Thyrotropin Receptor Cleavage at Site 1 Does Not Involve a Specific Amino Acid Motif but Instead Depends on the Presence of the Unique, 50 Amino Acid Insertion*

Kunihiko TanakaDagger §, Gregorio D. ChazenbalkDagger , Sandra M. McLachlanDagger , and Basil RapoportDagger

From the Dagger  Thyroid Molecular Biology Unit, Veterans Affairs Medical Center and the University of California, San Francisco, California 94121 and the § Nagasaki University School of Medicine, Nagasaki, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intramolecular cleavage of the single chain receptor at two separate sites. The region involved in cleavage at Site 2 has been identified, but previous mutagenesis studies failed to identify Site 1. We now report fortuitous observations on the effect of trypsin on the TSHR that localizes a small region harboring Site 1. Thus, as detected by immunoblotting and by 125I-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearing a glycan moiety from the C terminus of the A subunit. Based on the TSHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglycosylated A subunit relative to a series of C-terminal truncated TSHR ectodomain variants, places cleavage Site 1 in the vicinity of, or closely upstream to, residue 317. Remarkably, mutagenesis of every amino acid residue between residues 298-316 (present study) and 317-362 (previous data) did not prevent cleavage at Site 1. However, cleavage at this site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor family.

In summary, these data provide novel insight into TSHR intramolecular cleavage. Cleavage at Site 1 does not depend on a specific amino acid motif and differs from cleavage at Site 2 by involvement of a mechanism requiring the presence of the enigmatic TSHR 50-amino acid "insertion."

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The thyrotropin receptor (TSHR)1 is unique among the presently known members of the superfamily of G protein-coupled receptors for two reasons. First, ligand-mimicking autoantibodies to the TSHR arise commonly and are the direct cause of hyperthyroidism in Graves' disease (1, 2). Second, intramolecular cleavage of some, but not all, TSHR on the cell surface generates two subunits (A and B) that remain linked by disulfide bonds (3, 4). Surprisingly, subunit formation has recently been found to occur by cleavage at two separate sites, with the loss of a TSHR segment (C peptide) (5). Because of the pathophysiological importance of the TSHR, there is presently much interest in characterizing its cleavage sites. A TSHR segment involved in cleavage at the second, downstream site has recently been identified using a chimeric receptor approach (6). Cleavage at Site 2 is unrelated to the presence of a specific amino acid motif. Rather, cleavage is abrogated by the transposition into the TSHR of an N-linked glycosylation motif from the homologous region of the closely related, but noncleaving, luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) (6).

In contrast to Site 2, the upstream cleavage site (Site 1) has been much more difficult to localize. Relative to the non-cleaving LH/CGR, the TSHR contains a 50-amino acid "insertion" (TSHR residues 317-366) (7) that can be deleted without loss of ligand binding or receptor function (8). Observations that deletion of TSHR residues 317-366 did not prevent cleavage into A and B subunits and did not alter the size of the A subunit suggested a cleavage site closely upstream of residue 317 (9). However, mutation of a series of RK-rich motifs in this region failed to abrogate cleavage (10). The subsequent discovery of two TSHR cleavage sites (5), together with the estimated size of the deglycosylated A subunit polypeptide backbone (5, 11), raised the possibility that, contrary to the previous evidence, cleavage Site 1 did lie within the unique 50-amino acid region. However, extensive mutagenesis within this region failed to prevent cleavage at this site (6).

We now report fortuitous observations on the effect of trypsin on the TSHR that support and extend the original concept of a cleavage site closely upstream of residue 317. Extensive mutagenesis studies in this region now explain the previous paradoxical data. Thus, cleavage at Site 1 does not involve a specific amino acid motif. Instead, cleavage depends on the presence of the more downstream 50 amino acid insertion.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture-- TSHR-10,000 are a Chinese hamster ovary (CHO) cell line in which overexpression of the human TSHR (~2 × 106 receptors/cell) was attained by transgenome amplification using a dihydrofolate reductase minigene (12). These cells, as well as CHO cells expressing TSHR mutants (unamplified transgenomes), were propagated in Ham's F-12 medium supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), gentamicin (50 µg/ml), and amphotericin B (2.5 µg/ml).

