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J Biol Chem, Vol. 274, Issue 48, 33979-33984, November 26, 1999
From the Autoimmune Disease Unit, Cedars-Sinai Research Institute
and School of Medicine, UCLA, Los Angeles, California 90048
We studied cell surface thyrotropin receptor
(TSHR) by biotinylating proteins on the surface of metabolically
labeled, intact cells. In addition to TSHR cleaved into A and B
subunits, mature single-chain receptors with complex carbohydrate were
also present on the cell surface. A low A/B subunit ratio indicated
partial shedding of extracellular A subunits from transmembrane B
subunits. TSHR cleavage at upstream site 1 (within amino acid residues
305-316) would generate a B subunit of 51-52 kDa. However, only
smaller B subunits (40-46 kDa) were detected, corresponding to N
termini from residues ~370 (site 2) extending downstream to the
region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR
epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger
(45-52 kDa) than the majority of B subunits recovered with a
C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits.
Single-chain, mature TSHR do exist on the cell surface. The C peptide
lost during intramolecular cleavage disintegrates rapidly following
cleavage at upstream site 1 of the single-chain TSHR into A and B
subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane,
revealing a novel mechanism for A subunit shedding.
Graves' disease, one of the most common autoimmune diseases
affecting humans, is caused by autoantibodies that activate the thyrotropin (TSH)1 receptor
(TSHR) (reviewed in Ref. 1). Remarkably, functional autoantibodies do
not arise to the other, closely related members of the glycoprotein
hormone receptor family. A potential reason for this difference is the
unique subunit structure of the TSH receptor. Thus, unlike the other
glycoprotein hormone receptors, a variable proportion of TSHR on the
cell surface cleaves into an extracellular A subunit and a largely
transmembrane B subunit that remain linked by disulfide bonds (2-4).
This cleavage introduces the potential for antigenic stimulation of the
immune system, for two reasons. First, TSHR cleavage into A and
B subunits results in the deletion of an intervening "C peptide"
region (5, 6). Extracellular release of this polypeptide, either intact
or in fragments, could initiate or propagate an immune response to the TSHR. Second, there is the propensity for the A subunit itself to be
shed, at least in vitro (7, 8). Determination of the molecular basis for TSH receptor cleavage and shedding is, therefore, of potential clinical importance in understanding the pathogenesis of
Graves' disease.
Many uncertainties remain regarding the process of TSHR cleavage and
shedding, including the enzyme(s) involved (6, 7, 9) and the properties
of the shed A subunits (7, 8). The location and number of cleavage
sites has also been controversial. Initial evidence for a cleavage site
closely upstream of amino acid residue 317, obtained by TSH
cross-linking to a TSHR deletion mutant (3), was contradicted by
reports that amino acid residues 352-366 lie within the A subunit (10,
11). However, residues 352-366 cannot be part of the A subunit because
they are part of a C peptide region excised from the TSHR during
intramolecular cleavage (5), an observation recently confirmed (6).
Further support for an upstream cleavage site in the TSHR in the region of residues 305-316 was obtained in studies examining the effect of
trypsin on subunit structure (12), as well as by the identification in
transfected L cells of a minor B subunit component with an N terminus
at residue 314 (6).
Less clear, however, is the process by which the C peptide region
between the A and B subunits is lost. The observed deletion of this
segment (5), taken together with previous TSH cross-linking studies to
TSH-luteinizing hormone/chorionic gonadotropin chimeric receptors (13),
provided evidence for two separate TSHR cleavage sites, the second
(downstream) site being in the vicinity of amino acid residue 370 (14).
A recent refinement of this concept is that, rather than there being
two distinct cleavage sites, cleavage at upstream site 1 (approximately
residue 314) is primary and is followed by the sequential excision of
the C peptide region, terminating in the region of site 2 (6). However,
in this report, there is a discrepancy between the small size of the B
subunit polypeptide chains observed and their predicted size based on their N-terminal residues.
