From the Autoimmune Disease Unit, Cedars-Sinai Research Institute, Los Angeles, California 90048 and the University of California, Los Angeles School of Medicine, Los Angeles, California 90045
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ABSTRACT |
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Among the glycoprotein hormone receptors, only the thyrotropin receptor (TSHR) cleaves (at two sites) into disulfide-linked A and B subunits. A 50-amino acid insertion unique to the TSHR ectodomain (residues 317-366) plays no role in ligand binding or signal transduction, but its deletion abrogates cleavage at Site 1, closely upstream of the insertion. We sought to define the region within the 50-amino acid tract involved in TSHR cleavage at Site 1. Mutation of small segments within this region previously failed to prevent cleavage at Site 1. We, therefore, divided the 50-amino acid insertion into quartiles and deleted each one individually (TSHR residues 317-327, 328-338, 339-350, and 351-362). As determined by covalent cross-linking of 125I-TSH to intact cells expressing the mutant receptors, none of these deletions prevented TSHR cleavage at Site 1. Neither did larger deletions of quartiles 1 + 2, 2 + 3, and 3 + 4. However, qualitative differences in the extent of receptor cleavage suggested that quartiles 1 and 4 were playing a greater role in cleavage at Site 1 than were the middle two quartiles. In support of this hypothesis, deletion of these two discontinuous segments almost completely eliminated TSHR cleavage at Site 1.
In conclusion, intramolecular cleavage at Site 1 requires the presence
of the N-terminal and C-terminal quartiles of the 50-amino acid
insertion unique to the TSHR. Taken together with previous observations, our data suggest that this tract may provide a
discontinuous binding site for a protease that clips the TSHR at Site 1.
Three unusual features distinguish the thyrotropin
(TSH)1 receptor from the
other glycoprotein hormone receptors. First, stimulating autoantibodies
mimic the action of its ligand TSH and cause Graves' disease, one of
the most common autoimmune diseases affecting humans (reviewed in Ref.
1). Second, a variable proportion of TSH receptors (TSHR) on the cell
surface cleave into two subunits (A and B) that remain linked by
disulfide bonds (2-4). Intramolecular cleavage of the TSHR occurs at
two separate sites with the loss of a putative polypeptide fragment (C
peptide) (5). Finally, relative to the other glycoprotein hormone
receptors, the TSHR contains an insertion of 50 amino acids in
the vicinity of residues 317-366 (low homology makes the exact
boundaries difficult to define). This insertion, bordered by cysteine
residues and with characteristics of an hydrophilic external loop, can
be deleted without affecting ligand and autoantibody binding and
receptor activation (6).
Whether all three of these features are inter-related is an important
unanswered question that may yield clues regarding the pathogenesis of
Graves' disease. Characterization of the mechanism and sites of TSHR
cleavage is, therefore, of pathophysiologic interest. There is evidence
that TSHR cleavage (at an undetermined site) involves a matrix
metalloprotease (7). The putative TSHR C peptide has not been isolated
or characterized, possibly because it is degraded. Recently, a direct
association between TSHR cleavage and the 50-amino acid insertion has
been found. Thus, cleavage at Site 1 is dependent upon and appears to
occur closely upstream of, this 50-residue tract (8). However, there is
no specific motif for cleavage at Site 1 in that every amino acid in
this region can be replaced without abrogating cleavage. Cleavage at Site 2, which also lacks a specific motif, is not dependent on the
50-amino acid insertion but can be prevented by replacing TSHR amino
acid residues 367-369 with the N-linked glycosylation motif
at the corresponding location in the noncleaving
lutropin/choriogonadotropin receptor (9).
In the present study, we sought to define the region within the
50-amino acid insertion involved in TSHR cleavage at Site 1. Surprisingly, we found that almost the entire tract needs to be present
for cleavage to occur. However, cleavage is most dependent on the
N-terminal and C-terminal quartiles of the 50-amino acid segment.
Because there is no amino acid specificity at the cleavage site
itself, our data suggest that the 50-amino acid insertion in the TSHR
may function as a discontinuous binding site for a protease that clips
the TSHR at Site 1.
TSH Receptor Mutations--
All the following deletion mutations
were introduced into a TSHR unable to cleave at Site 2 (GQE367-369NET) (9); amino acid residues 317-327,
328-338, 339-350, 351-362, 317-338, 339-362, 328-350, and
317-328 + 351-362. DNA fragments containing these deletions were
generated by polymerase chain reaction using overlapping primers and
Pfu DNA polymerase (Stratagene, San Diego, CA) or, when
necessary, Amplitaq Gold (Perkin-Elmer). Templates were plasmids
containing the cDNA for the wild-type TSHR modified by the
introduction of three restriction sites (10) (upstream DNA fragment)
and the same receptor with the mutation GQE367-369NET (9)
(downstream DNA fragment). The joined DNA fragments were restricted
with AflII and SpeI and substituted for the
corresponding fragment in the wild-type TSHR cDNA with the 5'- and
3'-untranslated ends deleted in the expression vector pECE-Neo (11).