TSH Receptor Mutations-- All of the following mutations were introduced into a TSHR unable to cleave at Site 2 (GQE367-369NET) (6) as follows. (i) For RK312-313QQ (previously termed CS3)(10), the MluI-EcoRV fragment was substituted for the same fragment in TSHR-GQE367-369NET. (ii) The "D4" mutation involved the substitution of the D4 segment (amino acid residues 298-308) of the TSHR with the homologous region of the LH/CGR. For this, the MluI-EcoRV fragment from TSH-LHR-D4 (13) was used to replace the same fragment in TSHR-GQE367-369NET. (iii) The "D5" mutation from TSHR-LHR-D5, involving the substitution of TSHR residues 309-316 with those of the LH/CGR (13), was introduced into TSHR-GQE367-369NET by the same approach as for D4. (iv) An RK313-314QQ replacement in TSH-LHR-D5-GQE367-369NET (iii, above) was generated by PCR using overlapping primers and Pfu DNA polymerase (Stratagene, San Diego, CA). (v) Four receptor mutants involved alanine substitutions in the TSHR region between residues 305-316 (AAAA305-308, AAAA309-312, AAAA310-313, and AAAA314-316), and (vi) deletion of amino acid residues 317-366 were also introduced into TSHR-GQE367-369NET by PCR using the same approach. The nucleotide sequences of the PCR fragments were confirmed by the dideoxynucleotide termination method (14).

Plasmids were stably transfected with Superfect (Qiagen Inc., Chatsworth, CA) into CHO cells cultured as described above. Selection was with 400 µg/ml G418 (Life Technologies, Inc.). Surviving clones (>100/100-mm diameter culture dish) were pooled and propagated for further study.

Immunoprecipitation of Precursor-labeled TSHR-- TSHR-10,000 cells near confluence in 60-mm diameter culture dishes were rinsed with phosphate-buffered saline (PBS) and pre-incubated (0.5 h, twice) in Dulbecco's modified Eagle's-high glucose, 4500 mg/l, methionine- and cysteine-free medium containing 5% heat-inactivated FCS. The cells were then pulsed (1 h at 37 C) in 2 ml of fresh medium supplemented with 0.2 mCi/ml of [35S]methionine/cysteine (>1000 Ci/mmol, NEN Life Science Products, Wilmington DE). After aspiration of the medium and rinsing the cells once with PBS, chase was performed for 14-16 h (overnight) in standard, non-selective medium with 10% FCS. Cells were treated for the indicated times at 37 °C with 100 µg/ml trypsin (Sigma) in Krebs-Ringer Hepes buffer, with or without the addition of trypsin inhibitor (100 µg/ml, Sigma), exactly as described by Van Sande et al. (15). Cells were washed twice with PBS and scraped into 1 ml of ice-cold 20 mM Hepes, pH 7.2, 150 mM NaCl (buffer A) and processed as described previously in detail (12). Mouse monoclonal antibody (mAb) A10 or A11 (identical epitopes, kind gifts of Dr. Paul Banga, Kings College, London, UK), was used at a final dilution of 1:1000. Samples were resuspended in Laemmli sample buffer (16) with 0.7 M beta -mercaptoethanol (30 min at 50 °C) and electrophoresed on 10% SDS-polyacrylamide gels (Bio-Rad). Prestained molecular weight markers (Bio-Rad) 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, Rochester, NY).

