide linkage through cysteine (C) residues. R and K residues, potential tryptic cleavage sites, are shown within boxes. Amino acid numbering includes a 21-amino-acid signal peptide. Three structural effects of trypsin on TSHR on the surface of intact cells have previously been described: 1) loss of a murine mAb epitope (6 ); 2) accelerated cleavage of remaining uncleaved, single-chain TSHR into disulfide-linked A and B subunits (7 ); 3) clipping at the R/K cluster between amino acid residues 287–293 with loss of an approximately 2-kDa polypeptide fragment at the C terminus of the A subunit (shaded, half-moon) (7 ). This polypeptide fragment contains the glycan at N302.
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Which, if any, of the foregoing structural changes induced by trypsin is responsible for altered functional activity has not been established. Most attention has focused on loss of the TSHR segment containing the mAb epitope (6). However no structure-function relationship has been demonstrated for this phenomenon. Another possible explanation for the trypsin functional effect is that the single-chain, uncleaved TSHR is more constrained than the cleaved, two-subunit form of the receptor. This model could be likened to release of a spring with a consequent conformational alteration between A and B subunits. However, we recently established that TSHR intramolecular cleavage was by itself insufficient to release ectodomain constraint on TSHR constitutive activity (10). In the present study, we used a series of modified TSHR to examine further the structure-function relationship in trypsin-induced de-repression of ectodomain constraint.
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Materials and Methods
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Construction and expression of TSHR mutants and chimeric receptors. Mutant TSH receptor cDNAs: alanine substitutions (RQRK310-313AAAA; K371A, K401A, K415A; K565A; K651A; and K660A) were generated using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA). The template was the wild-type TSHR in the mammalian expression vector pECE-neo (11). The polymorphism (H601Y) in the serpentine region of our TSHR cDNA found to reduce constitutive activity (12, 13) had previously been corrected back to the wild type (10). Chimeric TSH-LH receptors TSH-LHR-11 and TSH-LHR-6 (substitutions depicted in Fig. 2A) were created previously (14) but required further modification to revert to wild type the H601Y polymorphism and remove most (1.3 kb) of the 3'-untranslated region that reduces the level of TSHR expression (15). Deletions were attained by restriction with XbaI, followed by gel purification and religation of the plasmid lacking the 1.3-kb fragment released by the enzyme. Nucleotide sequences of all mutations were confirmed by dideoxynucleotide sequencing.
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FIG. 2. Effect of trypsin on constitutive functional activity of chimeric TSH-LH receptors. A, Schematic representation of chimeric TSH-LH receptors. The ectodomain of the TSHR was divided into five arbitrary segments, A-E, which were replaced in various permutations with the corresponding segments of the LHR (14 ), two of which are depicted. For TSH-LHR-11, the entire ectodomains of the two receptors were transposed. For TSH-LHR-6, TSHR domains D and E (amino acid residues 261–418) were substituted. The absence in the LHR of the 50-amino-acid residue TSHR insertion (residues 317–366) is shown by the thin black line. B, Effect of trypsin on chimeric receptor constitutive activity in the absence of ligand. COS-7L cells transiently transfected with empty vector (vector) or vector containing the cDNA for the wild-type or chimeric TSH-LH receptors were pretreated for 2 min with 0.01% trypsin, either alone or together with 0.01% trypsin inhibitor. Cells were then incubated for a further 1 h in the presence of phosphodiesterase inhibitor before assay of intracellular cAMP levels (see Materials and Methods). Data represent the mean + SD of values obtained in duplicate wells, each measured in duplicate. The data shown are representative of four experiments.
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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. Where indicated (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 (Gibco, 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 amphotericin B (2.5 µg/ml).
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). The use of trypsin inhibitor plus trypsin as a control followed the protocol of Van Sande et al. (6), and in preliminary experiments we confirmed that this control provided basal cAMP values the same as in cells incubated in buffer alone. 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. In experiments in which TSH responsiveness was tested after exposure to trypsin, cells were incubated for 2 h at 37 C in F12 medium supplemented with 25 µM Rolipram, Brefeldin A, and Monensin, with or without 100 mU/ml bovine TSH (Sigma). 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). The cAMP levels were normalized for the concentration of cellular protein measured by the Bradford assay, and the data were expressed as picomoles cAMP per milligram of protein per well.
