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Department of Endocrinology (S.C.H.), Singapore General Hospital, Republic of Singapore 169608; and Institut de Recherche Interdisciplinaire (S.C.H., J.V.S., A.L., G.V., S.C.), Faculty of Medicine, Free University of Brussels B1070, Belgium
Address all correspondence and requests for reprints to: Dr. Gilbert Vassart, Institut de Recherche Interdisciplinaire, Faculty of Medicine, Free University of Brussels, Belgium. E-mail: gvassart{at}ulb.ac.be
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Abstract |
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s protein. Mutation of S281 in the
ectodomain is supposed to release this constraint, thereby causing
receptor activation. This defines S281 and its neighbors as a segment
important in intramolecular signal transduction. The functional
importance of this segment was explored by site-directed mutagenesis
experiments involving S281, as well as the two cysteine residues (C283,
C284) present immediately downstream. S281 was mutated to N, T, G, and
A in this study, and the functional characteristics of the mutants were
compared. We found that S281N, S281T, and S281G display stronger
constitutive activity than S281A mutant, suggesting that increase in
constitutive activity is related to the extent of disruption of the
local structure of the ectodomain. C283 and C284, the two consecutive
cysteines that are highly conserved in glycoprotein hormone receptors,
were mutated to serine, either alone (S281HSC or S281HCS) or in
combination (S281HSS) and were studied in two different TSH receptor
backgrounds. The mutated cysteine ectodomains were either linked to a
glycosylphosphatidylinositol anchor or the serpentine domain of
the wild-type holoreceptor. Glycosylphosphatidylinositol-anchored
ectodomain receptors showed good cell surface expression in CHO
cells, but only S281HCS was able to bind TSH specifically, illustrating
the importance of C283, or the putative disulphide bond, in maintaining
the conformation of the ligand binding site. In contrast, cysteine
mutants on an extracellular domain-holoreceptor background displayed
severely impaired membrane targeting and were poorly expressed in COS
cells. However, basal cAMP production, normalized to expression at the
plasma membrane, indicated significant increase in constitutive
activity of all three mutants, compared with the wild-type receptor.
Altogether, these findings support a model in which the ectodomain
would act as a silencer of the basal activity of the serpentine portion
of the receptor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, a number of the activating mutations have been identified in the extracellular loops of the serpentine portion of the receptor and even in the aminoterminal extracellular domain (ECD) responsible for the high-affinity binding of TSH (8, 11, 12). Serine 281 in the ECD, when mutated to three different residues (i.e. isoleucine, threonine, and asparagine), strongly increases the basal stimulation of adenylylcyclase by the receptor (11, 12). In addition, treatment of cells expressing the receptor with low concentrations of trypsin results in partial activation of the receptor, while removing an epitope from the ECD (13). From these observations, we have proposed that the ECD would contribute to the silencing of an inherently noisy serpentine portion of the TSHr (10, 11). This model implies that interaction(s) between specific regions of the ECD and the extracellular loops of the serpentine portion would exist, whereby this silencing effect would be exerted. Recent experiments dealing with expression of chimeric receptors have provided arguments in favor of this model (14 and our unpublished observations).
In the present study, the changes in constitutive activity of the TSHr upon mutation at the critical S281 to amino acids N, T, G, and A were compared. We found that increase in constitutive activity is related to the expected magnitude of the effects of the mutations on local structure, being minimal in the S281A mutant. In addition, we also explored the functional importance of two highly conserved cysteine residues of the ECD situated in the immediate vicinity of serine 281 (Cys 283 and Cys 284). Being close to a residue of the ECD whose mutation activates the receptor constitutively, we reasoned that they (or, more likely, the structure stabilized by the disulfide bridges they make) may contribute to the silencing effect of the ECD. Cysteines 283 and 284 were mutated to serine, either singly (S281HSC or S281HCS) or in combination (S281HSS) on the background of the holoreceptor or on the ECD expressed separately as a glycosylphosphatidylinositol(GPI)-anchored molecule (ECD-GPI) (15). The results point to an important structural role of C283 in allowing folding of the ECD into a structure capable of binding TSH. In addition, all three mutant receptor constructs (S281HSC, S281HCS, and S281HSS) displayed increase in their basal activity, thus providing additional support to the notion that a specific structure of the ECD is involved in keeping silent the serpentine portion of the receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cysteine mutant ECD-GPI constructs. An AatII-NdeI fragment from each cysteine mutant obtained above was inserted in the complementary DNA (cDNA) coding for the WT (S281HCC) TSHr ECD-GPI construct in pEFIN3 vector (Euroscreen, Brussels, Belgium). Transfection was done in CHO-K1 cells and followed by selection with Geneticin G418. Clones carrying high levels of receptor expression were isolated by limit dilution.