Immunoblots of TSHR Proteins-- TSHR-10,000 cells in 100-mm diameter dishes were treated for 2 min with trypsin in the presence or absence of trypsin inhibitor, as described above. Cells were then scraped and processed as described previously (5). Samples dissolved in Laemmli buffer with 0.7 M beta -mercaptoethanol were electrophoresed on 10% SDS-polyacrylamide gels using prestained molecular weight markers are described above. Proteins were transferred to ProBlott membranes (Applied Biosystems, Foster City, CA), which were processed as described previously (17). Membranes were incubated overnight (4 °C) with mAb A10 (final dilution of 1:1000).

Enzymatic Deglycosylation of TSHR Protein-- Where indicated, prior to the addition of Laemmli buffer, the protein A·IgG·TSHR complex (immunoprecipitations), the 10,000 × g crude membrane fraction (immunoblots), or concanavalin A-enriched TSHR-261, TSHR-289, and TSHR-309 ectodomain variants (18) were treated (10 min, 100 °C) in denaturing buffer containing 0.5% SDS, 1% beta -mercaptoethanol according to the protocol of the manufacturer (N.E. Biolabs, Beverly MA). N-glycosidase F and endoglycosidase H digestions were as described previously (5). Samples were then subjected to SDS-PAGE, as described above.

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 disuccinimidyl suberate (1 mM; Sigma) and processing as described previously in detail (5). After the addition of Laemmli buffer containing 0.7 M beta -mercaptoethanol (30 min at 50 °C), the samples were subjected to SDS-PAGE and autoradiography as described above.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Trypsin on Cell Surface TSHR Subunit Structure-- The availability of a CHO cell line (TSHR-10,000) expressing very large numbers of TSHR (~2 million receptors/cell) (12) made it feasible to study the effect of trypsin on the structure of the mature TSHR expressed on the surface of precursor-labeled intact cells. In control cells unaffected by trypsin (trypsin + trypsin inhibitor) (Fig. 1), immunoprecipitation revealed two forms of single subunit (uncleaved) TSHR, previously characterized as having complex (~115 kDa) and high mannose (~100 kDa) carbohydrate, respectively (5). In addition, the A (broad ~62 kDa) and B (~42 kDa) subunits were also evident in the TSHR that had undergone intramolecular cleavage. Light trypsinization of cell monolayers prior to solubilization for immunoprecipitation produced three striking effects. First, this treatment completely abolished the single chain TSHR with mature, complex carbohydrate (Fig. 1). The lack of effect on the single chain receptor with immature, high mannose carbohydrate is consistent with trypsin having access to only single chain receptors on the surface of intact cells. Second, trypsin markedly intensified bands migrating at ~ 50 kDa. The origin of bands of this size, which were observed previously (5, 11), has been enigmatic, and it has been suggested that they represent separate proteins co-purifying with the TSHR (11). Their reciprocal relationship with the single chain receptor, as well as their detection with a mAb to the extreme N terminus of the A subunit, establishes the ~50-kDa bands to be smaller than normal A subunits, truncated at their C termini, largely derived from cell surface, single chain receptor.


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Fig. 1.   Effect of trypsin on intact CHO cells expressing the human TSH receptor. TSHR-10,000 cell proteins were precursor labeled (1 h) followed by an overnight chase (see "Materials and Methods"). Thereafter, cells were exposed to trypsin for the indicated times in the presence or absence of trypsin inhibitor. Cells remained in monolayer culture throughout the procedure. After detergent extraction of the cells, TSHR were immunoprecipitated with mouse mAb A10 (23) to the extreme N terminus of the TSHR A subunit (epitope at residues 22-35, with the signal peptide being residues 1-21). Precipitates were subjected to SDS-polyacrylamide gel (10%) electrophoresis under reducing conditions followed by autoradiography. Identification of the indicated bands has been reported previously (5). Complex and high mannose refer to the type of glycan determined by differential sensitivity to endoglycosidase H and F. The apparently paradoxical detection of the B subunit with a mAb to the A subunit is explained by the disulfide linkage of these two subunits, subsequently separated by reduction. The lesser amount of B than A subunit is typical in such experiments and is probably secondary to loss of B subunit tethered to the A subunit during the stringent washing procedure.

