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Endocrinology Vol. 141, No. 10 3573-3577
Copyright © 2000 by The Endocrine Society

ARTICLES

Evidence that Cleavage of the Thyrotropin Receptor Involves a "Molecular Ruler" Mechanism: Deletion of Amino Acid Residues 305–320 Causes a Spatial Shift in Cleavage Site 1 Independent of Amino Acid Motif1

Kunihiko Tanaka, 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: rapaportB{at}cshs.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Some TSH receptors (TSHR) on the cell surface cleave into A and B subunits. Cleavage at upstream Site 1 is followed by the proteolytic excision of an intervening C peptide region terminating at a downstream Site 2. Although present evidence suggests that Site 1 lies between amino acid residues 303 and 317, the mechanism and exact amino acid(s) involved in cleavage are unknown. Previous amino acid substitutions at Site 1 failed to abrogate cleavage. We, therefore, performed deletion mutations within this region. Cleavage of cell surface TSHR, detected by 125I-TSH cross-linking to intact cells, was not prevented by deletion of four individual segments within the Site 1 cleavage region ({Delta}305–308, {Delta}309–312, {Delta}313–316, {Delta}317–320). However, deletion of the entire region ({Delta}305–320) reduced the extent of cleavage and shifted the cleavage site upstream of the glycan at amino acid residue N302. Elimination of this glycan (N302Q substitution) reversed the effect of deleting amino acid residues 305–320 on TSHR cleavage, suggesting that reduced cleavage at the new, upstream cleavage site was caused by steric hindrance by the glycan at N302. In summary, deletion, as opposed to mutagenesis, of the TSHR cleavage Site 1 region produces a spatial shift in TSHR cleavage Site 1 from downstream to upstream of the glycan at N302. These observations provide strong evidence that TSHR cleavage at this site does not occur at a particular amino acid motif and suggests that cleavage involves a "molecular ruler" mechanism involving cleavage at a fixed distance from a protease attachment site.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A LARGE proportion of TSH receptors (TSHR) on the surface of transfected mammalian cells cleave into disulfide-linked A and B subunits (reviewed in Ref. 1). Intracellular TSHR contain immature carbohydrate, do not cleave (2, 3), and largely undergo aborted synthesis and degradation (2). Cell surface cleavage is associated with deletion of an intervening C peptide region that corresponds approximately to a 50 amino acid insertion in the TSHR relative to the noncleaving LH/CG receptor (LH/CGR) (4, 5). TSHR cleavage appears to occur first at Site 1 upstream of the 50 amino acid insertion and is followed by progressive excision of the C peptide downstream to the Site 2 region (3, 5).

The exact site of cleavage at Site 1 remains unknown. Initial evidence suggested a TSHR cleavage site closely upstream of the TSHR 50 amino acid insertion (6). Relatively low homology between the TSHR and LH/CGR in this region precludes defining the precise limits of the insertion. Taking into account amino acid conservation, we have suggested the N terminus of the insertion to be A317 (7). Retention on the A subunit of an N-linked glycan at amino acid 302 establishes that cleavage occurs downstream of this residue (8). Therefore, cleavage Site 1 appears to lie between N302 and approximately A317. Direct sequencing of TSHR B subunits has determined numerous N termini and has provided evidence for cleavage "around S314" (5). On the other hand, mutagenesis of all residues in the cleavage region, including K313 and other potential subtilisin-related proprotein convertase endoproteolytic sites, have failed to prevent TSHR cleavage (8, 9).

The enzyme(s) and mechanism responsible for TSHR cleavage also remain uncertain. Inhibition of cleavage with a compound, BB-2116, suggested involvement of a matrix metalloprotease (MMP) (10). However, questions have arisen regarding the specificity of BB-2116, and further studies have indicated that the properties of the TSHR cleavage enzyme are distinct from known MMPs (5).

