(Received for publication, June 14, 1995)
From the
The assumption that a disulfide bond is present between two highly conserved cysteines in the extracellular loops of G protein-coupled receptors and is critical for receptor function has been cast in doubt. We undertook to determine whether a disulfide bond important for binding or activation is present in the thyrotropin-releasing hormone (TRH) receptor (TRH-R). Studies were performed with cells expressing wild-type (WT) and mutant receptors in the absence or presence of the reducing agent dithiothreitol (DTT). The affinity of WT TRH-R was 16-22-fold lower in the presence of DTT than in the absence of DTT. Mutant receptors were constructed in which Ala was substituted for conserved Cys-98 and Cys-179 of extracellular loops 1 and 2, respectively, and for the nonconserved Cys-100. C98A and C179A TRH-Rs did not exhibit high affinity binding. These mutant receptors were capable of stimulating inositol phosphate second messenger formation to the same extent as WT TRH-Rs but with a markedly lower potency. The affinities of C98A and C179A TRH-Rs, estimated from their potencies, were 4400- and 640-fold lower, respectively, than WT TRH-R. The estimated affinities of neither C98A nor C179A TRH-R were decreased by DTT. In contrast, the estimated affinity of C100A TRH-R was not different from WT TRH-R and was DTT sensitive. Moreover, the effect of mutating both Cys-98 and Cys-179 was not additive with the effects of the individual mutations. These data provide strong evidence that Cys-98 and Cys-179 form a disulfide bond. This interaction is not involved in receptor activation but is critical for maintaining the high affinity conformation of TRH-R.
Seven transmembrane-spanning, guanine nucleotide-binding (G)
protein-coupled receptors (GPCRs) ()constitute a large
family of cell surface regulatory molecules(1, 2) .
With rare exceptions (for example, the Mas oncogene and cannabinoid
receptors (3) ), members of this family contain a cysteine in
the putative first extracellular loop (EC-1) near the top of the third
transmembrane domain (TM-3) and another cysteine in EC-2. It has been
assumed that this pair of Cys residues forms a disulfide bond in most
GPCRs(2) . This linkage has been proposed to be important to
allow the receptor to attain a normal conformation during synthesis,
for normal expression on the cell surface or to maintain normal
function, in particular, normal binding and activation. Data consistent
with the presence of a disulfide bond between these two conserved Cys
residues have been reported for some receptors of the GPCR family, but
there is evidence that these residues do not always participate in this
linkage. Evidence in support of this disulfide linkage has been
primarily of two types. First, it has been shown that the binding
affinity of a number of GPCRs is decreased under reducing conditions.
This effect, however, may involve reductions of disulfides other than
this proposed bond. And second, substitution of one or the other of
these Cys residues has led to decreased binding affinity, expression,
or activation. These effects may be due to loss of the disulfide bond
or could be caused by an adverse effect of the substituting amino acid
(see below).
Rhodopsin, -adrenergic receptors, and
muscarinic acetylcholine receptors are the best studied GPCRs with
regard to the presence of an extracellular disulfide bond. In
experiments with mutants of rhodopsin in which one or both of the
cysteines were substituted by alanine(4) , findings were
consistent with the presence of a disulfide bond between the conserved
Cys residues. In contrast to previous studies in which the Cys residues
were substituted with Ser (see
below)(5, 6, 7) , these Cys to Ala mutants of
rhodopsin were expressed normally and bound retinal normally; Cys to
Ala mutants did exhibit a defect in the stability of an activated
intermediate, metarhodopsin II. In the
-adrenergic
receptor, there may be two extracellular disulfide bonds. Single
substitution with Val of all four Cys residues in the
-adrenergic receptor caused decreases in binding
affinity and receptor expression, but the residues linked by disulfide
bonds were not identified(8, 9) . In fact, data from a
series of
-adrenergic receptor mutants in which the
cysteines were substituted with Ala show there is no disulfide bond
between the two conserved cysteines(10) . The data from
experiments in which muscarinic acetylcholine receptors have been
mutated are more difficult to interpret because the conserved Cys
residues were substituted by Ser (11) (see below). (Findings
from experiments in which muscarinic receptors were labeled with a
disulfide-specific reagent are consistent with the presence of a
disulfide bond between the conserved
cysteines(12, 13) .) The data for other members of the
GPCR family are also consistent with the presence of this bond but are
not conclusive. For example, it has been reported that single
substitution of any of four extracellular cysteines with glycine in the
angiotensin II AT
receptor leads to a 10-fold decrease in
affinity(14) .
