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Thyroid Molecular Biology Unit (G.D.C., Y.N., A.K., J.C.J., S.M.M., B.R.), Veterans’ Administration Medical Center, and the University of California, San Francisco, California 94121; and Department of Pharmacology 1 (K.T., Y.N.), Nagasaki University School of Medicine, Nagasaki 852, Japan
Address all correspondence and requests for reprints to: Basil Rapoport, M.B. or Gregorio Chazenbalk, Ph.D., Veterans’ Administration Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, California 94121.
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Abstract |
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In summary, our study provides a surprising and novel finding for G protein-coupled receptors. Contrary to the prevailing concept of one cleavage site in the TSHR, we present evidence that there are, in fact, two such sites. The TSHR, like insulin, may release a C peptide during intramolecular cleavage into two subunits.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functional importance of TSHR cleavage is presently an enigma. Light trypsinization of cells expressing the TSHR leads to the loss of an epitope at amino acid residues 354–359 concomitant with receptor activation (8). On the other hand, TSH can activate chimeric TSH-LH/CG receptors that do not cleave into two subunits (9). Identification of the cleavage site in the TSHR ectodomain would be important for elucidating the structure-function relationship of the TSHR subunits. Deductions from studies of TSH cross-linking to TSHR mutants and chimeric TSH-LH/CG receptors (10) suggested a cleavage site closely upstream to amino acid residue 317 in the 418 residue ectodomain (numbering includes a putative 21 residue signal peptide). Other data obtained using a rabbit antiserum to the TSHR favored a cleavage site further downstream, in the vicinity of residue 366 (11). Mutagenesis of the three striking arginine- and lysine-rich clusters closely upstream to amino acid residue 317 did not prevent TSHR cleavage into two subunits, suggesting the absence of a role for classical subtilisin-related proprotein convertases (12). More recently, matrix metalloproteinases have been implicated in TSHR cleavage (13).
In the present study, we have used two new mammalian cell lines expressing the recombinant TSHR to refine further the site of cleavage in the ectodomain. One line (TSHR-10,000) (14) overexpresses the TSHR, thereby overcoming the handicap of a low signal to noise ratio experienced on direct immunodetection (without prior affinity purification) using conventional TSHR-expressing mammalian cell lines. A second cell line, TSHRmyc (15), contains a c-myc epitope tag in the region of the cleavage site. Data obtained with these cell lines provide a surprising and novel finding for G protein-coupled receptors; namely, there appear to be two, not one, cleavage sites in the human TSHR ectodomain. A corollary of this finding is that the TSHR, like insulin, may release a C peptide during intramolecular cleavage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 x 106 receptors
per cell). Overexpression was attained using a dihydrofolate reductase
minigene to amplify the stably transfected TSHR complementary DNA
(cDNA) transgenome. 2) TSHR-0 are CHO cells expressing the same TSHR
cDNA but without transgenome amplification (1.5 x
105 receptors per cell) (14, 16). 3) TSHRmyc are 293 human
embryonal kidney (HEK) cells stably expressing the unamplified gene for
an epitope-tagged human TSHR (15). Epitope-tagging was achieved by
replacing TSHR amino acids 338 to 349 with the human c-myc
peptide EEQKLISEEDLL. Cells were propagated in Ham’s F-12 medium (CHO
cells) or DMEM (293 HEK cells), supplemented with 10% FCS, penicillin
(100 U/ml), gentamicin (50 µg/ml), and amphotericin B (2.5
µg/ml).
Immunoprecipitation of precursor-labeled TSHR
Cells near confluence in 100-mm diameter culture dishes were
rinsed with PBS and preincubated (0.5 h, twice) in DME-H21 methionine-
and cysteine-free medium containing 5% heat-inactivated FCS. The cells
were then pulsed (1 h at 37 C) in 5 ml fresh medium supplemented
with 0.5 mCi of 35S-methionine/cysteine (>1000
Ci/mmol, DuPont-New England Nuclear, Wilmington, DE). After aspiration
of the medium and rinsing the cells once with PBS, chase was performed
for the indicated times in standard, nonselective medium with 10% FCS.