In the present study, we observed that deletion, as opposed to mutagenesis, of the TSHR cleavage Site 1 region shifts the cleavage site from downstream to upstream of the glycan at N302. These observations provide strong evidence that TSHR cleavage at this site does not occur at a particular amino acid motif and suggests that cleavage involves a "molecular ruler" mechanism (11) involving cleavage at a fixed distance from a protease attachment site.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TSH receptor mutations
1) TSHR {Delta}305–308, {Delta}309–312, {Delta}313–316, and {Delta}317–320. Four separate TSHR complementary DNA (cDNA) mutants were generated with deletions of the bases coding for amino acid residues 305–308, 309–312, 313–316, and 317–320, respectively. The plasmids containing these deletions were generated by PCR using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA). Templates were the wild-type TSHR cDNA with the 5'- and 3'-untranslated regions deleted; pTSHR-5'3'TR-NEO-ECE (12). After digestion with DpnI, plasmids were transformed into XL-1 Blue supercompetent cells (Stratagene).

2) TSHR {Delta}305–320. The cDNA coding for TSHR amino acid residues 305–320 was deleted by PCR using overlapping primers and Pfu DNA polymerase (Stratagene). The template was the cDNA for the wild-type TSHR modified by the introduction of three restriction sites (13). The joined DNA fragments were restricted with SalI and SpeI and substituted for the corresponding fragment in pTSHR-5',3'TR-NEO-ECE (see above) (12).

3) TSHR N302Q. The generation of this mutation has previously been reported (14), however, in the full-length (4 kb) TSHR cDNA. To enhance expression, the 5'- and 3'-untranslated regions were deleted by excising the AflII and XbaI fragment and substituting it for the corresponding fragment in the wild-type TSHR cDNA with the 5' untranslated region deleted; pTSHR-5'-NEO-ECE (12).

4) TSHR N302Q, {Delta}305–320. The codon for N302 was replaced with Q in TSHR {Delta}305–320 (see above) by site-directed mutagenesis. The nucleotide sequences of mutations, as well as PCR generated fragments and adjacent restriction sites, were confirmed by the dideoxynucleotide termination method (15).

Plasmids were stably transfected into CHO 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% FCS, penicillin (100 U/ml), gentamicin (50 µg/ml) and Amphotericin (2.5 µg/ml).

Covalent cross-linking of radiolabeled TSH
Confluent 100 mm diameter dishes of TSHR-expressing cells were incubated for 2.5 h at 37 C with ~ 5 µCi 125I-TSH followed by cross-linking with 1 mM disuccinimidyl suberate (Sigma) and processing as described previously in detail (4). Where indicated, for enzymatic deglycosylation, samples were treated (10 min, 100 C) in denaturing buffer containing 0.5% SDS and 1% ß-mercaptoethanol according to the protocol of the manufacturer (New England Biolabs, Beverly, MA). N-glycosidase F digestions were as described previously (4). 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 Laboratories, Inc., Hercules, CA). Prestained molecular weight markers (Bio-Rad Laboratories, Inc.) were included in parallel lanes. We precalibrated these markers against more accurate unstained markers to obtain the molecular weights indicated in the text. Radiolabeled proteins were visualized by autoradiography on Biomax MS x-ray film (Eastman Kodak Co., Rochester, NY). Quantitative densitometry was performed using Kodak 1D scanning software (Eastman Kodak, Rochester, NY). Background density, measured at a site within the lanes but distant from any bands, was subtracted to provide net density values. Statistical analysis was by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Deletion mutations within the cleavage Site 1 region
Previous studies indicated that substitution of all amino acid residues in the region of cleavage Site 1 (Fig. 1AGo) did not abolish TSHR cleavage into A and B subunits (8). To localize cleavage Site 1 more accurately, we considered that deletion rather than substitution mutagenesis might be more effective. Because deletion of the entire area might be incompatible with receptor expression, whereas individual amino acid deletions would be labor intensive and possibly noninformative, we initially generated four sequential TSHR deletion mutants between amino acid residues 305–320 (Fig. 1BGo), namely {Delta}305–308, {Delta}309–312, {Delta}313–316, and {Delta}317–320. Radiolabeled TSH cross-linking to the surface of intact, stably transfected CHO cells expressing these mutant receptors revealed in all cases that the majority of cell surface TSHR cleaved into A and B subunits (Fig. 1CGo). Thus, under reducing conditions, TSH cross-linked to the ligand-binding A subunit dissociated from the disulfide-linked B subunit. Consistent with previous observations (reviewed in Ref. 1), some single chain, uncleaved TSHR (no dissociation under reducing conditions) was also present on the cell surface.