Immunoblotting with the same monoclonal antibody (extreme N-terminal epitope) provided a "steady state" glimpse of the TSHR and its subunits in particulate fractions from these cells. By this detection method, as observed previously (5), the major form of TSHR identified in control (trypsin + trypsin inhibitor) cells is the mature A subunit with complex carbohydrate (endoglycosidase H-resistant and endoglycosidase F-sensitive) (Fig. 2). Only a small amount of single chain receptor (with high mannose glycan) is evident. Also in contrast to the immunoprecipitation studies, the B subunit is not detected because it is released by reduction before immunodetection. Consistent with the immunoprecipitation experiments, light trypsinization of intact cells prior to homogenization reduced most of the very broad ~50-62-kDa A subunit band to a focused band at ~50 kDa.


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Fig. 2.   Trypsin-induced reduction in A subunit size is greater for the glycosylated than for the deglycosylated A subunit. TSHR-10,000 cells in monolayer were treated for 2 min with trypsin in the presence or absence of trypsin inhibitor prior to preparation of 100-10,000 × g particulate fractions. Where indicated, aliquots were treated with endoglycosidase H or F (see "Materials and Methods"). The crude membrane preparations were then subjected to SDS-PAGE (10%) under reducing conditions, electroblotted onto membranes, and probed with a mouse mAb (A10) to the A subunit (amino acids 22-35).

Most important from the point of view of understanding TSHR subunit structure, the trypsin-induced reduction in A subunit size was much greater for the glycosylated A subunit (up to 12 kDa) than for the enzymatically deglycosylated A subunit (1-2 kDa) (Fig. 2). These data indicate that the small polypeptide fragment deleted by trypsin at the C terminus of the A subunit contains a glycan moiety. Incidentally, although not relevant to this deduction, a lesser amount of A subunit polypeptide of larger size (~40 kDa) was also evident (Fig. 2). The consistency of this observation in numerous experiments (5) speaks against incomplete deglycosylation and suggests, instead, a "big" A subunit, perhaps formed by intramolecular cleavage only at the downstream site (Site 2).

Confirmation of a glycan moiety on the 1-2-kDa tryptic fragment released by trypsin from the A subunit was obtained in 125I-TSH cross-linking studies to receptors on the surface of intact cells in monolayer. As reported previously (4, 12), TSH bound to both single chain TSHR and to the A subunit of the cleaved TSHR (~75 kDa receptor-ligand adduct). However, after light trypsinization (with the cells remaining in monolayer), TSH subsequently bound to receptors with two forms of the A subunit differing in size by ~8 kDa (~75-kDa versus ~ 67-kDa receptor-ligand adducts) (Fig. 3A). Again, after enzymatic deglycosylation, this large difference in A subunit size was greatly reduced (Fig. 3B).


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Fig. 3.   TSH cross-linking to TSHR on the surface of intact cells confirms that the TSHR fragment released by trypsin contains glycan. TSHR-10,000 cells in monolayer were treated for 2 min with trypsin in the presence or absence of trypsin inhibitor prior to 125I-TSH binding and cross-linking (see "Materials and Methods"). The 100-10,000 × g particulate fractions from these cells were subjected to SDS-PAGE (10%) under reducing conditions. Where indicated, aliquots of the crude membrane preparations were treated with endoglycosidase F (see "Materials and Methods"). Note the greater separation of the TSH-linked glycosylated A subunit doublet in panel A with more prolonged electrophoresis. In panel B, electrophoresis was shorter to visualize the deglycosylated TSH-A subunit adducts.

Examination of the potential N-linked glycosylation sites on the human TSHR reveals that Asn-302, the sixth and last such site, is the only possible contributor to the N-linked glycan moiety removed by trypsin from the C terminus of the TSHR A subunit (Fig. 4). The next, upstream glycan (Asn-198) would produce a polypeptide backbone of only 20 kDa, far smaller than that observed experimentally (see Fig. 2).