Covalent cross-linking of radiolabeled TSH and human chorionic gonadotropin
Highly purified bovine TSH (National Hormone and Distribution Program) was radiolabeled with 125I to a specific activity of about 60 µCi/mg using Bolton-Hunter reagent (4400 Ci/mmol; PerkinElmer Life Sciences, Boston, MA). Highly purified human chorionic gonadotropin (hCG) radiolabeled with 125I to a specific activity approximately 20 mCi/mg using Iodo-Gen (Pierce, Rockford, IL) was kindly provided by the laboratory of Dr. Glenn Braunstein, Cedars-Sinai Medical Center. Confluent 100-mm-diameter dishes of TSHR-expressing cells were incubated for 2.5 h at 37 C with approximately 5 µCi 125I-TSH or 125I-hCG followed by cross-linking with 1 mM disuccinimidyl suberate (Sigma) and processing as described previously in detail (8). After addition of Laemmli sample buffer (16) containing 0.7 M ß-mercaptoethanol (30 min at 50 C), the samples were electrophoresed on 7.5% or 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). Radiolabeled proteins were visualized by autoradiography on Biomax MS x-ray film (Eastman Kodak, Rochester, NY).
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Results
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Trypsin action on chimeric glycoprotein hormone receptors
Trypsin increases constitutive activity of TSHR expressed on the surface of intact cells. Given that there are 38 potential tryptic cleavage sites arginine (R) and lysine (K) residues in the TSHR ectodomain as well as three additional potential sites in the extracellular loops, we first wished to narrow the region at which tryptic digestion enhances constitutive activity. LH receptor functional activity is unaffected by trypsin treatment (6). We therefore examined the effect of trypsin on chimeric receptor TSH-LHR-11, which has the entire TSHR ectodomain (amino acid residues 1–418) replaced with the homologous residues of the LHR (14) (Fig. 2A
). The serpentine region, including the extracellular loops, are those of the TSHR. As observed previously (10), using reagents and conditions identical with those in the initial report of Van Sande et al. (6), trypsin increased cAMP levels in intact CHO cells expressing the wild-type TSHR on their surface, an effect prevented by trypsin inhibitor (Fig. 2B). In parallel cultures of cells expressing chimeric receptor TSH-LHR-11, trypsin had no effect on constitutive activity. These data suggest that tryptic cleavage in the TSHR ectodomain itself and not at basic residues in the extracellular loops (K565 in the second loop, K651, and K660 in the third loop) (Fig. 1) increases TSHR constitutive activity. Confirmation of this conclusion is provided in subsequent experiments.
Because all known trypsin-induced structural changes in the TSHR occur in the C-terminal segment of the ectodomain (described above; Fig. 1), we next tested the effect of trypsin on intact cells expressing chimeric receptor TSH-LHR-6. In this receptor, the N-terminal 260 amino acid residues are those of the TSHR and only residues 261–418 (arbitrary domains D and E) of the TSHR are substituted with the homologous residues of the LHR (Fig. 2A). As with TSH-LHR-11, trypsin treatment of intact cells did not increase constitutive activity of TSH-LHR-6 (Fig. 2B). These data localize the tryptic effect on TSHR constitutive activity to the region between amino acid residue 261 and ectodomain insertion into the plasma membrane.
A possible explanation for the inability of trypsin to increase constitutive activity in TSH-LHR-6 (and also TSH-LHR-11) was that the LHR segments in these chimeric receptors were resistant to tryptic digestion. To answer this question, we cross-linked radiolabeled ligand to intact cells expressing these receptors on their surface. Because of their different specificities for ligand (14) we cross-linked 125I-hCG to TSH-LHR-11 and 125I-TSH to TSH-LHR-6. Unlike the wild-type TSHR, neither of these chimeric receptors spontaneously cleave into disulfide linked A and B subunits (17). Electrophoresis of cross-linked adducts under reducing conditions revealed only single, full-length polypeptide chains (Fig. 3, A, lane 1, B, lane 2). In contrast, two forms of TSHR were evident on the cell surface (Fig. 3B, lane 1). As established previously (18), the approximately 120-kDa ligand-receptor adduct contains the single chain TSHR and the approximately 75-kDa adduct contains the ligand-binding A subunit of the cleaved TSHR released from the membrane spanning B subunit by disulfide bond reduction (see Fig. 1).