Cells were washed twice with PBS and scraped into 1 ml ice-cold 20
mM HEPES, pH 7.2, 150 mM NaCl (buffer A)
containing the protease inhibitors phenylmethylsulfonyl fluoride (PMSF)
(100 µg/ml), leupeptin (1 µg/ml) aprotinin (1 µg/ml), and
pepstatin A (2 µg/ml) (all from Sigma Chemical Co., St. Louis, MO).
The cells were pelleted (5 min, 100 x g), washed twice
with PBS and resuspended in buffer A containing 1% Triton X-100. After
90 min at 4 C with occasional vortexing, the mixture was centrifuged
for 45 min at 100,000 x g and the supernatant was
diluted 1:4 in immunoprecipitation buffer (20 mM HEPES, pH
7.2, 300 mM NaCl, 0.1% SDS, 0.5% Nonidet-P40, 2
mM EDTA). The solubilized cell proteins were precleared for
1 h at 4 C with approximately 150 µg normal mouse serum IgG
prebound to 25 µl packed and washed protein A-agarose (Sigma). The
protein A was removed by centrifugation (3 min at 10,000 x
g) in a microcentrifuge. Mouse monoclonal antibodies (mAb)
were then added, as described in the text. A10 and A11 (kind gifts of
Dr. Paul Banga, London, UK) both recognize TSHR amino acid residues
22–35 (17) and were used at a final dilution of 1:1000. The
anti-myc mAb 9E10 (obtained from ATCC) was used at a final
dilution of 1:500. T3–365, a mAb to the TSHR B subunit (kindly
provided by Drs. E. Milgrom and H. Loosfelt, Le Kremlin-Bicetre,
France) and a mAb to human thyroid peroxidase (kindly provided by Dr.
Scott Hutchison, Nichols Institute, San Juan Capistrano, CA) were used
at a dilution of 1:1000. After 3 h at 4 C, 25 µl packed and
washed protein A-agarose was added and the tubes were tumbled for
1 h at 4 C. The protein A was recovered by centrifugation for 3
min at 10,000 x g (4 C), washed 5 times with 1 ml
immunoprecipitation buffer, and then once with 10 mM Tris,
pH 7.4, 2 mM EDTA and 0.5% SDS. Finally, the pellet was
resuspended in Laemmli sample buffer (18) with 0.7 M
ß-mercaptoethanol (30 min at 50 C) and electrophoresed on 7.5% or
10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). Prestained mol wt
markers (Bio-Rad) were included in parallel lanes. We precalibrated
these markers against more accurate unstained markers to obtain the mol
wts indicated in the text. Radiolabeled proteins were visualized by
autoradiography on Kodak XAR-5 x-ray film (Eastman Kodak, Rochester,
NY).
Immunoblots of TSHR proteins
Stably transfected TSHR-10,000 cells (in two 100-mm diameter
dishes) were resuspended by incubation in Ca2+- and
Mg2+-free PBS with 0.5 mM EDTA. The cells were
pelleted (5 min, 100 x g, 4 C), resuspended in 1.5 ml
of 10 mM Tris-HCl, pH 7.4, containing the protease
inhibitors described above and homogenized with a Polytron homogenizer
(Brinkman Instruments, Westbury, CT). After centrifugation for 10 min
at 500 x g (4 C), the supernatant was recentrifuged
for 20 min at 10,000 x g (4 C). The pellet was
resuspended in 0.1 ml of the same buffer, after which Laemmli buffer
with 0.7 M ß-mercaptoethanol was added (30 min at 50 C)
and the sample electrophoresed on 10% SDS-polyacrylamide gels.
Prestained mol wt markers are described above. Proteins were
transferred to ProBlott membranes (Applied Biosystems, Foster City,
CA), which were processed as described previously (19). Membranes were
incubated overnight (4 C) with mAb A10 or A11 to the A subunit, or with
mAb to the B subunit; T3–495 (TSH-R1; Transbio, Boulogne, France) or
T3–365 (final dilutions of 1:1000). After rinsing, the membranes were
incubated for 1 h at room temperature with alkaline phosphatase
conjugated goat antimouse immunoglobulin G (1:400 dilution) (Cappel,
Durham, NC). The signal was developed with nitroblue tetrazolium and
5-bromo, 4-chloro, 3-indolyl phosphate in 100 mM Tris-HCl
buffer, pH 9.5, containing 100 mM NaCl and 5 mM
MgCl2.