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Fig. 4.   Schematic representation of the TSHR, with emphasis on the regions of spontaneous and trypsin-induced cleavage. Site 1 indicates the region involved at the TSHR upstream cleavage site, as deduced from the present study. The horizontal shaded bar is included as a quantitative reference to indicate the maximum size (2 kDa) of the polypeptide fragment released by trypsin from the C terminus of the A subunit (see Fig. 2). Cleavage by trypsin upstream of the glycan at residue 302 must occur at the indicated cluster of R and K residues. The next potential cleavage site upstream trypsin site (Arg-273) would yield a tryptic fragment (encompassing Asn-302) of 3 kDa, far greater than observed experimentally. Site 2 refers to the amino acid triplet that, when substituted with the corresponding N-linked glycosylation motif at the homologous region of the LH/CGR (GQE367-369NET), abrogates cleavage (6).

Incidentally, the presence of both normal and small A subunits after trypsinization (Fig. 3A) was consistently observed in these cross-linking experiments and contrasted with the far lower proportion of normal size A subunit on immunoblotting (see Fig. 2). An explanation for this phenomenon that we favor is that the 2-3-h TSH binding period prior to covalent cross-linking permits trafficking to the cell surface of intracellular TSHR left unscathed by the effect of trypsin only on cell surface receptors.

TSHR A Subunit Size-- Information on the precise size of the TSHR A subunit polypeptide chain backbone would be vital in designing further mutagenesis experiments to localize TSHR cleavage Site 1. Our previous focus on residues downstream of residue 317 (6) was based on observations by us (5) and others (11, 19) that the A subunit polypeptide backbone was ~35 kDa in size relative to standard molecular mass markers. In light of the experiments described above and because extensive mutagenesis downstream of residue 317 had previously failed to abrogate cleavage at Site 1, we reevaluated the size of the deglycosylated A subunit polypeptide relative to a series of TSHR ectodomain variants, truncated at their C termini (Fig. 5) (18). These ectodomain variants provide ideal molecular mass markers because they contain amino acid residues identical to the TSHR A subunit with the exception of a 6 histidine residue tail. After endoglycosidase F digestion, immunoblotting indicated that the TSHR A subunit was clearly larger that TSHR-261-6H and appeared to be intermediate in size to TSHR-309-6H and TSHR-289-6H (Fig. 5). Thus, even if the 6H residue tail retards migration disproportionately to its size (e.g. 2 kDa), cleavage Site 1 is unlikely to be further downstream than TSHR residue 325. 


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Fig. 5.   The polypeptide backbone of the deglycosylated TSHR A subunit, cleaved spontaneously at Site 1, is smaller than that of deglycosylated ectodomain variant TSHR-309-6H. TSHR-261-6H, TSHR-289-6H, and TSHR-309-6H, shown schematically below, were generated in CHO cells and were partially purified from conditioned medium using concanavalin A (18). These samples, together with a 100-10,000 × g particulate fraction of TSHR-10,000 cells were treated with endoglycosidase F, subjected to SDS-PAGE (10%) under reducing conditions, electroblotted onto membranes, and probed with a mouse mAb (A10) to the A subunit (amino acids 22-35) (see "Materials and Methods").

Mutagenesis in the Vicinity of TSHR Cleavage Site 1-- Information that the TSHR A subunit included residue Asn-302, together with evidence that the A subunit polypeptide backbone was smaller than previously thought, as well as the inability of mutagenesis downstream of residue 317 to prevent cleavage at Site 1, prompted us to perform further mutagenesis in the region between residues 302 and 316. To use a "readout" of loss of cleavage by TSH cross-linking to TSHR on the surface of intact cells, all mutations (Fig. 6) were performed on a background of a TSHR unable to cleave at Site 2 (GQE367-369NET) (6).


