From the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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All G protein-coupled receptors are predicted to
consist of a bundle of seven transmembrane helices (I-VII) that are
connected by various extracellular and intracellular loops. At
present, little is known about the molecular interactions that are
critical for the proper assembly of the transmembrane receptor core. To address this issue, we took advantage of the ability of coexpressed N-
and C-terminal m3 muscarinic receptor fragments to form
functional receptor complexes (Schöneberg, T., Liu,
J., and Wess, J. (1995) J. Biol. Chem. 270, 18000-18006). As a model system, we used two polypeptides, referred to
as m3-trunk and m3-tail, that were generated by "splitting" the m3
muscarinic receptor within the third intracellular loop. We initially
demonstrated, by employing a sandwich enzyme-linked immunosorbent assay
strategy, that the two receptor fragments directly associate with each
other when coexpressed in COS-7 cells. Additional studies with N- and
C-terminal fragments derived from other G protein-coupled receptors
showed that fragment association was highly receptor-specific. In
subsequent experiments, the sandwich enzyme-linked immunosorbent assay
system was used to identify amino acids that are required for proper
fragment (receptor) assembly. Point mutations were introduced into
m3-trunk or m3-tail, and the ability of these mutations to interfere
with efficient fragment assembly was examined. These studies showed
that three highly conserved proline residues (located in transmembrane
helices V, VI, and VII) are essential for proper fragment association
(receptor assembly). Interestingly, incubation with classical
muscarinic agonists and antagonists or allosteric ligands led to
significant increases in the efficiency of fragment association
(particularly upon substitution of the conserved proline residues),
indicating that all of these ligands can act as "anchors" between
the m3-trunk and m3-tail fragments. The approach described here should
be generally applicable to gain deeper insight into the molecular
mechanisms governing G protein-coupled receptor structure and assembly.
All G protein-coupled receptors
(GPCRs)1 are predicted to
share a common three-dimensional fold consisting of seven transmembrane (TM) helices (TM I-VII) linked by alternating intracellular (i1-i3) and extracellular (o2-o4) loops (Fig. 1) (1-3). TM I-VII are thought
to be sequentially arranged in a ringlike fashion, thus forming a
tightly packed TM receptor core (4, 5). Residues located on the inner
surfaces of different TM helices are known to be involved in the
binding of a great number of ligands that act on GPCRs (2-6).
Moreover, ligand-induced conformational changes in the TM receptor core
are thought to be intimately involved in receptor activation (7-9).
However, detailed structural information about the molecular
interactions that allow TM I-VII to assemble in the proper orientation
and geometry is not available at present, primarily due to the lack of
high resolution structural data for any GPCR.
Several studies have shown that GPCRs, like other polytopic
transmembrane proteins (10), can be assembled from two or more independently stable receptor fragments (11-19). For these studies, GPCRs were "split" in various intracellular and extracellular loops
by using recombinant DNA techniques. When the resulting fragment pairs
were coexpressed in cultured cells, high affinity ligand binding and
ligand-dependent G protein activation were observed in
several cases (11-19). The most straightforward explanation for these
findings is that GPCRs consist of multiple autonomous folding domains,
probably due to the ability of individual TM helices to properly fold
independent of the structural context within the full-length receptor
protein. Taken together, these findings strongly support the notion
that the folding of GPCRs (as has been proposed for other polytopic
transmembrane proteins; Ref. 10) occurs in two consecutive steps. In
step I, individual TM helices are established across the lipid bilayer,
which, in step II, are then assembled, by specific helix-helix
interactions, to form a functional receptor protein.
Over the past few years, we have carried out studies with split m3
muscarinic (12, 14) and V2 vasopressin receptors (16) to gain deeper
insight into the molecular mechanisms governing GPCR structure and
assembly. In one study (14), we split the rat m3 muscarinic receptor in
all three intracellular (i1-i3) and all three extracellular loops
(o2-o4). Coexpression in COS-7 cells of three of the six resulting
polypeptide pairs (sites of split: i2, i3, or o3) led to the appearance
of a significant number of specific high affinity radioligand binding
sites (14). In addition, fragment pairs generated by splits within the
i3 or o3 regions still retained the ability to activate G proteins in an agonist-dependent fashion. Moreover, immunofluorescence
microscopic studies using intact and permeabilized cells showed that
the different receptor fragments were individually stable and were
properly inserted (in the correct orientation) into lipid bilayers
(14).