Overall, a major problem in evaluating this somewhat confusing
compendium of data is that previous studies on the immunodetection or
immunopurification of TSHR subunits have been performed on thyroid
tissue or cell homogenates that include TSHR products in the
degradation and synthetic pathways as well as TSHR on the cell surface
(4-6, 10, 15-20). Although in one study, surface-radiolabeled TSHR
were examined, subunit forms of the receptor, in particular the B
subunit, could not be clearly identified (16). In the present study, we
have succeeded in using a cell surface biotinylation approach to
examine the properties of both TSHR subunits expressed exclusively on
the cell surface.
Cell Lines and Culture
TSHR-10,000 is a Chinese hamster ovary (CHO) cell line
overexpressing the human TSHR (~2 × 106
receptors/cell) (20). Overexpression was attained by transgenome amplification using a dihydrofolate reductase minigene approach. The
TSHRmyc-10,000 CHO cell line was generated by the same method. To
accomplish this, we transferred the cDNA for an epitope-tagged TSHR
(TSHR amino acids 338-349 replaced with the human c-myc peptide EEQKLISEEDLL) (21) into the plasmid pSV2-DHFR-ECE (22). Cells 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).
Biotinylation and Extraction of Metabolically Labeled TSHR on
the Cell Surface
TSHR-10,000 or TSHRmyc-10,000 cells in 60-mm diameter culture
dishes were pulsed (1 h at 37 °C) with 0.2 mCi/ml
[35S]methionine/cysteine in Dulbecco's modified
Eagle's, high glucose (4500 mg/l), methionine- and cysteine-free
medium containing 5% heat-inactivated FCS, as described previously
(20). The cell monolayers were washed twice with ice-cold PBS, pH 8.0, and the biotin cross-linker sulfosuccinimidyl-2-(biotinamide)
ethyl-1,3'-dithiopropionate (Pierce; 0.5 mg/ml in PBS, pH 8.0) was
added for 20 min on ice. The solution was removed and the cross-linking
procedure was repeated once. After aspiration, remaining reactive
sulfosuccinimidyl-2-(biotinamide) ethyl-1,3'-dithiopropionate was
blocked by addition of 50 mM NH4Cl in PBS, pH
8.0, for 10 min on ice with occasional agitation.
Extraction of TSHR was as described previously (20). In brief, cells
were washed twice with PBS and scraped into 1 ml of ice-cold buffer A
(20 mM Hepes, pH 7.2, 150 mM NaCl, and 100 µg/ml phenylmethylsulfonyl fluoride and 1 µg/ml leupeptin (both
from Sigma)). The cells were pelleted, rinsed, and resuspended in
buffer A containing 1% Triton X-100. After 2 h at 4 °C with
occasional vortexing, the mixture was centrifuged for 1 h at
100,000 × g and the supernatant was retained for
immunoprecipitations. Where indicated, in an alternative procedure
(23), 1.2 ml of buffer containing 10 mM Tris, pH 7.4, 50 mM NaCl, 1% Triton X-100, and 0.5% bovine serum albumin
was added directly to the cell monolayer without scraping. After
rocking for 2 h at 4 °C, the supernatant was recovered and
centrifuged as described above.
Immunoprecipitation of TSHR and Separation in Biotinylated and
Non-biotinylated Fractions
The immunoprecipitation procedure used has been described
previously in detail (20). Briefly, solubilized cell proteins were
diluted 1:1 in buffer B (20 mM HEPES, pH 7.2, 300 mM NaCl, 0.1% SDS, 0.5% Nonidet P-40, and the protease
inhibitors described above) and were precleared with normal mouse serum
IgG prebound to 25 µl of packed and washed protein A-agarose (Sigma).