The nucleotide sequences of the polymerase chain reaction-generated
fragments, and adjacent restriction sites were confirmed by the
dideoxynucleotide termination method (12). The construction of TSHR
with deletion of residues 317-366 and the GQE367-369NET
substitution (TSHR
Plasmids were stably transfected into Chinese hamster ovary (CHO) cells
with Superfect (Qiagen, Santa Clarita CA). 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.
Cells were cultured in Ham's F-12 medium supplemented with 10% fetal
calf serum (fetal calf serum), penicillin (100 units/ml), gentamicin
(50 µg/ml), and amphotericin B (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 of 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 sample buffer (13) containing 0.7 M
Deletion of the entire TSHR 50 amino acid insertion (residues
317-366) (8), but not mutagenesis of 3-5 amino acid residue blocks
within this region (9), prevents cleavage at Site 1. On this basis, we
reasoned that deleting intermediate length blocks of amino acid
residues would broadly localize the region involved in Site 1 and would
then allow us to focus on smaller segments. We, therefore, divided the
50-amino acid insertion into quartiles and deleted each one
individually (TSHR residues 317-327, 328-338, 339-350, and
351-362). Four residues (363-366) most distal to cleavage Site 1 were
left intact to preserve a restriction site necessary for plasmid
constructs. However, previous studies on chimeric TSH-LH/CG receptors
indicated that deletion of the 46 N-terminal residues was sufficient to
prevent cleavage at Site 1 (9). Because of the presence of two cleavage
sites, to be informative (readout of a noncleaving receptor), deletions
were performed on a background of a TSHR mutant
(GQE367-369NET) in which cleavage at the more downstream
Site 2 is eliminated (9). Contrary to expectations, the sequential
deletion of each of the four quartiles in the TSHR 50-amino acid
insertion (Fig. 1A) did not
prevent TSHR cleavage into two subunits. Thus, covalent cross-linking
of 125I-TSH to monolayers of intact CHO cells expressing
these deletion mutants revealed complexes of ligand with both single
polypeptide chain (uncleaved) and two subunit (cleaved) receptors (Fig.
1B).
INTRODUCTION
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Abstract
Introduction
References
MATERIALS AND METHODS
317-366-NET) has been described previously (6,
8).
-mercaptoethanol (30 min at 50 °C), the samples were
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 Co.).
RESULTS
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Fig. 1.
Deletion of individual quartiles in the TSHR
50-amino acid insertion does not abrogate cleavage at Site 1. Panel A, schematic representation of the amino acid residues
deleted (317-327, 328-338, 339-350, and 351-362). The clear
segments indicate the 50-amino acid region, and the
horizontal lines are the deletions. Note that four amino
acids (363-366) remain to preserve a restriction site for plasmid
constructs. Deletions were performed on a background of a TSHR mutant
(GQE367-369NET) in which cleavage at downstream Site 2 is
eliminated (9) (see also Fig. 5). Abrogation of cleavage at Site 1 can
then be determined, because elimination of both cleavage sites creates
a largely noncleaving TSHR. Panel B, radiolabeled TSH
cross-linking to cell-surface TSHR. CHO cells expressing the wild-type
TSHR express both cleaved (two subunit) and uncleaved, single
polypeptide chain forms of the receptor on the cell surface (Ref. 3;
reviewed in Ref. 1). Similarly, for the above deletion mutants, cleaved
and single chain holoreceptors were detected by autoradiography after
covalent cross-linking of 125I-TSH to monolayers of intact
CHO cells ("Materials and Methods"). TSH cross-links primarily to
the A subunit of the cleaved receptor, which is separated from the B
subunit under denaturing and reducing conditions used for
polyacrylamide gel electrophoresis (7.5%) of the ligand-receptor
complexes.
Based on the above results, we turned to larger, rather than smaller, deletions in the TSHR 50-amino acid insertion. For this purpose we constructed three TSHR with deletions of quartiles 1 and 2 (amino acid residues 317-338), quartiles 2 and 3 (residues 328-350), and quartiles 3 and 4 (residues 339-362) (Fig. 2A). Again, all deletions were made on a background of a receptor with the mutation (GQE367-369NET) that eliminates cleavage at Site 2. As with the smaller deletions, radiolabeled TSH cross-linking to the surface of intact cells indicated that none of these much larger deletions prevented TSHR cleavage at Site 1 (Fig. 2B).