In this study, we have used two m3 muscarinic receptor fragments,
referred to as m3-trunk (containing TM I-V) and m3-tail (containing TM
VI and VII), as model systems to learn more about the molecular
mechanisms underlying GPCR assembly. The m3-trunk and m3-tail
polypeptides were generated by splitting the rat m3 muscarinic receptor
within the i3 loop (Fig. 1; Refs. 12 and 14).
Initially, we established a sandwich ELISA that allowed us to monitor
the association between the two m3 receptor fragments with high
sensitivity. We demonstrated that the strength of the ELISA signals
strictly correlated with the amount of expressed receptor fragments. In
addition, by including fragments derived from other GPCRs in this
analysis, we verified that fragment assembly was highly
receptor-specific.
Subsequently, we used the sandwich ELISA to identify m3 receptor
residues located within the TM receptor core that are required for
proper receptor assembly. Toward this goal, we coexpressed m3-trunk and
m3-tail polypeptides that contained point mutations within individual
TM helices and studied fragment association via ELISA. These studies
showed that three proline residues that are highly conserved among
GPCRs of the rhodopsin family are critical for efficient fragment assembly.
Another question that we addressed was whether muscarinic ligands
(agonists, antagonist, or allosteric ligands) were able to affect the
interaction between the m3-trunk and m3-tail fragments. We found that
most ligands were able to stabilize the interaction between the
m3-trunk and m3-tail polypeptides, as evidenced by increased ELISA
signals in the presence of ligands. This stabilizing effect was most
pronounced when fragment association was impaired due to Pro The approach described here provides new insights into the molecular
mechanisms governing GPCR assembly and should be applicable to other
classes of GPCRs. The outlined experimental strategy offers the unique
opportunity to study, in a quick and reliable fashion, the structural
role of essentially every amino acid within a given GPCR.
DNA Constructs--
All mutant muscarinic receptor constructs
were derived from Rm3pcD-N-HA, a mammalian expression plasmid coding
for the rat m3 muscarinic receptor containing a 9-amino acid
hemagglutinin (HA) epitope tag (YPYDVPDYA) at its N terminus (14).
Previous studies showed that the presence of the HA tag had no
significant effect on the ligand binding and G protein coupling
properties of the wild type m3 receptor (14). The construction of two
pcD-based expression vectors coding for m3-trunk (rat m3 receptor
residues 1-272) and m3-tail (rat m3 receptor residues 388-589) (see
also Fig. 1) has been described previously (12, 14). The m3-trunk polypeptide, like the full-length m3 receptor, carried an HA epitope tag at its N terminus. To allow proper translation of the m3-tail fragment, an in-frame translation start codon was inserted immediately upstream of codon 388 (12). Point mutations that had been generated previously in Rm3pcD (20-24) were introduced into m3-trunk-pcD or
m3-tail-pcD by simple subcloning procedures.
The construction of pcD-based expression vectors coding for V2-trunk,
V2-tail, and GnRH-trunk has been described previously (16, 17).
V2-trunk and GnRH-trunk are fragments derived from the human V2
vasopressin (16) and gonadotropin-releasing hormone (GnRH) receptors
(17), respectively, and are structurally homologous to m3-trunk.
Similar to m3-trunk, V2-trunk and GnRH-trunk were modified by an
N-terminal HA epitope tag. The V2-tail polypeptide is derived from the
human V2 vasopressin receptor and is structurally homologous to m3-tail
(16). The different receptor fragments are composed as follows (amino
acid numbers are given in parentheses): V2-trunk (1-241), V2-tail
(242-371), and GnRH-trunk (1-284).
Since it proved difficult to reliably detect the full-length m3
receptor (Rm3-N-HA) via Western blotting analysis, a construct was
generated in which the central portion of the i3 loop (amino acids
Ala274-Lys469) was deleted as described
previously (25). The resulting plasmid codes for a modified version of
the m3 receptor, which, for the sake of simplicity, is referred to as
m3'(wt) receptor. Radioligand binding and second messenger assays
showed that the m3'(wt) receptor displayed ligand binding and G protein
coupling properties similar to the wild type m3 receptor
(25).2
The identity of all mutant constructs was verified by restriction
endonuclease analysis and DNA sequencing.
Cell Culture and Transfections--
COS-7 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, 100 µg/ml
penicillin, and 100 units/ml streptomycin, at 37 °C in a humidified
5% CO2 incubator. For transfections, 2 × 106 cells were seeded into 100-mm dishes. Cells were
transfected about 20-24 h later by using a DEAE-dextran method (26).
In the case of the m3'(wt) receptor, cells were transfected with 4 µg
of plasmid DNA/dish. In cotransfection experiments involving receptor
fragments, 4 µg of each plasmid were used per dish, unless indicated otherwise.