Mouse monoclonal antibodies were added (16 h at 4 °C), and immune
complexes were recovered using protein A-agarose. The following
antibodies were used: (i) to TSHR A subunit amino acid residues
147-229 (A9; Dr. Paul Banga, King's College, London, United Kingdom;
1:1,500 final) (24), (ii) to the TSHR B subunit (T3-365; Dr. Edwin
Milgrom, Hôpital de Bicetre, Le Kremlin Bicetre, France; 2 µg/ml) (4), or (iii) to the c-myc epitope introduced into the TSHR at
residues 338-349 (21) (9E10; American Type Tissue Culture; 1:500).
After extensive washing in buffer B, immunoprecipitated TSHR were
removed from the protein A by adding 3 µl of 10% SDS and 150 µl of
buffer B (30 min at 50 °C). After centrifugation for 3 min at
10,000 × g at 4 °C, the supernatant was diluted
with 150 µl of buffer B, and incubated for 2 h with 25 µl of
pre-washed streptavidin-agarose (Pierce) and centrifuged. The
supernatant containing non-biotinylated TSHR was retained. The beads
were washed four times with buffer B and once with PBS and resuspended in Laemmli sample buffer with 0.7 M Enzymatic Deglycosylation of TSHR Protein
Biotinylated TSHR bound to streptavidin-agarose or TSHR not
bound to the streptavidin-agarose beads were incubated (10 min, 100 °C) in denaturing buffer containing 0.5% SDS, 1%
Attempts to Purify the TSHR C Peptide Region Released into
the Culture Medium
Metabolic Labeling and Immunoprecipitation--
TSHRmyc-10,000
cells near confluence in 60-mm diameter culture dishes were
metabolically labeled as described above, with the following
modifications. Cells were pulsed for 3 h at 37 °C in 2 ml of
Earle's balanced salt solution with 5% dialyzed FCS and 5 µCi/ml
L-[U-14C]amino acid mixture (Amersham
Pharmacia Biotech, Buckinghamshire, United Kingdom). After rinsing,
cells were cultured in standard F12 medium with 10% FCS. The medium
was harvested after 16 h, and 9E10 mouse monoclonal antibody to
the c-myc epitope was added, as described above.
Affinity Purification--
23 mg of purified 9E10 IgG was added
to 2 g of pre-washed CNBr-Sepharose 4B (Amersham Pharmacia
Biotech) and tumbled for 2 h at room temperature. The beads were
then blocked with 0.1 M Tris-HCl, rinsed successively with
0.1 M acetate buffer, pH 4.0, 0.5 M NaCl and
with 0.1 M Tris-HCl, pH 8.0, 0.5 M NaCl. F-12
medium with 10% FCS (1200 ml) harvested from 10-cm diameter dishes of TSHRmyc-10,000 cells was applied (1.5 ml/min) to the 9E10-agarose affinity column. After rinsing the column with 0.1 M
ammonium acetate, pH 7.5, protein was eluted with 1 M
acetic acid, pH 2.2. The fractions were evaporated to dryness under
vacuum, resuspended in Laemmli sample buffer with 0.7 M
Cell Surface TSH Receptors Identified with an Antibody to the A
Subunit--
In order to examine the feasibility of distinguishing
between cell surface and intracellular TSHR, we first metabolically labeled TSHR-expressing CHO cells with [35S]methionine
and cysteine and then biotinylated proteins on the surface of these
intact cells in monolayer culture. TSHR in detergent extracts of these
cells were immunoprecipitated with an antibody to the extracellular A
subunit and were then separated on the basis of their binding, or lack
of binding, to streptavidin (Fig. 1A). Polyacrylamide gel
electrophoresis under reducing condition revealed single-chain,
uncleaved TSHR of 120 kDa with complex carbohydrate (endoglycosidase
H-resistant) only in the biotinylated pool of receptors (Fig.
1B). In a reciprocal manner, single-chain TSHR of ~100 kDa
with immature, high mannose glycan (endoglycosidase H-sensitive) were
detected only in the non-biotinylated receptor pool (Fig.
1C). These data indicate the efficiency and selectivity of
biotinylation of cell surface versus intracellular TSHR.
Further, they establish that single-chain, uncleaved TSHR with mature, complex glycan do occur on the cell surface.