Serine and cysteine mutant ECD-holoreceptor constructs. After restriction enzyme digestion with XhoI and EcoRV, mutated serine and cysteine ECDs from the above described constructs were ligated into the full-length TSHr cDNA in pBluescript SK+, which had an EcoRV site engineered at the CEDIM (408–412) motif. A XbaI-KpnI fragment from each mutant was inserted into the pcDNA3 vector (Invitrogen, Groningen, The Netherlands). For the cysteine ECD-holoreceptors, transient transfection was done with 2 µg DNA for each construct in 300,000 COS 7, using FUGENE 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol, whereas transfection of serine mutants was performed by the DEAE-dextran method followed by a dimethylsulfoxide shock (16). Cells transfected with the empty vector or the cDNA of TSHr served as negative and positive controls, respectively.
Characterization of serine and cysteine mutants
Flow cytofluorometry. Monoclonal antibodies (mAb) BA8 and
15.2 were used for the experiments as described previously
(15). BA8 recognizes a conformational epitope on the TSHr
(15) localized between amino acids 21 and 164
(localization determined by FACS with TSHr/LHr and human
TSHr/rat TSHr chimeric receptors; our unpublished results),
whereas 15.2 (Costagliola, S., unpublished results) recognizes a linear
epitope located close to the C-terminal portion of the ECD from the
amino acid residues 371–400.
TSH binding assay. For TSH binding, cells were washed twice
with binding assay buffer (NaCl-free HBSS containing 277 mM
sucrose, 5% BSA, and 25 mM HEPES, pH 7.4) and incubated
with 100,000 cpm/ml 125I-labeled TSH (58
µCI/µg, BRAHMS Diagnostics, Berlin, Germany) for 4 h at room
temperature, with or without unlabeled TSH (Sigma-Aldrich Corp., Bornem, Belgium). The cells were then washed twice
with cold (4 C) buffer, subsequently solubilized with 1
M NaOH, and the radioactivity was determined in a 
counter.
cAMP determination. These were done only on COS cells transfected with the mutant ECD-holoreceptors, after 1 h incubation in the presence of 25 µM Rolipram (Laboratoire Logeais, Paris, France) with or without TSH, as detailed previously (13).
Computation of specific constitutive activity (SCA) and relative SCA (RSCA). Given that the transfection efficiency for each construct is constant for a given batch of cells, the SCA is calculated by: SCA = (AR-AV)/(FR-FV), where AR and AV are the cAMP of cells transfected with the mutant construct and vector, respectively, and FR and FV are the corresponding mean fluorescence unit obtained with FACS analyses.
RSCA, which is normalized to the WT TSHr, is obtained by: RSCA = SCAR/SCAWT
Western blotting
Preparation of receptor.
Six dishes, each containing 300,000
cells transfected with mutant ECD-holoreceptor constructs, were
detached with 5 mM EDTA and 5 mM EGTA in PBS
and spun down at 280 x g to obtain a cell pellet,
which was resuspended and homogenized in a Potter-Elvehjem glass
homogenizer with a Teflon pestle in 1250 µl lysis buffer (100
mM
(NH4)2SO4, 20
mM Tris at pH 7.5, and 10% glycerol) containing
protease inhibitors (Complete, Roche Molecular Biochemicals). The lysate was then centrifuged at 500 x
g for 10 min, and the supernatant was recovered for further
ultracentrifugation at 30,000 x g for 30 min. Two
hundred microliters of lysis buffer, containing 1%
N-dodecyl-ß-D- maltoside
(Anatrace, Maumee, OH), was added to the pellet; and the
suspension was incubated for another 30 min, at 4 C, under constant
rotation to allow thorough mixing. Final centrifugation was carried out
at 100,000 x g for 1 h, and the supernatant was
stored at -80 C for further use. All procedures described were
performed at 4 C.
Immunoblotting.