Preparation of Membrane Lysates--
Approximately 70 h
after transfections, cells were washed once with phosphate-buffered
saline (PBS), scraped into 1.5 ml of sterile water, collected in
Eppendorf tubes, and centrifuged for 30 min at 20,000 × g (4 °C). Pellets were resuspended in 1 ml of buffer A
(PBS supplemented with 0.05% Tween 20, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A,
and 0.2% digitonin), followed by a 30-min incubation on ice and a 30-min spin at 20,000 × g (4 °C) (to remove soluble
as well as peripheral membrane proteins). Pellets were resuspended in
0.2 ml of buffer B (same composition as buffer A except that the
digitonin concentration was increased to 4% and 1% sodium
deoxycholate was added), followed by a 3-h incubation at 4 °C (under
rotation) and a 30-min centrifugation at 20,000 × g
(4 °C). Supernatants containing solubilized receptors/receptor
fragments were used for further experiments.
Western Blotting Analysis--
Receptor/receptor fragment
expression was monitored via immunoblotting. 12 µl of membrane
lysates (prepared as described in the previous paragraph) were mixed
with 3 µl of 15% SDS (final SDS concentration: 3%) and incubated
for 3 h at room temperature. Subsequently, samples were mixed with
Laemmli buffer (27), heated for 5 min at 65 °C, and then subjected
to 10% or 15% SDS-PAGE. Proteins were transferred onto nitrocellulose
membranes via electroblotting. Membranes were blocked overnight with
PBS containing 5% bovine serum albumin (BSA) and then incubated with
primary antibody (12CA5 anti-HA mouse monoclonal antibody (Boehringer
Mannheim) or anti-C-m3 rabbit polyclonal antibody; see Fig. 1) at a
concentration of 1 µg/ml in PBS-T-BSA (PBS containing 0.05% Tween 20 and 1% BSA) for 1 h. Bound antibody was then probed with a
secondary antibody conjugated to horseradish peroxidase (goat
anti-mouse IgG or monkey anti-rabbit IgG antibody (Amersham Pharmacia
Biotech); final dilution in PBS-T-BSA: 1:3,000). Immunoreactive
proteins were visualized using an enhanced chemiluminescence detection
kit (Amersham Pharmacia Biotech). The intensities of immunoreactive
bands were quantitated by scanning densitometry using the program Scion
Image (Scion Corp.).
Sandwich ELISA--
96-well microtiter plates (Nunc-Immuno
Plate, MaxiSorpTM) were coated with the anti-C-m3 (a rabbit polyclonal
antibody directed against the C-terminal segment of the rat m3
muscarinic receptor) or the anti-C-V2 antibodies (a rabbit polyclonal
antibody directed against the C-terminal 29 amino acids of the human V2
vasopressin receptor; Ref. 16) by incubating plates overnight at room
temperature (under shaking) with 100 µl of antibody solution/well
(antibody concentration: 5 µg/ml). Plates were then washed twice with
PBS containing 0.05% Tween 20 (PBS-T; 200 µl/well), incubated with 5% BSA in PBS (200 µl/well) for 1 h at room temperature, and
washed again twice with PBS-T (200 µl/well). In parallel, 50 µl of
membrane lysates prepared from transfected cells as described above
(containing solubilized receptors/receptor fragments) were mixed with
50 µl of PBS-T and incubated for 1 h at 37 °C with 50 µl of
the 12CA5 monoclonal antibody (final concentration: 1 µg/ml).
Subsequently, samples were mixed with 50 µl of anti-mouse IgG
antibody (Amersham Pharmacia Biotech; final dilution: 1:3,000) linked
to horseradish peroxidase, and incubated for another 1 h at
37 °C. Samples were then added to antibody-coated microtiter plates
(see above), followed by a 2-h incubation at 37 °C. After this
incubation step, plates were washed with PBS-T (four times, 200 µl/well). Subsequently, enzymatic reactions were carried out at room
temperature by adding H2O2 and
o-phenylenediamine (2.5 mM each in 0.1 M phosphate-citrate buffer, pH 5.0) (100 µl of each
reagent/well). Reactions were stopped after 30 min by the addition of
50 µl of 1 M H2SO4 containing 0.05 M Na2SO3. Color development
was measured bichromatically at 490 and 630 nm (background) using the
BioKinetics reader (EL 312, Bio Tek Instruments, Inc.).
In a subset of experiments, sandwich ELISA measurements were carried
out in the presence of different classes of muscarinic ligands
(agonists, antagonists, or allosteric ligands). Ligands were included
during the preparation of membrane lysates and all steps of the
sandwich ELISA, except for the final color reaction.