In addition to the single-chain receptors, dissociated cell surface A
and B subunits were also evident under the same reducing conditions.
The A subunits, derived from the single-chain receptors (17), contain
complex carbohydrate whereas the largely transmembrane or intracellular
B subunits are non-glycosylated (panel A). Only trace amounts of non-biotinylated, cleaved receptor subunits are present within the cell (panel B). The size of
the deglycosylated polypeptide core of the cell surface A subunit
(approximately 34 kDa) is consistent with an upstream intramolecular
cleavage site ("site 1") in the vicinity of amino acid residues
305-316 (3, 5, 6, 12). In contrast to the more sharply defined A
subunit polypeptide core revealed by endoglycenase F treatment, non-glycosylated B subunits on the cell surface isolated with an
antibody to the A subunit were evident as a diffuse band of approximately 40-46 kDa (Fig. 1). In most experiments (as in Fig. 1),
this diffuse band was a poorly defined doublet.
Cell Surface TSH Receptors Identified with an Antibody to the B
Subunit--
Using an antibody to the A subunit, it is not possible to
detect B subunit cleavage or degradation downstream of amino acid residue 408. This is because there are only three cysteine residues (Cys-390, -398, and -408) on the B subunit prior to its predicted insertion into the plasma membrane at residue 418 (Fig.
2). Which of these cysteines are involved
in B subunit tethering to the A subunit is not definitively established
(reviewed in Ref. 1), but loss of the ultimate B subunit Cys-408 would
certainly be incompatible with a retained A subunit (Fig. 2). On the
other hand, immunoprecipitation with an antibody to the B subunit would determine the size(s) of B subunits regardless of their attachment to
the A subunit.
We, therefore, compared the sizes of B subunits of TSHR on the surface
of intact cells as detected by immunoprecipitation with antibodies to
the TSHR A and to the B subunits (Fig.
3A). The size range of B
subunits was similar regardless of whether they were isolated with an
antibody to the A or B subunit. The lower limit of the B subunit smear
(approximately 40 kDa) indicates that B subunit cleavage does continue
downstream to the vicinity of the ultimate Cys-408 or even to the
plasma membrane (Fig. 3B). Loss of Cys-408 would lead to A
subunit shedding. An additional observation was that the intensity of
the B subunit band relative to the A subunit band was greater when B
subunits were isolated with the B subunit antibody as opposed to the A
subunit antibody. Although the intensity of precursor-labeled A and B
subunit bands must be interpreted in the light of a nearly 2:1 excess
of methionine and cysteine residues in the B versus the A
subunit, these data are consistent with the existence on the cell
surface of some B subunits without attached A subunits, i.e.
A subunit shedding.
Detection of "Big" B Subunits--
Before the C peptide region
is lost, TSHR cleavage at upstream site 1 would create a big B subunit
still containing the C peptide region with a predicted size of 51-52
kDa (Fig. 3B). No B subunits of this size were detected in
the preceding experiments using cells expressing the wild type TSHR. In
order to facilitate study of the TSHR C peptide region, we generated a
cell line overexpressing the TSHR with a c-myc epitope at amino acid
residues 338-349, within the C peptide region. Clear differences were
observed between cell surface TSHR subunit forms immunoprecipitated
from the same precursor-labeled material using monoclonal antibodies
(mAb) to the c-myc epitope (9E10) and to the B subunit (T3-365;
epitope in the B subunit C-terminal region, amino acid residues
640-764) (Fig. 3C). First, much less (4-fold) radioactivity
was recovered with 9E10 than with the mAb to the B subunit. Second,
consistent with previous observations on an unamplified cell line
expressing the same receptor, loss of the c-myc epitope-containing C
peptide region resulted in proportionately greater recovery of
single-chain versus two-subunit forms of the TSHR (5).