A number of GPCRs have been studied by mutation of the conserved cysteines to serines. The data from these experiments are difficult to interpret because the hydrophilic nature of the hydroxyl group of serine may have adversely affected receptor conformation. This idea is based on findings with rhodopsin. It was observed that substitution of the conserved Cys residues with Ser in rhodopsin resulted in more marked deleterious effects than substitution with Ala (4) in that Cys to Ser mutants exhibited abnormal levels of expression and glycosylation and an inability to bind retinal(5, 6, 7) . Thus, studies of, for example, muscarinic acetylcholine (11) and thyroid-stimulating hormone receptors (15, 16) in which mutant receptors with Ser substitutions demonstrated loss of binding must be interpreted cautiously.
Thus, it cannot be concluded that conserved Cys residues in EC-1 and EC-2 form a disulfide bond in all GPCRs. Moreover, if a disulfide bond is present in a GPCR it is not necessarily critical for attainment of the native conformation, level of expression, or function of the receptor.
The TRH receptor (TRH-R) is a member of the GPCR family(17) . In previous studies, binding of TRH has been found to decrease under disulfide bond reducing conditions(18, 19) . The studies presented here were undertaken to determine whether the conserved Cys at position 98 (Cys-98) of EC-1 and the conserved Cys-179 of EC-2 of TRH-R are covalently linked by a disulfide bond and whether this linkage is necessary for normal TRH-R function. Our data, based on single and double mutations of these Cys residues and on the effects of the reducing agent dithiothreitol (DTT) on these mutants, offer strong evidence that a disulfide bond is present between Cys-98 and Cys-179 and, furthermore, that this linkage is critical for maintaining the high affinity conformation of TRH-R.
To begin to assess the importance of disulfide bonds in TRH-R
for binding, the effect of the reducing agent DTT was studied. WT
TRH-Rs were stably expressed in AtT-20 pituitary
cells(23, 24) , and binding affinities for MeTRH were
measured in the absence or presence of DTT (Fig. 1A).
In the absence of DTT, the K for MeTRH was 1.3
nM, and the affinity was lowered 16-fold in the presence of
DTT. For WT TRH-Rs transiently expressed in COS-1 cells, the K
for MeTRH was 2.4 nM in the absence of
DTT and was lowered 22-fold in the presence of DTT (Fig. 1B, Table 1). These data, from two
different cell lines, indicate the importance of a reducible disulfide
bond(s) in TRH-R for high affinity binding.
Figure 1:
Effect of DTT on binding to WT
TRH-Rs. Competition binding for [H]MeTRH by
unlabeled MeTRH in the absence or presence of DTT as described under
``Experimental Procedures'' is shown. WT TRH-Rs were
expressed stably in AtT-20 cells (A) or transiently in COS-1
cells (B). Duplicate determinations were obtained in two to
four experiments.