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FIG. 3. Covalent cross-linking of radiolabeled ligand to chimeric TSH-LHR. Monolayer cultures of intact CHO cells stably expressing the indicated receptors were incubated with radiolabeled ligand for 2.5 h followed by chemical cross-linking (see 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. Control represents cells pretreated for 2 min with 0.01% trypsin together with 0.01% trypsin inhibitor before adding the ligand. Trypsin indicates pretreatment for the same period with trypsin alone. The lanes are numbered for description in the text (see Results). A, Cells expressing TSH-LHR-11 (Fig. 2A) covalently cross-linked with 125I-hCG. B, Cells expressing the wild-type TSHR (WT-TSHR) and TSH-LHR-6 covalently cross-linked with 125I-TSH. Note that ligand cross-linking reveals only the uncleaved receptor and the A subunit of the cleaved receptor. The B subunit is not visible because it does not bind ligand and is released from the A subunit by disulfide bond reduction (29 ). In the case of TSH cross-linking, free ligand migrates close to radioactivity at the gel front and has run off the gel. Free hCG is larger than TSH and is therefore visible on the autoradiograph. The data shown are representative of two (TSH-LHR-11) or three (TSH-LHR-6) experiments.
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Trypsin treatment of the cells before ligand cross-linking produced similar structural effects on all three receptors. The effects on the wild-type TSHR have been reported previously (7). Trypsin converted residual uncleaved, single-chain TSHR on the cell surface into the two-subunit form (Fig. 3B, lane 3). In addition, trypsin reduced the size of the TSHR A subunit (Fig. 3B; compare lanes 1 and 3). This effect has been found to be consequent to deletion of an approximately 2-kDa peptide fragment (containing an N-linked glycan) at the C terminus of the A subunit (7) (Fig. 1). Most important from the perspective of the present study, despite the lack of a functional effect of trypsin on chimeric receptors TSH-LHR-11 and TSH-LHR-6 (Fig. 2B), trypsin largely converted these receptors into two-subunit forms, with ligand-binding A subunits (Fig. 3A, lane 2, B, lane 4). The larger A subunit of the TSH-LHR-6 relative to the wild-type TSHR A subunit (Fig. 3B, lane 4 vs. lane 3) is consistent with two additional N-linked glycan introduced by the chimeric transposition (19).
Mutation of potential trypsin cleavage sites
The inability of trypsin to increase constitutive activity of chimeric receptors TSH-LHR-11 and TSH-LHR-6 implicates R and K residues within TSHR ectodomain juxtamembrane region (amino acid residues 261–418). Nevertheless, because of the importance of the serpentine region of the TSHR in signal transduction (e.g. Ref.20), we wished to definitely exclude potential tryptic cleavage sites in the extracellular loops. We therefore mutated to alanine the three R and K residues in these loops (K565 in loop 2, K651 in loop 3, and K660 in loop 3) (Fig. 1). Constitutive cAMP levels generated by these TSHR mutants (after subtraction of baseline cAMP levels in vector-alone transfected cells) were considerably lower than for the wild-type TSHR (Fig. 4). However, the proportionate increase induced by trypsin (2- to 3-fold) was similar for all mutants and the wild-type TSHR. We turned next to the basic residues in the N terminus of the B subunit, K371, K401, and K415 (Fig. 1). Despite the simultaneous mutation of all three residues, trypsin increased TSHR constitutive activity (Fig. 5). Continuing to address the remaining R and K residues in a counterclockwise manner, we have previously excluded K337 and K339 as being implicated in the trypsin phenomenon. The stimulatory effect of trypsin persisted despite deletion by mutagenesis of the entire C peptide region containing these residues (10).