Finally, by normalizing the radioactivity applied to each lane to
compensate for the lesser recovery of radioactivity with the anti-myc
mAb, we were able for the first time to visualize B subunits recognized
with this antibody. This small proportion of B subunits expressed on
the surface of intact TSHRmyc-10,000 cells were, in most part, larger than the B subunits isolated with the mAb to a more downstream epitope,
extending (as predicted) up to ~51 kDa in size (Fig. 3C).
Inability to Purify an Intact C Peptide from Culture
Medium--
We also used the TSHRmyc-10,000 cell line in an attempt to
purify a C peptide from culture medium. Conditioned medium harvested from this cell line (3 days, 1,200 ml) was applied to an affinity column containing mAb 9E10 to the c-myc epitope. Eluted material was
subjected to polyacrylamide gel electrophoresis and examined both by
Coomassie Blue staining and by immunoblotting using monoclonal antibody
9E10. No polypeptide fragments were observed (data not shown). In
separate experiments, no C peptide could be immunoprecipitated from
medium harvested from TSHRmyc-10,000 cells metabolically labeled with
14C-labeled amino acids (there are no cysteine or
methionine residues within the C peptide region) (data not shown).
Taken together, these data support the concept that the C peptide
region is not excised as a single fragment.
Influence of TSH Receptor Extraction Procedure on B Subunit
Structure--
A wide spectrum of B subunit sizes has been identified
in TSHR extracted from thyroid tissue or transfected mammalian cells (4-6, 10, 11, 17, 25). In all of these studies, TSHR extraction has
involved tissue or cell homogenization, commonly after freezing and
thawing. However, the TSHR is a labile protein and the method used for
TSHR preparation markedly affects its ligand binding properties. Thus,
only a small fraction of the TSH binding capacity on intact cells is
recovered after cells are scraped off the culture dish, a reduction not
prevented by conventional protease inhibitors. Recently, direct
detergent extraction of cell monolayers (without scraping, freezing, or
homogenization) was found to greatly enhance recovery of TSHR capable
of ligand binding (23). We, therefore, examined the effect of cell
scraping on the size of TSHR B subunits expressed on the surface of
stably transfected CHO cells.
After metabolic labeling and biotinylation of TSHR-10,000 cell surface
proteins, the cells were processed in two different manners prior to
TSHR immunopurification with an antibody to the B subunit and selection
of cell surface receptors with streptavidin: (i) our conventional
method of first harvesting the cells by scraping before detergent
extraction, and (ii) direct application of detergent to CHO cell
monolayers without other manipulation of the cells. With direct
solubilization, in some (four of six) experiments, the 40-46-kDa B
subunit smear was biased toward the higher molecular mass form (Fig.
4). Conversely, after cell scraping, the
B subunits were either uniformly distributed between the higher and
lower molecular weights, or were biased toward the lower size. However, in other experiments, direct detergent solubilization did not bias
toward recovery of larger B subunits (see, for example, the TSHRmyc-10,000 cells in Fig. 3C).
The present data are the first to examine the properties of TSHR,
including their A and B subunits, expressed on the surface of intact
cells. Analysis of TSHR carbohydrate moieties establishes the efficacy
of the cell surface biotinylation approach that we used to distinguish
between cell surface and intracellular receptors. Previous studies on
TSH cross-linking to TSHR on the surface of intact cells (for example,
Refs. 2 and 3) could distinguish between single-chain receptors and the
A subunits of cleaved receptors, but the ligand-receptor complexes were
not amenable to fine resolution of their sizes and the non-ligand
binding B subunit could not be detected. Immunopurification of TSHR
does provide more detailed information (for example, Ref. 4). However,
such studies have been performed on cell and thyroid homogenates, which
include mixtures of cell surface receptors and intracellular receptors at different stages of the synthetic and degradative pathways. Intracellular products are reported to be particularly abundant in
transfected cells expressing the recombinant TSHR (17).
The data on cell surface TSHR expression clarify a number of puzzling
or controversial issues. One issue that has been the subject of debate
for many years is whether or not mature, single-chain TSHR exist on the
cell surface. This possibility was raised by the detection of
single-chain TSHR on TSH cross-linking to intact cells (2, 3).