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Fig. 6.   Amino acid substitutions introduced into the vicinity of cleavage Site 1 in the TSHR. The receptors shown in bold letters do not cleave into subunits.

Prior to appreciating the presence of two cleavage sites in the TSHR, we had observed TSHR cleavage after RK312-313QQ substitution in the wild-type TSHR (10). We, therefore, first reexamined the effect of mutagenesis of this potential cleavage site in combination with the GQE367-369NET mutation (TSHR-RK312-313QQ-NET). Cleavage at Site 1 was not abolished. Thus, both single chain and two-subunit forms of the TSHR were detected by 125I-TSH cross-linking (Fig. 7). We next broadly targeted the region between residues 298 and 316 using receptors TSH-LHR-D4-NET and TSH-LHR-D5-NET that involve replacement of every amino acid, except two (Ser-305 and Gln-307), with the homologous residues of the non-cleaving LH/CGR (Fig. 6). Cleavage at Site 1 was unaltered (Fig. 7). Because of the presence of an RK motif in the LH/CGR in TSH-LHR-D5-NET, we replaced these residues (TSH-LHR-D5-RK313,314QQ-NET), again without affecting cleavage at Site 1. For "insurance," we substituted Ala residues for all TSHR residues between 305 and 316, including the two residues not previously altered (Ser-305 and Gln-307). None of the mutations prevented TSHR cleavage at Site 1. 


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Fig. 7.   Radiolabeled TSH cross-linking to CHO cells stably expressing the receptors described in Fig. 6. Cross-linked products were subjected to PAGE (7.5%) under reducing conditions followed by autoradiography. Note that the ligand, 125I-TSH, binds to the uncleaved, single chain TSH holoreceptor or to the ligand-binding A subunit in the cleaved TSHR, indicating the presence of both cleaved and uncleaved TSHR on the cell surface. The mass of the hormone ligand complex includes one subunit of the ligand, which itself contains two subunits linked by disulfide bonds. Under reducing conditions, only one ligand subunit (~ 14 kDa) remains covalently linked to the TSHR.

Finally, having mutated every single TSHR residue between 298 and 362 (present study and Ref. 6), it was apparent that cleavage at Site 1 did not involve amino acid specificity at the site itself. We, therefore, hypothesized that the mechanism of cleavage at Site 1 was likely to involve a separate region in the TSHR. One potential region was the 50-amino acid segment unique to the TSHR (residues 317-366). Support for this concept was our previous abrogation of Site 1 cleavage by replacing, on a GQE367-369NET background, the entire TSHR "D" domain (residues 261-362) (20) with the corresponding region of the LH/CGR (6). The corresponding LH/CGR D domain lacks most of the 50-amino acid TSHR "insertion". Previously, we had deleted the entire 50-amino acid insertion in TSHR without abrogating receptor cleavage into two subunits (4). However, this deletion had been performed in the wild-type TSHR prior to our discovery of two cleavage sites. Therefore, in the present study, we deleted residues 317-366 in a receptor unable to cleave at Site 2 (TSHR-Delta 50-NET). Cleavage at Site 1 was largely abolished (Fig. 7), confirming our hypothesis.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intramolecular cleavage of the TSHR has been very difficult to study. First, it has only recently been learned that there are two, not one, cleavage sites in the formation of the A and B subunits (5). This information was essential for mutagenesis studies to define the individual cleavage sites. Second, it is now evident that neither cleavage at Site 1 nor cleavage at Site 2 depends on the presence of a specific amino acid motif. Thus, we now show that every amino acid residue in the region of Site 1, including the RK-rich region at residues 310-313, can be altered without preventing cleavage. Similarly, in the case of Site 2, we previously observed that cleavage was unaffected by amino acid substitutions, but was only abolished by the transposition of a glycan motif present at the same site in the LH/CGR, but not in the TSHR. The nonspecificity of the amino acids at both cleavage Sites 1 and 2 suggests that external factors are involved in the cleavage mechanism. For Site 2, we hypothesize that a proteolytic enzyme, possibly a metalloprotease (21) with "relaxed" specificity (22), may be equally adept at cleaving the TSHR and the LH/CGR, but is prevented from doing so in the case of the LH/CGR by steric hindrance from the glycan moiety.