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At this juncture, on re-evaluating data from repeated experiments showing an inability to prevent cleavage at Site 1, we noted that there appeared to be quantitative differences in the degree of TSHR cleavage occurring with the different deletions (see for example Figs. 1 and 2). Densitometric analysis confirmed this impression (Fig. 3). Thus, deletion of the middle two quartiles (residues 328-350) had no effect on the ratio of uncleaved to cleaved receptors relative to the TSHR without deletions. In contrast, deletion of either quartiles 1 and 2 (residues 317-338) or quartiles 3 and 4 (residues 339-362) significantly reduced the extent of TSHR cleavage into two subunits (p < 0.025 and p < 0.05, respectively).
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These data suggested that quartiles 1 and 4 were playing a greater role in cleavage at Site 1 than were the middle two quartiles. To test this hypothesis, we generated a new TSHR with deletions of discontinuous quartiles 1 and 4 (residues 317-327 and 351-362) (Fig. 4A). In confirmation of this hypothesis, the effect of these two discontinuous deletions was to almost completely eliminate TSHR cleavage at Site 1 (Fig. 4B). As observed previously with deletion of the entire 50-amino acid segment (8), prevention of cleavage at Site 1 by outer quartile deletion is not absolute, with a trace of cleaved receptor still being evident.
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DISCUSSION |
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Only the TSHR, and not the other closely related glycoprotein hormone receptors, undergoes proteolytic cleavage at the cell surface. However, mutagenesis studies have been unable to define a specific amino acid motif at either of the two cleavage sites in the TSHR ectodomain (8, 9). TSHR cleavage must, therefore, be caused by a proteolytic enzyme with relaxed specificity at its catalytic site yet which interacts preferentially with the TSHR and not with the other glycoprotein hormone receptors. This likelihood is supported by evidence for the involvement of a matrix metalloproteinase in TSHR cleavage at the cell surface (7, 14). Matrix metalloproteinases have broad amino acid sequence specificities (for example, Refs. 15-17).
The concept that the specificity of TSHR cleavage relates to the binding of a protease to this receptor rather than to a protease with a specific catalytic site is supported by the observation that transposition of an N-linked glycosylation site from the noncleaving LH/CG receptor to the corresponding region of the TSHR abrogates cleavage at Site 2 (9). Thus, a glycan moiety could provide steric hindrance to the binding of a protease. Similarly, it is possible that the 50-amino acid insertion could contain a protease binding site because deletion of this tract eliminates cleavage at Site 1 (8). It is intriguing, however, that each of these two modifications to the TSHR appears to eliminate cleavage at only one of the two sites. These data suggest that either two distinct proteases are responsible for TSHR cleavage, or alternatively, cleavage involves two separate binding sites for the same protease.
Another possible role for the 50-amino acid insertion in TSHR cleavage is that this segment has intrinsic protease activity and clips the receptor at an adjacent site. The present data do not support this hypothesis. Thus, every quartile of the 50-amino acid insertion can be deleted without abrogating TSHR cleavage. Deletion of a catalytic element is unlikely to leave TSHR cleavage at Site 1 unaffected or only partially affected.
We suggest a model (Fig. 5) that integrates the present data with the following previously known features of the TSHR ectodomain. (i) The insertion (residues 317-366) is a very hydrophilic structure that can be deleted without affecting the conformational integrity of the TSHR (6) or loss of of the ligand binding A subunit (6, 18), which is linked by disulfide bridges to the membrane-spanning B subunit; (ii) although the precise residues at cleavage Sites 1 and 2 are unknown, these sites appear to lie close to each end of the 50-amino acid insertion (8, 9, 18); and (iii) for the A and B subunits to remain linked after cleavage, the cysteine residues involved in linkage must lie outside the two cleavage sites. Indeed, the cysteines most likely to link the A and B subunits are closely upstream of cleavage Site 1 (Cys-283, Cys-284, Cys-301 on the A subunit) and downstream of cleavage Site 2 (Cys-390, Cys-398, Cys-408 on the B subunit) (reviewed in Ref. 1).
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Taken together, these data suggest that the 50-amino acid insertion is
an external loop on the surface of the TSHR, with its first and fourth
quartiles being drawn together by disulfide bonding. Consequent to
intramolecular cleaving, all or most of this insertion is removed like
a polyp clipped at its base. The present observations raise the
possibility that the first and fourth quartiles (the base of the polyp)
constitute a binding site for a protease that clips the receptor at
Site 1. Further cleavage at Site 2, whether by the same or a different
protease, releases the polyp from the receptor in the form of a C
peptide, which either remains intact or is degraded.
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ACKNOWLEDGEMENTS |
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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 United States Department of Agriculture, and the University of Maryland School of Medicine for kindly providing the highly purified bovine TSH for radioiodination.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK19289.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 should be addressed: Cedars-Sinai Medical
Center, 8700 Beverly Blvd., Suite B-131, Los Angeles, CA 90048. Tel.:
310-855-4774; Fax: 310-652-0578.
The abbreviations used are: TSH, thyrotropin; TSHR, TSH receptor; CHO, Chinese hamster ovary.
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REFERENCES |
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