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  		Figure 1. TSHR deletion mutants within the cleavage Site 1 region. A, Schematic representation of the TSHR ectodomain region in which deletion mutations were performed within the region of cleavage Site 1. This cleavage site becomes the C terminus of the ligand binding extracellular A subunit. TSHR cleavage is also associated with the excision of a C peptide region that corresponds approximately to a 50 amino acid residue insertion in the TSHR relative to the noncleaving LH/CG receptor. This excision creates at Site 2 the N terminus of the largely transmembrane and intracellular B subunit. The TSHR A and B subunits remain tethered by disulfide bonds. B, Specific amino acid deletions in the upstream cleavage site region. Deletions are indicated by dots. C, Radiolabeled TSH covalent cross-linking to four TSHR mutants with sequential deletions in the region of upstream cleavage Site 1 ({Delta}305–308, {Delta}309–312, {Delta}313–316, and {Delta} 317–320). Covalent cross-linking of 125I-TSH to cell surface receptors was performed with monolayers of intact Chinese hamster ovary (CHO) cells stably transfected with the deletion mutations (see Materials and Methods). TSH cross-links to the extracellular A subunit of the cleaved receptor, which is separated from the B subunit under denaturing and reducing conditions used for PAGE (7.5%) of the ligand-receptor complexes. As observed previously, cell surface TSHR exist as both cleaved (two subunit) and uncleaved, single polypeptide chain forms (reviewed in Ref. 1 ). Autoradiography was for 5 h.

 
The lack of effect of sequential four amino acid residue deletions within the cleavage Site 1 region led us to delete the entire region ({Delta}305–320). Even this receptor expressed normally on the cell surface and cleaved receptors were clearly evident (Fig. 2AGo). However, this larger deletion mutation produced two effects. First, the proportion of uncleaved to cleaved receptors was increased relative to the wild-type TSHR; that is, the extent of cleavage was reduced. Quantitative densitometric analysis of 7 separate experiments revealed uncleaved to cleaved ratios for the wild-type TSHR and for TSHR{Delta}305–320 to be 0.43 ± 0.08 SEM and 3.38 ± 0.70 (mean ± SEM; P < 0.001), respectively (Fig. 2CGo).



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  		Figure 2. Deletion of the entire cleavage Site 1 region reduces the extent of TSHR cleavage into A and B subunits. Radiolabeled TSH was covalently cross-linked to TSHR with amino acid residues 305–320 deleted. Cross-linking was performed after TSH binding to receptors on the surface of stably transfected CHO cells, followed by PAGE (10%) of ligand-receptor complexes under denaturing and reducing conditions to separate A and B subunits of cleaved receptors. A, Radiolabeled TSH cross-linking to the wild-type TSHR and the deletion mutant ({Delta}305–320). Cell extracts were applied directly to the gel (control) or following enzymatic deglycosylation with N-glycenase F (Endo F). All samples (control and Endo F treated) were heated at 95 C in Laemmli sample buffer (16 ). Autoradiography was for 88 h. B, A second experiment similar to that shown in A except that electrophoretic migration was longer. Autoradiography was for 48 h. C, Quantitative densitometry from seven separate experiments. Data shown are ratios of the densities of the the single chain, uncleaved TSHR vs. those of the A subunits of the cleaved receptors. Bars indicate the mean + SEM. *, P < 0.001; Student’s t test.