To determine which
cysteines were contributing to this effect, the conserved Cys-98 and
Cys-179, as well as the nonconserved Cys-100, were mutated to alanine,
expressed transiently in COS-1 cells, and assayed for binding (Table 1). The affinity of C100A TRH-R for MeTRH was the same as
WT TRH-R. In contrast, C98A and C179A TRH-Rs showed no specific binding
with up to 10 nM [H]MeTRH. To estimate
the affinity of these presumably low affinity receptors, the
EC
values of TRH for stimulation of IP formation were
measured. The maximal extents of stimulation of WT, C98A, C100A, and
C179A TRH-Rs were the same, which is consistent with the idea that the
efficacies of these receptors are similar. Therefore, relative
potencies were used to estimate relative affinities(25) . The
EC
of TRH in cells expressing WT TRH-R was 0.84
nM. The EC
values of C98A, C100A, and C179A
TRH-Rs were 4400-, 2-, and 640-fold higher than that of WT TRH-R (Fig. 2A, Table 1). This indicated that C98A and
C179A TRH-Rs are expressed on the cell surface and that Cys-98 and
Cys-179 are critical for high affinity binding. In contrast, Cys-100
does not appear to be important for high affinity binding. To determine
whether there was an interaction between Cys-98 and Cys-179, the double
mutant C98A/C179A TRH-R was expressed in COS-1 cells and the EC
of TRH was measured. If mutation of Cys-98 and Cys-179 were
affecting the same bond, one would expect the effect of the double
mutation to be similar to the effects of the individual mutations. This
is what was found. The EC
for C98A/C179A TRH-R was the
same as that for C98A TRH-R, indicating that mutation of either Cys-98
or Cys-179 resulted in loss of the same bond.
Figure 2:
Stimulation of inositol phosphate
formation by TRH in COS-1 cells expressing WT or mutant TRH-Rs.
Cysteines were mutated to alanine (A) or serine (B).
Duplicate determinations were obtained in two to eleven experiments. A: , WT;
, C100A;
, C179A;
, C98A;
, C98A/C179A. B:
, WT;
, C179S;
,
C98S;
, C98S/C179S.
To confirm these
findings, Cys-98 and Cys-179 were mutated to Ser. The maximal
stimulations by TRH in COS-1 cells expressing C98S, C179S, and
C98S/C179S TRH-Rs were 64 ± 9, 53 ± 10, and 97 ±
5% of that in cells expressing WT-TRH-Rs. The EC values
for TRH in cells expressing C98S, C179S, and C98S/C179S TRH-Rs were
200,000-, 21,000-, and 430,000-fold higher than that of WT TRH-R (Fig. 2B, Table 1). Therefore, as for the
mutations to Ala, single mutations of Cys-98 or Cys-179 to Ser resulted
in loss of high affinity binding. The results with the doubly mutated
receptor were consistent with the presence of a disulfide bond between
Cys-98 and Cys-179 because the estimated affinity of C98S/C179S TRH-R
was similar to C98S TRH-R and was much less than the additive losses of
C98S and C179S TRH-Rs that would be expected if these mutations
affected different aspects of TRH-R. The recovery of a WT level of
maximal stimulation by the double Ser mutant is consistent with an
interaction between Cys-98 and Cys-179 also. That is, the two Ser
residues in C98S/C179S TRH-R may form a hydrogen bond that could
function in place of the native disulfide bond and restore efficacy to
the double mutant receptor.
To obtain additional evidence that a
disulfide bond was present between Cys-98 and Cys-179, we measured the
effects of DTT on the EC values of TRH for stimulation of
IPs in cells expressing mutant receptors. In AtT-20 cells expressing WT
TRH-Rs, the EC
in the presence of DTT was 120-fold higher
than in the absence of DTT (0.66 (0.42-1.0) nMversus 78(53-120) nM) demonstrating that
effects of DTT on affinity could be assessed through measurement of IP
formation. The effects of DTT on estimated affinities of WT and mutant
receptors expressed in COS-1 cells were then measured (Table 1).
In the presence of DTT, the EC
values of WT and C100A
TRH-Rs were 12- and 9.3-fold higher, respectively, compared with
stimulation in the absence of DTT, indicating the presence of a
disulfide bond important for binding. In contrast, the EC
values of C98A and C179A TRH-Rs were lower, 30 and 61%,
respectively, in the presence of DTT than in the absence of DTT. This
indicates that the critical disulfide bond present in WT TRH-R is
absent in C98A and C179A TRH-R. This is consistent with there being a
disulfide link specifically between Cys-98 and Cys-179 in WT TRH-R that
is important for high affinity binding.
