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."
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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
-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
-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%
-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
-mercaptoethanol (30 min at
50 °C), the samples were subjected to SDS-PAGE and autoradiography
as described above.
 |
RESULTS |
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.
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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).
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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.
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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).
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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").
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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.
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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.
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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-
50-NET). Cleavage at
Site 1 was largely abolished (Fig. 7), confirming our hypothesis.
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DISCUSSION |
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.
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.