Whether or not the proteases responsible for TSHR cleavage in CHO cells have the same specificity as the proteases causing TSHR cleavage in thyrocytes is an important question that will be answered when the respective proteases are identified and compared. However, in our view, there are unlikely to be great differences because of the very similar TSHR subunit structure observed in the recombinant TSHR in CHO cells and the TSHR in a well differentiated rat thyroid cell line (3).

The novel feature about cleavage at Site 1 (present report) is the involvement of the 50 amino acid segment that is present only in the TSHR and not in the other, non-cleaving glycoprotein hormone receptors. This 50-residue segment does not play a role in cleavage at Site 2 because deletion of this entire segment, which leads to non-cleavage at Site 1, does not abrogate TSHR cleavage (4). After deletion of residues 317-366, intramolecular cleavage of the holoreceptor can only be abrogated when cleavage at Site 2 is prevented. The mechanism by which the TSHR 50 residue insertion contributes to cleavage at Site 1 will be of interest in future studies. We suggest two hypotheses. First, this segment may contain a binding site for a presently unidentified protease. Second, the segment may, itself, contain intrinsic proteolytic activity for an adjacent region (Site 1) in the TSHR.

It should be noted that the boundaries of the TSHR 50 amino acid insertion (residues 317-366) (7) are inexact because of low homology in adjacent regions between the TSHR and LH/CGR. Evidence that TSHR cleavage Site 1 (present study) and Site 2 (6) coincide relatively closely to residues 317-366, together with the previous observation that TSHR residues 317-366 can be deleted without loss of receptor function (8), supports the definition of these boundaries and suggests that these residues largely correspond to a C peptide that must be excised and released during TSHR intramolecular cleavage at two sites. Purification of the putative C peptide for determination of its amino acid composition and N terminus residues is an important, but presently unfeasible, goal. Thus, TSHR intramolecular cleavage cannot be studied in systems conducive to high level TSHR expression (prokaryotic or insect cells), but is only evident in mammalian cells. The mammalian cell TSHR has never been purified. Moreover, the C peptide is no longer present in such receptors after cleavage. Prohibitively small quantities of C peptide are released into relatively large volumes of culture medium.

In summary, novel information on the process of TSHR intramolecular cleavage provides new insight into the subunit structure of the TSHR. The data open the way to future studies on the functional importance of TSHR cleavage, as well as the potential role of the unusual TSHR subunit structure in the pathogenesis of Graves' disease.

    ACKNOWLEDGEMENTS

We thank Dr. Paul Banga, Kings College, London, UK for providing excellent mAb to the TSHR, as well as the National Hormone and Distribution Program, the NIDDK, National Institutes of Health, the Center for Population Research of the NICHD, National Institutes of Health, 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 radioiodination.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK19289 and DK48216.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and requests for reprints should be addressed: Veterans Affairs Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement St., San Francisco, CA 94121.