We conclude there is a disulfide bond between the conserved Cys-98 of EC-1 and the conserved Cys-179 of EC-2 in TRH-R and that this bond is necessary to constrain TRH-R in a high affinity conformation. These conclusions are based on the following observations: 1) high affinity TRH binding is decreased by DTT in WT TRH-Rs; 2) substitution of either of the conserved Cys residues decreased estimated TRH binding affinity whereas substitution of nearby Cys-100 did not affect binding affinity; 3) estimated binding affinity was not decreased by DTT in mutants in which Cys-98 or Cys-179 were substituted by Ala; 4) substitution of both Cys residues by Ala did not decrease estimated affinity more than substitution of Cys-98 alone; and 5) the double mutant C98S/C179S TRH-R, in which both cysteines were substituted with serines, was capable of stimulating a higher level of IP formation than C98S or C179S TRH-Rs. This last point is consistent with the idea that single Ser substitutions are more deleterious to receptor expression or efficacy than the double mutant because in the double mutant the two Ser residues may form a hydrogen bond that could simulate the function of the lost disulfide bond. Similarly, single substitutions of conserved Cys residues with Ser in the parathyroid hormone/parathyroid hormone-related peptide receptor were found to cause greater decreases in expression of this receptor than that observed with the double mutant(26) .
In contrast to the effect on WT TRH-Rs, pretreatment with the reducing agent DTT did not lower the potencies of C98A or C179A TRH-Rs but increased them. This suggests that DTT affects another disulfide bond(s) in TRH-R or in another cellular protein(s) in addition to the disulfide link between Cys-98 and Cys-179. As reduction of this disulfide bond(s) increases the apparent potency of TRH, the effect of breaking the Cys-98/Cys-179 disulfide bond is underestimated. This would explain, in part, the greater decrease in estimated affinity, which is based on relative potencies, noted with Cys-98 or Cys-179 mutants than that observed upon incubating WT TRH-R with DTT. Other factors that may account for these differences may be the more deleterious effects of either a methyl group (in Ala) or a hydroxyl group (in Ser) rather than free sulfhydryl groups (in 2 Cys) and differences between preventing formation of a disulfide bond during synthesis and folding of the receptor (in the substituted mutants) compared with reduction of a disulfide bond in a correctly folded WT TRH-R after its insertion into the plasma membrane.
Several
different roles have been ascribed to the putative disulfide bond
between EC loops of GPCRs. It is possible that this disulfide linkage
may form during the early stages of receptor synthesis and be necessary
for the normal folding and insertion into the membrane of these cell
surface proteins. There is, however, no direct data to support this
idea and, in particular, the timing of formation of this disulfide bond
during synthesis has not been determined in any GPCR. In rhodopsin, the
EC disulfide bond appears not to be required for binding the ligand
retinal but for the stability of an activated intermediate,
metarhodopsin II(4) . In TRH-R (this paper), the EC disulfide
between these conserved Cys residues is necessary to constrain the
receptor in a conformation that can attain high affinity binding. By
contrast, two other EC disulfide linkages may be used to maintain a
high affinity form of -adrenergic
receptors(10) . The conserved EC disulfide bond, however, is
not required for full apparent efficacy in GPCRs.
In conclusion, we have provided strong evidence for a disulfide bond between the conserved Cys-98 of EC-1 and the conserved Cys-179 of EC-2 in TRH-R and have demonstrated the critical importance of this disulfide link for high affinity binding of TRH. Based on our previous studies showing the binding pocket for TRH to be within the TM bundle (27) , we propose that the extracellular disulfide link is essential for constraining the TM helices of TRH-R in the proper binding conformation.