Disulfide-linked, two-subunit receptors were well recognized (4, 26).
However, high affinity TSH binding to the single-chain TSHR comparable
to the cleaved receptor led to the suggestion that the former were
physiological (3). This concept has been vigorously disputed, and these
observations have been considered an artifact of transfected cells (4,
6, 17, 27). It is proposed that single-chain TSHR precursors with
immature carbohydrate have been "mistaken for the mature receptor"
(6) and that such precursors are readily detected in transfected cells
because of their abnormal abundance consequent to TSHR overexpression
overwhelming the mechanism for glycan maturation and TSHR processing
(17, 27). The present data clearly indicate that single-chain TSHR on
the cell surface have mature, complex carbohydrate and are consistent
with the previous observation of high affinity TSH binding to
single-chain TSHR on the cell surface (3). Moreover, single-chain
receptors capable of TSH binding are not confined to transfected cells,
but are also observed in a well differentiated rat thyroid cell line
(2). Finally, the same proportion of single-chain versus
cleaved TSHR on the cell surface is observed over a 100-fold range in
the level of expression in transfected cells (20), excluding the
suggested limitation in synthetic capacity.
Turning to the process of TSHR cleavage, the impressive accomplishment
of de Bernard et al. (6) in affinity-purifying large quantities of TSHR B subunits from thyroid tissue and from transfected mouse L cells confirms previous findings that an epitope within a C
peptide segment is excised from the TSHR (5) with an upstream cleavage
site in the vicinity of amino acid residues 305-316 (3, 12). Our
inability to purify an intact C peptide, similar to the experience of
this group (6), supports the concept that the C peptide region is
removed through a process of progressive cleavage or degradation.
Regarding the region downstream of the excised C peptide segment, B
subunits purified from homogenates of thyroid tissue and transfected
mouse L cells reveal multiple N termini, the dominant ones being
between amino acid residues 366 and 378 (6), consistent with previous
mutagenesis data suggesting the approximate location of cleavage
"site 2" (14). Taken together, these data suggest that, in contrast
to our previous interpretation of available evidence, C peptide
excision may not occur by means of two distinct cleavage sites. Rather,
as reported by de Bernard et al. (6), cleavage is likely to
occur initially upstream at site 1 and is followed by progressive
cleavage or N-terminal degradation of the B subunit downstream to the
region of site 2. Regardless of whether there are one or two primary cleavage sites, the end result is the same, namely removal of a C
peptide region between sites 1 and 2.
Our present data provide new insight into other aspects of TSHR
cleavage that have been difficult to understand, particularly regarding
the size of the B subunit, an important issue in understanding the
cleavage process (see Fig. 3B). Thus, primary cleavage of the TSHR at site 1 (approximately at residue 310) would generate an
initial B subunit with a calculated mass of 51-52 kDa. After removal
of the C peptide region culminating approximately at amino acid residue
366-378, the calculated B subunit size is 45 kDa. It is unclear why no
45-kDa B subunits were observed by de Bernard et al. (6).
Indeed, the majority of the B subunit fragments on which they were able
to determine N-terminal sequence (see above) were 38-39 kDa in size.
In contrast, our data on cell surface B subunit size do conform with
the expected sizes of B subunits with N termini at site 1 and site 2. Possible explanations for the discrepancy between N-terminal sequences
and B subunit sizes in the de Bernard study include inaccurate
molecular size markers and B subunit degradation. Because B subunits
were purified from cell or tissue homogenates, it cannot be certain
that N-terminal sequence analysis was on cell surface B subunits.
Indeed, in transfected mouse L cells, there is a large excess of
immature TSHR molecules that undergo rapid degradation (17).