Three microliters of Laemli sample buffer
(5x) containing SDS and ß-mercpathoethanol as a reducing agent, were
added to 10 µl receptor protein prepared as above and denatured at 40
C for 1 h. The sample was then run on 7% acrylamide gel and
probed with the mAb 28 (culture supernatant diluted 1:50), which
recognizes a linear epitope at the N-terminal of the ECD from amino
acid residues 31–50 (Costagliola, S., to be published). The proteins
were visualized with an antimouse IgG horseradish peroxidase conjugate
and the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech, Benelux, The Netherlands).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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illustrates alignment of
the region of glycoprotein hormone receptors downstream of the last
leucine repeat. As can been seen, the CCAF tetrapeptide, two residue
downstream of Ser281 (numbering of the TSHr), is almost invariant in
the TSH, LH/CG, and FSH receptors, as well as in related orphan
receptors of mammalian, drosophila, or nematode origins. Their
proximity with the functionally important Ser281 residue, whose
mutation activates the receptor constitutively, and their conservation
at the C-terminal frontier downstream to the leucine repeats of the
ECDs in all members of the glycoprotein hormone receptor family,
suggested that Cys 283 and Cys284, or the structure they stabilize via
disulfide bonding, would be important for intramolecular signal
transduction. It was thus decided to mutate them to serine, alone or in
combination, and to explore the effect of such mutations both on the
ability of the ECD to fold normally and bind TSH, and on the basal
activity of the receptor. An interplay has been demonstrated between
the ectodomain and the serpentine domain, explaining the gain in
affinity for TSH binding displayed by constitutively active TSHr
mutants (17). To avoid possible interference resulting
from this interaction, the effect of Cys 283 and Cys284 mutations was
tested on the ECD expressed as a GPI-anchored molecule, i.e.
devoid of the serpentine domain. The same mutations were engineered on
the background of the holoreceptor to explore their effects on basal
receptor activity.
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, A and B). Representative clones were
selected that displayed expression levels corresponding to the average
of the mixes. They were evaluated by FACS analyses with two different
mAb: mAb BA8 recognizes a conformational epitope located in the
aminoterminal portion of the ECD (15), and mAb 15.2
recognizes a linear epitope at the C-terminal end of ECD. When compared
with S281HCC (WT), S281HSS showed the highest level of receptor
expression, followed by S281HCS and S281HSC (Fig. 2B). Only the S281HCS
mutant showed sizable specific binding of TSH that was displaceable
with unlabeled hormone (Fig. 3A).
Displacement of 125I-labeled TSH bound to this
mutant receptor by unlabeled hormone was similar to the WT receptor,
with dissociation constants of 1.6 mUI/ml and 2.1 mUI/ml, respectively
(Fig. 3B). Mutating Cys 283 (as in S281HSC) abolished this binding
ability, emphasizing the importance of Cys 283 in maintaining
conformation of the binding site. It was also apparent from the above
findings that Cys 284 is involved to a lesser extent in this functional
aspect of the TSHr. Although well expressed on the cell surface, the
double-mutant S281HSS showed only marginal TSH binding capability (Fig. 3A). In all cases, recognition of the mutants was similar with both
mAb, suggesting that the conformational changes did not involve
destruction of the conformational epitope of mAb BA8.
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). mAb 15.2, whose linear epitope
is located just upstream of the first transmembrane segment, was able
to recognize the cysteine mutants better than BA8 (Fig. 4B). This could
be explained, in part, by the difference in the epitopes recognized by
both antibodies. Conformational changes in the ECD as a result of
cysteine mutation would have diminished the binding of BA8
(conformational epitope) without affecting recognition by 15.2.
However, FACS studies on permeabilized cells showed that mAb BA8 was
able to recognize the mutant receptors and that most of these receptors
were sequestrated intracellularly (Fig. 4C). Western blots of mutant
receptors prepared from transfected cells also indicated that the
majority of these mutant receptors were approximately100 kDa in size,
which corresponded to the single-chain TSHr with immature, high mannose
glycan, whereas the 120-kDa protein, representing the mature TSHr with
complex carbohydrate (18, 19), was barely detectable (Fig. 5). Cells transfected with cysteine
mutant ECD-holoreceptor constructs did not bind TSH and showed no
response to maximal stimulation with TSH (100 mU/ml) (data not shown).
However, despite the low level of receptor expression at the cell
membrane, cells transfected with cysteine mutants showed increased cAMP
production above that of vector, indicating constitutive activity of
these receptors (Table 1). When
normalized to the level of expression (as measured by FACS on
nonpermeabilized cells), constitutive activity of the mutants was
higher than that of the WT receptor (Fig. 6). However, constitutive activity did
not reach the level achieved by an S281N mutant (Fig. 6).