1 TSHR, thyrotropin receptor; TSH, thyrotropin; LH/CGR, luteinizing hormone/chorionic gonadotropin receptor; CHO, Chinese hamster ovary; FCS, fetal calf serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Adams, D. D., and Purves, H. D. (1956) Proc. Univ. Otago Sch. Med. 34, 11-12
  2. Kriss, J. P., Pleshakov, V., and Chien, J. R. (1964) J. Clin. Endocrinol. Metab. 24, 1005-1028
  3. Furmaniak, J., Hashim, F. A., Buckland, P. R., Petersen, V. B., Beever, K., Howells, R. D., Rees Smith, B. (1987) FEBS Lett. 215, 316-322[CrossRef][Medline] [Order article via Infotrieve]
  4. Russo, D., Chazenbalk, G. D., Nagayama, Y., Wadsworth, H. L., Seto, P., Rapoport, B. (1991) Mol. Endocrinol. 5, 1607-1612[Abstract]
  5. Chazenbalk, G. D., Tanaka, K., Nagayama, Y., Kakinuma, A., Jaume, J. C., McLachlan, S. M., Rapoport, B. (1997) Endocrinology 138, 2893-2899[Abstract/Free Full Text]
  6. Kakinuma, A., Chazenbalk, G. D., Tanaka, K., Nagayama, Y., McLachlan, S. M., Rapoport, B. (1997) J. Biol. Chem. 272, 28296-28300[Abstract/Free Full Text]
  7. Nagayama, Y., Kaufman, K. D., Seto, P., and Rapoport, B. (1989) Biochem. Biophys. Res. Commun. 165, 1184-1190[Medline] [Order article via Infotrieve]
  8. Wadsworth, H. L., Chazenbalk, G. D., Nagayama, Y., Russo, D., Rapoport, B. (1990) Science 249, 1423-1425[Medline] [Order article via Infotrieve]
  9. Russo, D., Nagayama, Y., Chazenbalk, G. D., Wadsworth, H. L., Rapoport, B. (1992) Endocrinology 130, 2135-2138[Abstract]
  10. Chazenbalk, G. D., and Rapoport, B. (1994) J. Biol. Chem. 269, 32209-32213[Abstract/Free Full Text]
  11. Misrahi, M., Ghinea, N., Sar, S., Saunier, B., Jolivet, A., Loosfelt, H., Cerutti, M., Devauchelle, G., and Milgrom, E. (1994) Eur. J. Biochem. 222, 711-719[Abstract]
  12. Chazenbalk, G. D., Kakinuma, A., Jaume, J. C., McLachlan, S. M., Rapoport, B. (1996) Endocrinology 137, 4586-4591[Abstract]
  13. Nagayama, Y., and Rapoport, B. (1992) Endocrinology 131, 548-552[Abstract]
  14. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract]
  15. Van Sande, J., Massart, C., Costagliola, S., Allgeier, A., Cetani, F., Vassart, G., and Dumont, J. E. (1996) Mol. Cell. Endocrinol. 119, 161-168[CrossRef][Medline] [Order article via Infotrieve]
  16. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  17. Rapoport, B., McLachlan, S. M., Kakinuma, A., and Chazenbalk, G. D. (1996) J. Clin. Endocrinol. Metab. 81, 2525-2533[Abstract]
  18. Chazenbalk, G. D., Jaume, J. C., McLachlan, S. M., Rapoport, B. (1997) J. Biol. Chem. 272, 18959-18965[Abstract/Free Full Text]
  19. Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B., and Milgrom, E. (1992) Proc. Natl. Acad. Sci. U. S. A. 895, 3765-3769
  20. Nagayama, Y., Wadsworth, H. L., Chazenbalk, G. D., Russo, D., Seto, P., Rapoport, B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 902-905[Abstract]
  21. Couet, J., Sar, S., Jolivet, A., Hai, M.-T., Milgrom, E., and Misrahi, M. (1996) J. Biol. Chem. 271, 4545-4552[Abstract/Free Full Text]
  22. Black, R. A., Durie, F. H., Otten-Evans, C., Miller, R., Slack, J. L., Lynch, D. H., Castner, B., Mohler, K. M., Gerhart, M., Johnson, R. S., Itoh, Y., Okada, Y., Nagase, H. (1996) Biochem. Biophys. Res. Commun. 225, 400-405[CrossRef][Medline] [Order article via Infotrieve]
  23. Nicholson, L. B., Vlase, H., Graves, P., Nilsson, M., Molne, J., Huang, G. C., Morgenthaler, N. G., Davies, T. F., McGregor, A. M., Banga, J. P. (1996) J. Mol. Endocrinol. 16, 159-170[Abstract]


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