In our study on cell surface receptors, a B subunit "smear" between
site 1 (52 kDa) and site 2 (45 kDa) was evident only in a small
proportion of B subunits purified with the anti-myc antibody whose
epitope is in the C peptide region. The majority of B subunits were
evident as a broad biphasic band of 40-46 kDa. These observations indicate that after cleavage at upstream site 1, N-terminal removal of
the C peptide region is not a gradual and progressive process. Rather,
the C peptide region appears to disintegrate rapidly. This
disintegration pauses downstream at site 2. The lower part of the
biphasic B subunit band (~40 kDa) suggests that, after slowing at
site 2, N-terminal clipping or degradation of the B subunit
re-accelerates to the vicinity of the plasma membrane. Continued
degradation would lead to A subunit destabilization (with decreased
ligand binding) (23) and, ultimately, A subunit shedding (Fig. 2), as
is evident from the greater proportion of B subunits immunoprecipitated
using the B subunit antibody versus the A subunit antibody
(Fig. 3A). Shedding of the A subunit may, therefore, not
require protein disulfide isomerase (9). Whether continued B subunit
N-terminal degradation beyond site 2 to its membrane insertion is
physiological or an artifact is uncertain. The TSHR is a labile protein
(8, 23). Further, in some (but not all experiments) in which TSHR were
extracted directly with detergent without first scraping the cells,
there was a lesser amount of the smaller B subunit component.
Conversely, culture of cells in serum-poor medium, a condition
previously used to study the mechanism of A subunit shedding (7, 9),
shifts the size of TSHR B subunits toward their lower molecular weight forms (8).
In conclusion, the present study characterizes TSHR (including their A
and B subunits) on the cell surface. Single-chain TSHR with mature
carbohydrate do exist on the cell surface. The C peptide region lost
from the TSHR during intramolecular cleavage is not released intact,
but disintegrates rapidly following cleavage of the single-chain TSHR
into A and B subunits at upstream site 1. This N-terminal
disintegration pauses at site 2, but then progresses further downstream
to the vicinity of the plasma membrane, revealing a novel mechanism
for shedding of the A subunit.
We thank Dr. Paul Banga and Drs. Edwin
Milgrom and Hugue Loosfelt for generous gifts of their excellent
monoclonal antibodies to the TSHR.
*
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.
The abbreviations used are:
TSH, thyrotropin;
TSHR, thyrotropin receptor;
CHO, Chinese hamster ovary;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody;
FCS, fetal calf
serum.
Subunit Structure of Thyrotropin Receptors Expressed on the
Cell Surface*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol (30 min at 50 °C) to release the bound, biotinylated TSHR. Aliquots of both biotinylated and non-biotinylated TSHR 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 (Bio-Rad) to
obtain the molecular weights indicated in the text. Radiolabeled proteins were visualized by autography on Biomax MS x-ray film (Eastman
Kodak Co.).
-mercaptoethanol. Enzymatic deglycosylation was performed according
to the protocol of the manufacturer (New England Biolabs).
N-Glycosidase F digestion (100 units for 2 h at
37 °C) was in 50 mM sodium phosphate, pH 7.5, 1%
Nonidet P-40. Endoglycosidase H digestion (50 units for 2 h at
37 °C) was in 50 mM sodium citrate, pH 5.5. Samples were then subjected to SDS-polyacrylamide gel electrophoresis as described above.
-mercaptoethanol (30 min at 50 °C), electrophoresed on 16.5%
SDS-polyacrylamide gels, and electrophoretically transferred to
polyvinylidene difluoride membranes (Bio-Rad). Membranes were incubated
overnight (4 °C) with 9E10 (final dilution;1:500). After rinsing,
the membranes were incubated for 1 h at room temperature with
alkaline phosphatase-conjugated goat anti-mouse IgG (1:400) (Cappel,
Durham, NC). The signal was developed with nitro blue 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunoprecipitation of TSH receptors (TSHR)
with an antibody to the A subunit. Panel A,
TSHR-expressing CHO cells (TSHR-10,000) were metabolically labeled with
[35S]methionine/cysteine, and then proteins on the
surface of intact cell monolayers were biotinylated. Cells were
harvested by scraping, and TSHR were extracted with detergent and
immunoprecipitated with a monoclonal antibody to the A subunit (A9;
epitope between amino acid residues 147 and 229) (24). TSHR were then
separated with Sepharose-streptavidin beads into streptavidin-adherent
(biotinylated) and non-adherent (non-biotinylated) pools. Aliquots of
each were enzymatically deglycosylated with endoglycosidase F
(Endo F) and endoglycosidase H (Endo H).