 
The second effect of the deletion mutation TSHR{Delta}305–320 was that the size of the 125I-TSH-A subunit complex was slightly smaller (~ 68 kDa) than in the wild-type TSHR (~74 kDa). This difference in size of the ligand-A subunit adduct was abolished by enzymatic deglycosylation with N-glycenase F (Fig. 2AGo). This phenomenon was observed in three separate experiments, the second of which, with a longer electrophoretic run, is shown in Fig. 2BGo. The same disparity in size has been observed previously following trypsinization of cell surface TSHR and has been deduced to result from the loss of the N-linked glycan at amino acid residue 302 at the A subunit C terminus (an epitope at the N terminus remains intact) (8). These data suggest that deletion of residues 305–320 shifts TSHR cleavage at Site 1 from downstream to upstream of N302 (Fig. 1AGo).

Role of the N-linked glycan at amino acid residue 302 on diminished TSHR cleavage induced by {Delta}305–320
We hypothesized that the glycan at TSHR residue N302, possibly through steric hindrance of a protease, could contribute to reduced receptor cleavage following deletion of amino acid residues 305–320. If this hypothesis is correct, elimination of the N-linked glycan at residue 302 would reverse the reduction in TSHR cleavage produced by deletion of residues 305–320. This phenomenon was, indeed, observed (Fig. 3Go). Thus, in three experiments, the ratio of uncleaved to cleaved TSHR was no different between the wild-type TSHR (0.32 ± 0.17; mean + SEM) and the TSHR with the concurrent deletion of residues 305–320 together with the N302Q mutation (0.34 ± 0.10). These ratios were similar to those for the mutation of N302Q alone without the 305–320 deletion (0.24 ± 0.01). In contrast, as observed in seven previous experiments (Fig. 2Go), the ratio of uncleaved to cleaved receptors in TSHR {Delta}305–320 was significantly increased vs. TSHR {Delta}305–320-N302Q (2.03 ± 0.23; P < 0.005) (Fig. 3BGo).



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  		Figure 3. N-linked glycan at amino acid residue 302 is responsible for reduced TSHR cleavage induced by {Delta} 305–320. A, Radiolabeled TSH was covalently cross-linked to intact, stably transfected CHO cells expressing the wild-type TSHR and the following TSHR mutants: 1) {Delta}305–320, deletion of TSHR amino acids 305–320; 2) N302Q, TSHR amino acid N302 replaced with Q to abolish the motif for N-linked glycosylation; 3) {Delta}305–320, N302Q, TSHR containing both the foregoing mutations. Cell extracts (treated at 50 C) were applied to 7.5% polyacrylamide gels. Autoradiography was for 31 h. B, Quantitative densitometry from three separate experiments. Data shown are ratios of the densities of the the single chain, uncleaved TSHR vs. those of the A subunits of the cleaved receptors. Note that the data on the ratios of the wild-type TSHR and {Delta}305–320 provide additional information on these two receptors not included in Fig. 2CGo. Bars indicate the mean + SEM. *, P < 0.005 for {Delta}305–320 vs. {Delta}305–320, N302Q.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cleavage of the TSHR is a complex phenomenon. A number of approaches have been used to understand this process, each having advantages and disadvantages. We have used covalent cross-linking of radiolabeled TSH to intact cells because it is the only method available to us that can provide quantitative information on the proportion of mature TSHR that are cleaved vs. those that remain uncleaved on the surface of intact cells. In our experience, adequate detection of metabolically labeled TSHR by immunoprecipitation is impractical without generation of stable cell lines overexpressing the TSHR by means of transgenome amplification, a laborious process taking approximately 1 yr and not readily applicable for numerous mutant receptors (17). Large-scale affinity purification of TSHR B subunits and determination of their N-terminal amino acid sequences has been a technical tour de force (5). However, the quantities of TSHR monoclonal antibodies required for such studies are not generally available. Moreover, cell surface TSHR as well as intracellular synthetic and degradative products are recovered.