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FIG. 4. Removal by mutagenesis of potential tryptic cleavage sites in the extracellular loops of the TSHR serpentine region does not abolish trypsin-enhanced constitutive activity. COS-7L cells were transiently transfected with the empty vector (vector), the cDNA for the wild-type TSHR, and the TSHR containing the indicated substitutions of alanine for lysine. After 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 (see Materials and Methods). Data represent the mean + SD of values obtained in quadruplicate wells, each measured in duplicate. Statistical significance (t test); *, P < 0.001; **, P = 0.004; ***, P = 0.025. The data shown are representative of three experiments.
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FIG. 5. Removal by mutagenesis of potential tryptic cleavage sites in the extracellular portion of the B subunit do not abolish trypsin-mediated TSHR activation. COS-7L cells were transiently transfected with the empty vector (vector), the cDNA for the wild-type TSHR, and a TSHR mutant with substitutions of alanine for lysine at residues 317, 401, and 415. After 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 (see Materials and Methods). Data represent the mean + SD of values obtained in duplicate wells, each measured in duplicate. The data shown are representative of seven experiments.
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Further upstream, entering the region of the A subunit, is a cluster of basic residues comprising R310, R312, and K313 (Fig. 1). Once again, the simultaneous mutation of these residues did not abrogate the tryptic effect on basal TSHR activity (Fig. 6). Finally, and by exclusion, only one cluster of R and K residues remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293 (Fig. 1). Moreover, cleavage at any one of these residues would delete the glycan containing peptide at the C terminus of the A subunit (Fig. 1), reproducing a known structural effect of trypsin (7). However, this cluster of basic residues cannot be tested directly because even limited mutation (two of four residues) is incompatible with TSHR cell surface expression (21).
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FIG. 6. Mutation of potential tryptic cleavage sites at the C terminus of the A subunit do not prevent trypsin-enhanced constitutive activity. COS-7L cells were transiently transfected with empty vector (vector) or vector containing the cDNA for the wild-type TSHR and a TSHR with amino acid residues 310–313, KKIR, mutated to AAAA. 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 (see 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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The TSHR ectodomain partially constrains ligand-independent, constitutive activity of the TSHR, a feature of pathophysiological importance. How the ectodomain exerts this effect is unknown. Certainly, deletion by mutagenesis of all or most of the ectodomain reduces constraint (4, 5). However, removal of the ectodomain is not essential. Ligand-independent TSHR constitutive activity can also be enhanced by ectodomain mutations (spontaneous or introduced) (22, 23, 24) as well as by mutations in the extracellular loops of the serpentine region (3). Consequently, a reasonable hypothesis is that the ectodomain constrains the serpentine region of the TSHR by interacting with the exoloops (5, 22, 24). Similar conclusions have been drawn for other members of the glycoprotein hormone receptor family (25).
Trypsin also can enhance TSHR constitutive activity. A trypsin-induced increase in thyroid tissue cAMP content (26) or adenylyl cyclase activity (27) was first observed two decades ago. Only later, with expression of the recombinant TSHR in nonthyroidal cells, was this tryptic effect proven to be on the TSHR and, in particular, on the ectodomain (6). Clearly, localizing the functional tryptic cleavage site(s) would be of heuristic value in elucidating the inverse agonist property of the TSHR ectodomain. The first clue regarding this location was trypsin-induced loss of a mAb epitope including TSHR amino acids 354–359 (6). However, this observation provides less precise localization than originally anticipated. Unknown at the time, spontaneous intramolecular TSHR cleavage into disulfide-linked A and B subunits removes a C peptide region of approximately 50 amino acid residues that contains the mAb epitope (Fig. 1) (8, 9). Not all cell surface receptors undergo intramolecular cleavage (28) and trypsin completes the process (Fig. 3) (7). Trypsin clipping at any site within the TSHR disulfide-linked loop could therefore lead to loss of the C peptide region with the mAb epitope. A corollary is that the mAb epitope can be removed only concomitant with TSHR intramolecular cleavage into disulfide-linked A and B subunits (see Fig. 1).