Enzymatic deglycosylation of TSHR protein
The Protein A/IgG/TSHR complex or the 10,000 x
g crude membrane fraction (see above) was incubated (10 min,
100 C) in denaturing buffer containing 0.5% SDS, 1%
ß-mercaptoethanol. Enzymatic deglycosylation was performed according
to the protocol of the manufacturer (N.E. Biolabs, Beverly, MA).
N-glycosidase F digestion (100 U for 2 h at 37 C) was
in 50 mM Na phosphate, pH 7.5, 1% NP-40. Endoglycosidase H
digestion (50 U for 2 h at 37 C) was in 50 mM Na
citrate, pH 5.5. Samples were then subjected to SDS-PAGE, as described
above.
Covalent cross-linking of radiolabeled TSH
Highly purified bovine TSH (5 µg, 30 U/mg protein) was
radiolabeled with 125I to a specific activity of
approximately 80 µCi/µg protein using the Bolton-Hunter reagent
(4400 Ci/mmol; DuPont-New England Nuclear) according to the protocol of
the manufacturer, followed by Sephadex G-100 chromatography (20).
Confluent 100-mm diameter dishes of TSHR-expressing cells were
incubated for 2 h at 37 C with 5 µCi 125I-TSH in 5
ml modified Hanks’ buffer (without NaCl), supplemented with 280
mM sucrose and 0.25% BSA (binding buffer). Unbound
125I-TSH was removed by rinsing the cells three times with
ice-cold binding buffer. Disuccinimidyl suberate (DSS; 1
mM; Sigma) in 10 mM Na phosphate buffer, pH
7.4, containing the protease inhibitors described above was then added
for 20 min at room temperature. The cross-linking reaction was
terminated by the addition of 20 mM ammonium acetate (final
concentration).
After cross-linking, the cells were rinsed twice with PBS and scraped into 10 mM Tris, pH 7.5, containing the same protease inhibitors. Cells were homogenized using a Polytron homogenizer and centrifuged for 5 min at 4 C (500 x g). The supernatant was centrifuged (15 min, 10,000 x g, 4 C) and the pellet was resuspended in 50 µl 10 mM Tris, pH 7.5. After the addition of Laemmli buffer containing 0.7 M ß-mercaptoethanol (30 min at 42 C), the samples were subjected to 10% SDS-PAGE and autoradiography as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Two forms of single subunit (uncleaved)
TSHR were: 1) approximately 115 kDa in size (complex carbohydrate
resistant to endoglycosidase H) and 2) approximately 100 kDa in size
(immature, high mannose carbohydrate sensitive to endoglycosidase H)
(Fig. 1). N-glycosidase F digestion removed both forms of
carbohydrate, exposing an approximately 84-kDa polypeptide backbone.
Cleaved (two subunit) TSHR was also present. The extracellular A
subunit was visible as a broad approximately 62 kDa band with complex
carbohydrate, which upon deglycosylation became a more focused 35-kDa
band. The largely transmembrane B subunit could not be visualized in
these immunoprecipitation experiments (Fig. 1A), presumably because of
its detachment from the antibody-bound A subunit and consequent loss
during the very stringent washing procedure. The specificity of mAb for
the TSHR was evident in control experiments using untransfected CHO
cells and with a mAb to thyroid peroxidase (TPO) (Fig. 1B).
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, A and B). Thus, in addition to single subunit forms
of the TSHR, a primary B subunit band of approximately 42 kDa was
evident, as well as some minor bands of slightly greater size.
Incubation with endoglycosidases F and H did not affect the mobility of
the B subunit bands, consistent with their lack of N-linked
glycosylation sites (Fig. 2A). Specificity of mAb T3–495 and T3–365
was confirmed on immunoblotting with untransfected CHO cells (Fig. 2B).