The present study, using covalent cross-linking of radiolabeled TSH to a series of TSHR mutants provides new insight into the mechanism of TSHR cleavage. First, the data strongly support our previous puzzling inability to inhibit TSHR cleavage in the Site 1 region by amino acid substitution (8, 9). Thus, TSHR cleavage can still occur (albeit to a lesser extent) when this entire region (amino acid residues 305–320) is deleted. TSHR cleavage, therefore, joins a number of other membrane-associated proteins, such as pro-TGF-{alpha} (18), pro-TNF-{alpha} (19, 20), angiotensin-converting enzyme (21), amyloid precursor protein (22) and acetylcholine receptor inducing activity (23), with no amino acid specificity at the site(s) of cleavage. The shift to a different cleavage site following removal of a primary cleavage site, observed in the present study with the TSHR, has also been reported for pro-TGF-{alpha} (18) and pro-TNF-{alpha} (19, 20).

A second point of information from the deletion of residues 305–320 in the wild-type TSHR is that this modification reduces the extent of TSHR cleavage into A and B subunits. These data support the concept that cleavage at upstream Site 1 is primary and is followed by progressive excision or degradation of the C peptide region downstream to the vicinity of Site 2, as suggested by the B subunit amino terminal sequencing data of Milgrom and co-workers (5). Previously, only the concurrent deletion of the 50 amino acid insertion (8), or segments thereof (24), and the introduction of a glycan at residue N367 could abrogate TSHR cleavage. Third, reduced TSHR cleavage consequent to deletion of amino acid residues 305–320 is likely to be caused by steric hindrance by the naturally occurring glycan at N302 adjacent to cleavage Site 1. Thus, the effect of this deletion is reversed by the conservative substitution of a single amino acid that eliminates this N-linked glycan motif.

Identification and characterization of the enzyme(s) responsible for TSHR cleavage would be an important advance. This enzyme is not thyroid specific (cleavage occurs in transfected nonthyroidal cells) yet only cleaves the TSHR and not the closely related gonadotropin receptors. As mentioned above, initial studies using a matrix metalloprotease (MMP) inhibitor (BB-2116) suggested the involvement of an MMP (10, 25). However, recent data question the specificity of BB-2116 inhibition of the TSHR cleavage mechanism (5). Nevertheless, MMPs have broad amino acid sequence specificities (for example 26, 27, 28), consistent with the lack of amino acid specificity at the TSHR cleavage site(s). Moreover, the ever expanding repertoire of these enzymes makes interpretation of data obtained with a particular inhibitor difficult. Remarkably, the TSHR ectodomain anchored to the cell surface by a glycosylphosphatidyl inositol (GPI) tail does not appear to cleave (29, 30). These data suggest that the TSHR protease(s) may interact with the transmembrane, serpentine component of the TSHR, a property more in line with membrane-type MMPs, a number of which have been described in recent years (reviewed in Ref. 31). Protein cleavage at a certain distance from the membrane independent of the amino acid sequence has been described as a "molecular ruler mechanism" (11). TSHR cleavage by a presently unknown membrane-type MMP would be compatible with a spatial shift in a TSHR cleavage site caused by deletion of amino acid residues 305–320.


   Acknowledgments
 
We thank the National Hormone and Distribution Program, the National Institute of Diabetes and Digestive and Kidney Diseases, the Center for Population Research of the National Institute of Child Health and Human Development, The Agricultural Research Service of the U.S. Department of Agriculture, and the University of Maryland School of Medicine for kindly providing the highly purified bovine TSH for radio-iodination.


   Footnotes
 
1 This work was supported by NIH Grant DK-19289. Back

Received May 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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L. Q. Nguyen, O. K. Arseven, H. Gerber, B. S. Stein, J. L. Jameson, and P. Kopp
Cloning of the Cat TSH Receptor and Evidence Against an Autoimmune Etiology of Feline Hyperthyroidism
Endocrinology, February 1, 2002; 143(2): 395 - 402.
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R. Latif, P. Graves, and T. F. Davies
Oligomerization of the Human Thyrotropin Receptor. FLUORESCENT PROTEIN-TAGGED hTSHR REVEALS POST-TRANSLATIONAL COMPLEXES
J. Biol. Chem., November 21, 2001; 276(48): 45217 - 45224.
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