Two other possible mechanisms by which trypsin could induce its effect on TSHR functional activity have previously been excluded. First, trypsin could delete a large segment of the ectodomain from a major proportion of cell surface TSHR, thereby mimicking the deletion mutagenesis studies described above. Strong contrary evidence is that trypsin pretreatment enhances constitutive activity to the same extent as mutational deletion (4, 5, 6) but has no effect on the cAMP response to TSH stimulation (6). We have confirmed this observation and also extended the finding to the action of thyroid-stimulating autoantibodies (Chen, C.-R., S. M. McLachlan, and B. Rapoport, unpublished data). Both agonists require the TSHR ectodomain for functional activity. A second possible explanation was that tryptic activation of the TSHR was less dependent on the exact site of clipping than on conversion of the single chain into the two-subunit form, with release of intramolecular tension and subsequent conformation changes in the serpentine region. This mechanism was excluded by inserting a thrombin recognition motif in a mutant TSHR that cannot spontaneously cleave into subunits. Thrombin treatment restored TSHR intramolecular cleavage into disulfide-linked A and B subunits but did not increase TSHR constitutive activity (10).
The present study provides new insight into the mechanism by which trypsin enhances TSHR constitutive activity. First, the data obtained with chimeric TSH-LH receptors clearly localize the functional tryptic site to the C-terminal region of the ectodomain (amino acid residues 261–418), consistent with the previously observed loss of a mAb epitope (6). Second, mutagenesis of all potential tryptic sites, R and K residues) in the TSHR extracellular loops excludes these loops from playing a role in trypsin activation. Third, in the remainder of the ectodomain C-terminal region, all R and K residues downstream of by R293 can also be eliminated from contention.
Finally, only one R/K cluster (K287, K290, K291, R293) remains as a potential functional tryptic cleavage locus that would still leave disulfide-linked A and B subunits. This R/K cluster is not amenable to simultaneous mutagenic substitution (21), perhaps because it is the dominant group of basic residues in the TSHR ectodomain. Nevertheless, and most important, this site contains the only residues in the ectodomain at which clipping will delete an approximately 2-kDa polypeptide, N-linked glycan-bearing fragment at the C terminus of the A subunit, a previously established structural effect of trypsin action (see Fig. 1) (7). Tryptic clipping at this locus will all also reproduce the observed phenomenon of converting all remaining single-chain TSHRs into the two-subunit form, with A and B subunits remaining linked by disulfide bonds. This cleaved form of the TSHR will lack the C-peptide region containing the mAb epitope (8, 9). Moreover, removal by trypsin of the approximately 2-kDa polypeptide (approximately residues 287–313) would explain why targeted restoration of cleavage by thrombin in a noncleaving TSHR mutant did not increase constitutive activity (10). The thrombin recognition motif was inserted at the site of spontaneous cleavage downstream of the 2-kDa polypeptide (see Fig. 1). Consequently, unlike trypsin, thrombin cleavage would leave the approximately 2 kDa polypeptide intact.
All these data taken together provide strong evidence that tryptic cleavage at one or more of the R and K residues between amino acids 287 and 293 leads to de-repression of TSHR ectodomain constraint on functional activity. This finding raises the possibility that the C terminus of the A subunit (approximately residues 287–313) plays a role in the inverse tethered ligand property of the TSHR ectodomain. We note, too, the proximity of this potential tethered ligand to amino acid residues S281, C283, and C284, all of which contribute to the modulation of TSHR constitutive activity (24).
In conclusion, using trypsin as a tool, our study provides insight into the mechanism of TSHR ectodomain constraint on constitutive function. Tryptic cleavage within an R/K cluster at amino acid residues 287–293 is likely to contribute to de-repression of ectodomain constraint. The data further suggest that the C terminus of the A subunit functions as a suppressor (or tethered inverse agonist) of TSHR constitutive activity.
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Footnotes
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This work was supported by NIH Grant DK19289.
Abbreviations: CHO, Chinese hamster ovary; hCG, human chorionic gonadotropin; LHR, LH receptor; mAb, monoclonal antibody; TSHR, TSH receptor.
Received April 8, 2003.
Accepted for publication May 19, 2003.
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Abstract
Introduction