Panel B, biotinylated TSHR. Panel
C, non-biotinylated TSHR. Autoradiography was for 18 days.
View larger version (21K):
[in a new window]
Fig. 2.
Schematic representation of cysteine residues
involved in A subunit linkage to the B subunit. Although it is not
known precisely which cysteines are paired, available evidence suggests
a critical role for Cys-283 and Cys-284 on the A subunit and Cys-398
and Cys-408 on the B subunit (reviewed in Ref. 1). Cys-301 and Cys-390
contribute to the stability of the cleaved receptor, but their mutation
does not lead to loss of the A subunit (28). N-terminal degradation of
the B subunit leading to loss of Cys-408 would be incompatible with
retention of the A subunit. Immunoprecipitation of the cleaved TSHR
with an antibody to the A subunit would not, therefore, co-isolate B
subunits degraded downstream of Cys-408.
View larger version (23K):
[in a new window]
Fig. 3.
Cell surface biotinylated TSH receptors
identified with an antibody to the B subunit. A, TSHR
biotinylated on the surface of TSHR-10,000 cell monolayers were
immunoprecipitated exactly as described in the legend to Fig. 1, except
that an antibody to the B subunit was used (T3-365; epitope within
amino acid residues 640-764) (4). For comparison, immunoprecipitation
with an A subunit antibody (A9) is shown in a parallel lane.
Autoradiography was for 5 days. B, expected size of TSH
receptor B subunit intermediates formed by incomplete removal of the C
peptide region. Available evidence indicates that cleavage at upstream
site 1 occurs between amino acid residues 305-316. The cleaved TSHR
lacks a segment (C peptide region) downstream of cleavage site 1. N-terminal degradation of the B subunit downstream to site 2, and
possibly beyond site 2, would generate B subunit polypeptides of the
indicated calculated mass based on their amino acid composition.
C, detection of big B subunits on the cell surface. Intact
cells expressing TSHR with a c-myc epitope substituting for residues
338-349 within the C peptide region (TSHRmyc-10,000) were
metabolically labeled and their surface proteins biotinylated. Cell
monolayers were extracted directly with buffer containing detergent
without first removing the cells by scraping (see "Materials and
Methods"). TSHR were immunoprecipitated from the same detergent
extract using two different mAbs; 9E10 to the c-myc epitope and T3-365
whose epitope lies within amino acid residues 640-764. Cell surface
receptors were recovered with streptavidin-agarose and subjected to
polyacrylamide gel electrophoresis and autoradiography. The same amount
of radioactivity was applied to each lane, compensating for the lesser
(4-fold) radioactivity recovered with the c-myc mAb. Cell surface TSHR
immunoprecipitated with mAb T3-365 from wild type (wt) TSHR-expressing
cells are included to indicate the similar TSHR subunit sizes in the
two cell lines, when isolated with the same antibody. Autoradiography
was for 4 days.
View larger version (41K):
[in a new window]
Fig. 4.
Influence of TSH receptor extraction
procedure on B subunit structure. TSHR-10,000 overexpressing the
TSHR were metabolically labeled and proteins on the surface of intact
cells in monolayer were biotinylated. Cell proteins were then extracted
in one of two ways: (i) harvesting cells by scraping followed by
extraction with detergent, and (ii) addition of detergent directly to
the cell monolayer without scraping. TSHR were immunoprecipitated with
a monoclonal antibody to the B subunit, and biotinylated, cell surface
TSHR were then separated and subjected to polyacrylamide gel
electrophoresis and autoradiography. Exposure in the experiment shown
was for 20 days.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence and reprint requests 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.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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