As in the immunoprecipitation experiments, the size of the A subunit
could clearly be determined by immunoblotting with mAb A10 to the TSHR
amino terminus. The approximately 62-kDa mature A subunit contained
complex carbohydrate (endoglycosidase H resistant) (Fig. 2C). Most
important, deglycosylation with N-glycosidase F confirmed an
approximately 35 kDa A subunit backbone. Lesser fragments of
approximately 39 and 42 kDa were also evident. Again, the specificity
of mAb A10 to the A subunit was confirmed on immunoblots with
untransfected CHO cells and on immunoblotting of TSHR-10,000 cells with
a mAb to TPO (Fig. 2D).
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42 kDa) was only
approximately 77 kDa. A 35-kDa polypeptide backbone for the TSHR A
subunit would place the cleavage site in the region of amino acid
residue 330, taking into account the absence of the signal peptide.
Further, an approximately 42-kDa size for the nonglycosylated B subunit
would be consistent with a holoreceptor cleavage site at about residue
380. It, therefore, appeared that a "C peptide" fragment in the
vicinity of residues 330–380 could be missing from the cleaved TSHR
ectodomain (Fig. 3). This deduction is inconsistent with
the prevailing concept of a single cleavage site in the TSHR
ectodomain.
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). Detection by a mAb to c-myc of only the
single subunit forms of the TSHR, and not the cleaved A subunit, would
support the concept that a portion of the ectodomain is lost during
intramolecular cleavage. Cleavage within the c-myc epitope
could also lead to loss of this epitope. The two cleavage site
hypothesis would also predict detection in TSHRmyc cells of the A
subunit with mAb A10 to the amino terminus of this subunit, or, even
more definitively, by cross-linking with radiolabeled TSH.
The TSHRmyc cells do not contain an amplified transgenome and express
fewer receptors (100,000 per cell) (15) than TSHR-10,000 cells.
Nevertheless, both anti-myc mAb 9E10 and mAb A10 were
equally effective in detecting the single chain forms of the TSHR in
these cells (Fig. 4A). It contrast, it was more
difficult to detect the diffuse, glycosylated TSHR A subunit band in
the TSHRmyc cells than in the TSHR-10,000 cells. However, after
deglycosylation with N-glycosidase F, the 35-kDa A subunit
in the TSHRmyc cells became more focused and was clearly visible on
immunoprecipitation with mAb A10 to the TSHR amino terminus. In
contrast, the A subunit was not detected in the same material with the
anti-myc mAb 9E10. Because of the importance of this finding, a similar
experiment is also shown in which the deglycosylated A subunit in
TSHR-10,000 cells is included (Fig. 4B). The 35 kDa deglycosylated A
subunit band detected by mAb A10 in the TSHRmyc and TSHR-10,000 cells
was not an artifact because no such band was detected by
immunoprecipitation with mAb 10 of precursor-labeled, untransfected HEK
cells, nor did a nonrelevant mAb (to TPO) detect this band in TSHRmyc
cells (Fig. 4C). Finally, we applied the most sensitive method (in our
hands) to confirm the presence of the TSHR A subunit in TSHRmyc cells,
namely the covalent cross-linking of radiolabeled TSH to the surface of
intact cells in monolayer culture. By this means, the A subunit was
clearly evident in TSHRmyc cells (Fig. 5). Further, the
proportion between the A subunit and the uncleaved, single subunit
receptor detected by TSH cross-linking was the same in the TSHRmyc
cells as in a cell line expressing similar numbers of wild-type TSHR
(TSHR-0) (16).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Only the simultaneous substitution of domains D and
E abrogated cleavage. These findings are consistent with the D and E
domains each containing a cleavage site.