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and 11, 12), we
explored whether these amino acid substitutions were merely disrupting
a silencing interaction of the ECD with the serpentine (loss of
structure), or if a gain of structure was involved. Ser 281 was mutated
to Ala and Gly, and the basal activity of the mutant receptors was
tested by transfection in COS cells. Alanine substitution is expected
to affect minimally the backbone structure, whereas disrupting
side-chain-mediated polar or ionic interactions. On the contrary,
substitution by glycine may induce significant change in the backbone
conformation by introducing a local flexibility (20). Both
mutants were well expressed at the cell surface (Table 2). Whereas
the S281G mutant displayed increase in constitutive activity of the
same magnitude as the cysteine mutants, basal activity of the S281A was
similar or even lower when compared with the WT receptor (Table 2 and
Fig. 6
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As we have shown clearly in our experiments involving ECD-holoreceptor
mutant, most of the mutated receptors failed to traffic to cell surface
and were sequestrated intracellularly. The mutant receptors trapped
internally were predominantly approximately 100 kDa in size,
corresponding to forms linked to immature, high mannose glycans
(19). Scarce amount of the mature holoreceptor (
120
kDa) could only be detected on immunoblotting, which agreed with the
results of FACS analyses. Therefore, the failure of ligand binding in
these instances is likely attributable to lack of receptor expression
rather than modification of the ligand binding site. Expression of the
same mutants on a GPI-anchor background promoted the efficient
targeting of some of them to the cell surface, thus allowing, for the
first time, assessment of the relative importance of Cys 283 and 284 in
TSH binding. Keeping Cys 283 intact in the ECD (as in S281HCS-GPI) is
compatible with specific binding of TSH, with dissociation constant
similar to the WT receptor. Mutation of this residue (in S281HSC-GPI)
abolished ligand binding and emphasized the critical role of Cys 283 in
maintaining the conformation of the TSH binding site.
Precise interpretation of these experiments and of those of previous studies (21, 22, 23) would require knowledge of the partnering cysteine residues involved in individual disulfide bonds. The difference in behavior of our mutants, when expressed on a GPI or holoreceptor background, could be secondary to differences in the nature of illegitimate disulfide bonds that may take place in the mutants. Apart from the mutated residues, the GPI-based constructs contain all cysteines present in the WT ECD. In the holoreceptor-based mutants, the additional cysteines present in the first and second extracellular loops (which are thought to make a disulfide bond in most GPCRs) might participate in illegitimate bonds, thus causing misfolding and trapping of the mutant proteins in the endoplasmic reticulum. It would be of interest to extend the present study by testing all combinations of S281HCS, S281HSC, and S281HSS mutations with mutations of Cys494 and Cys569 in the first and second exoloops, respectively.
The TSHr displays significant basal activity, when expressed by
transfection in heterologous cells (24, 25), and this
constitutive activity can be increased by a wide spectrum of mutations
(1, 2, 3, 4, 5, 6, 7, 8, 9). The activating mutations affecting residues of the
serpentine portion of the receptor are in agreement with the current
model for GPCR activation, wherein equilibrium would exist between
constrained inactive conformation and relaxed active conformation
(26). The existence of activating mutations affecting a
residue of the ECD, Ser281, led us to propose that, in the TSHr, the
ECD would contribute to stabilization of an inactive conformation of
the serpentine by exerting a silencing effect via interaction with the
exoloops (11). This model predicts that disruption of the
WT structure of the ECD, or mere removal of the ECD, would lead to
receptor activation in the absence of TSH. In this context, mutations
causing disruption of disulphide bond(s) in the vicinity of Ser281, via
mutations of Cys 283 and/or Cys 284, would be expected to trigger
receptor activation. Indeed, all three cysteine mutant
ECD-holoreceptors displayed constitutive activity at levels higher than
the WT. Also, when expressed alone as a truncated structure devoid of
its aminoterminal ECD, the serpentine portion of the TSHr displays
increase in constitutive activity, when compared with the WT
holoreceptor [ (14) and our unpublished data]. In both
cases, however, the basal activity did not reach the level achieved by
S281N. An attractive possibility would be that mutations affecting
Ser281 would cause a conformational change, acting as a switch,
transforming domain of the ECD with a silencing effect on the
serpentine into a structure endowed with stimulatory activity. This
model would account for the observation that mutations affecting Ser281
of the TSHr [or Ser277 of the LHr (27)] cause increase
in constitutive activity, in relation with the expected effects of the
amino acid substitutions on disruption of the local structure.
Substitutions by alanine are virtually neutral, with no stimulatory
effect on basal activity; whereas substitutions by glycine, threonine,
or asparagine (Fig. 6) or, more so, by isoleucine lead to stronger
activation (12, 27). The mutations with more drastic
effects on structure would trigger the switch more efficiently. The
model leads to the suggestion that activation of the receptors by their
natural agonists could involve a similar mechanism, switching a
silencing effect of the unliganded ECD into a stimulatory effect
induced by hormone binding. Alternative hypotheses have been proposed,
involving a direct stimulatory effect of the -subunits of
glycoprotein hormones on the serpentine portion of the receptor
(28). Further work will explore the relevance of the above
model in the cases of the noisy TSHr and the silent LHr and FSH
receptor and will attempt identification of the residues involved both
in the putative switch domain of the ECD and its interacting partner in
the exoloops of the serpentine domain.
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Acknowledgments |
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Footnotes |
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2 A fellow of the Health Manpower Development Programme of
Ministry of Health, Republic of Singapore, and supported by a grant
from NMRC (NMRC 0347/1999).
Received January 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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