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A third line of evidence suggesting two cleavage sites in the TSHR
ectodomain is the present observation of the selective loss of a
strategically situated c-myc epitope in the two subunit, but
not in the single chain, form of the TSHR. There are three possible
explanations for this phenomenon: 1) loss of a peptide fragment
containing the c-myc epitope during intramolecular cleavage
at two different sites; 2) cleavage at a single cleavage site within
the c-myc epitope leading to loss of antibody recognition;
3) a combination of these two events (two cleavage sites, one of these
within the c-myc epitope). Of these possibilities, cleavage
at a single site within the c-myc epitope is
unlikely, for a number of reasons. Thus: 1) single cleavage within
amino acid residues 338–349 (the c-myc epitope) would
generate a B subunit of 46–47 kDa, clearly larger than the actual size
(42 kDa) observed experimentally (see Fig. 2, A and B); 2) Chimeric
receptor TSH-LHR-4 (24) and a deletion mutant (residues 317–366) of
the wild-type TSHR (25) lack the region in which the c-myc
epitope was inserted (Fig. 6), yet both receptors still cleave into two
subunits (7, 10); 3) The c-myc epitope sequence differs
substantially from that of the wild-type receptor. Persistent cleavage
in such a highly mutated region would, therefore, indicate lack of
amino acid sequence specificity for a TSHR cleavage site. For all these
reasons, there are likely to be two cleavage sites in the TSHR
ectodomain, the more upstream of which may, or may not, be with the
region of the c-myc epitope.
The unsuspected possibility of two cleavage sites in the TSHR explains
why we were previously misled into deducing that TSHR cleavage occurred
closely upstream, rather than closely downstream, of amino acid residue
317 (7, 10, 12). We made this erroneous deduction because TSHR cleavage
into A and B subunits was still evident on TSH cross-linking to a
receptor with residues 317–366 deleted by mutagenesis (7, 25), a
finding that would exclude a single, but not two, cleavages sites in
this region (Fig. 6). Moreover, the A subunit in this TSHR mutant was
similar in size to that of the wild-type TSHR. In retrospect, cleavage
a few residues upstream or downstream of pivotal residue 317 would be
difficult to discern from a relatively large (74 kDa) cross-linked
product.
It is of interest that the c-myc epitope lies within a 50-amino acid segment (residues 317–366) that we observed to be unique to the TSHR when compared with other glycoprotein hormone receptors (3). Although the precise boundaries of this 50-amino acid insertion are uncertain (because of low homology among the receptors in adjacent regions), this TSHR segment has been the subject of intense study. The very hydrophilic nature of residues 317–366 led us to speculate that it was a projection on the exterior of the TSHR molecule, perhaps important in ligand specificity (25). Surprisingly, however, its deletion had no effect on TSH binding or on TSH-mediated signal transduction (25). The deduced superficial topography of TSHR residues 317–366 was also the reason for selection of this region for c-myc epitope tagging. Other investigators have used synthetic peptides corresponding to portions of this region for generating antisera to the TSHR. One peptide in particular (residues 352–367) is highly immunogenic and is reported to be recognized by TSHR autoantibodies in the majority of Graves’ sera (26, 27). Further, an antiserum to a closely related synthetic peptide (residues 352–366) recognizes the A subunit in FRTL-5 rat thyroid cells, but very poorly in transfected COS cells (11). It will be interesting to correlate these findings with the possible absence of a part of this region in the cleaved TSHR.
In summary, multiple lines of evidence, taken together, suggest that there are two cleavage sites in the ectodomain of the TSHR. To our knowledge, the TSHR would be the first member of the family of G protein coupled receptors whose ectodomain appears to contain multiple cleavage sites. Purification and characterization of the putative TSHR C peptide would provide proof for the two cleavage site hypothesis. However, it is not presently feasible to detect the release of a small TSHR polypeptide fragment into the culture medium from the TSHRmyc cell line that is not overexpressing the TSHR. Nevertheless, the evidence for two cleavage sites in the TSHR ectodomain provides an impetus to future studies on the structure-function of the TSHR subunits.
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Footnotes |
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2 Visiting scientist at the University of California, San
Francisco.
3 Supported, in part, by the Molecular Medicine program, University
of California, San Francisco.
Received December 18, 1996.
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Y. Osuga, S.-G. Liang, J. S. Dallas, C. Wang, and A. J. W. Hsueh Soluble Ecto-Domain Mutant of Thyrotropin (TSH) Receptor Incapable of Binding TSH Neutralizes the Action of Thyroid-Stimulating Antibodies from Graves' Patients Endocrinology, February 1, 1998; 139(2): 671 - 676. [Abstract] [Full Text] [PDF]
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-helix and ß-sheet and is based
on the three-dimensional structure of ribonuclease A inhibitor (