Ligand Binding Assays--
Radioligand binding studies were
carried out using membranes prepared from transfected cells. For the
preparation of cell membranes, pellets from the first centrifugation
step described under "Preparation of Membrane Lysates" were
resuspended in 5 ml of binding buffer, homogenized with a polytron
homogenizer (two times for 30 s), and recentrifuged for 30 min at
23,500 × g (4 °C). The resulting pellets were
resuspended in binding buffer (composition: 136 mM NaCl, 5 mM KCl, 10 mM HEPES, 5 mM sodium phosphate buffer, 1 mM MgCl2, pH 7.4).
Equilibrium dissociation constants (KD) and number
of binding sites (Bmax) were determined in
saturation binding experiments using
N-[3H]methylscopolamine
([3H]NMS; 79 Ci/mmol; NEN Life Science Products) as a
radioligand (concentration range used: 0.05-1.5 nM).
Incubations were carried out at room temperature for 1 h (volume
of the incubation mixture: 0.8 ml). Bound and free radioligand were
separated by filtration through Whatman GF/C glass fiber filters.
Nonspecific binding was determined in the presence of 1 µM atropine. Binding data were analyzed by nonlinear
regression analysis using the program Lotus 1-2-3 (IBM Corp.).
Generation of a Polyclonal m3 Muscarinic Receptor
Antibody--
A polyclonal antibody was raised (in rabbits) against a
keyhole limpet hemocyanin-conjugated synthetic peptide corresponding to
the C-terminal 18 amino acids of the rat m3 muscarinic receptor (Fig.
1). This antibody (referred to as anti-C-m3) was purified by affinity
chromatography on a peptide column according to standard procedures and
specifically recognized the m3 muscarinic receptor protein.2
Drugs--
The following drugs were used: alcuronium chloride
(La Roche), atropine sulfate (Sigma), carbamylcholine chloride
(carbachol; Sigma), ( Use of a Sandwich ELISA to Study the Association between N- and
C-terminal m3 Muscarinic Receptor Fragments--
In this study, we
have investigated the interaction between an N-terminal m3 muscarinic
receptor fragment (referred to as m3-trunk) and the corresponding
C-terminal m3 receptor polypeptide (referred to as m3-tail). The
structures of these two polypeptides that were generated by splitting
the rat m3 receptor within the i3 loop are given in Fig.
1. As a full-length receptor control, we
used a modified version of the m3 receptor (referred to as m3'(wt)), which lacked the central portion of the i3 loop (amino acids
Ala274-Lys469) (25). The m3'(wt) receptor
displayed ligand binding and G protein coupling properties similar to
the unmodified m3 receptor (25).2 However, in contrast to
the wild type m3 receptor,2 the m3'(wt) receptor could be
detected easily via immunoblotting (see below) and was therefore
included as a control throughout all experiments.
Non-transfected COS-7 cells were unable to bind significant amounts of
the radiolabeled muscarinic antagonist, [3H]NMS. In
contrast, radioligand binding studies carried out with membranes
prepared from COS-7 cells cotransfected with m3-trunk and m3-tail
revealed the appearance of a significant number of high affinity
[3H]NMS binding sites (KD = 130 ± 11 pM; Bmax = 58 ± 4 fmol/106 cells) (the corresponding values for m3'(wt) were:
KD = 290 ± 8 pM;
Bmax = 134 ± 5 fmol/106
cells). The most straightforward explanation for this observation is
that the m3-trunk and m3-tail polypeptides can directly associate with
each other to form a functional receptor protein. To provide more
direct evidence for this notion, we employed the sandwich ELISA
strategy summarized in Fig. 2 (for
further details, see "Experimental Procedures"). In brief, this
ELISA takes advantage of the ability of the anti-C-m3 rabbit polyclonal
antibody (which is directed against the C-terminal segment of the rat
m3 muscarinic receptor; Fig. 1) to capture m3-trunk/m3-tail complexes,
using 96-well plates coated with the anti-C-m3 antibody and lysates prepared from COS-7 cells cotransfected with m3-trunk and m3-tail. While m3-tail allows binding of the fragment complex to the anti-C-m3 antibody, the associated m3-trunk fragment (which carried an HA epitope
tag at its N terminus; Ref. 14) was detected with the 12CA5 anti-HA
mouse monoclonal antibody, followed by the addition of anti-mouse IgG
antibody linked to horseradish peroxidase and the determination of
enzymatic activity by a simple color reaction (for details, see
"Experimental Procedures").
The results of a typical sandwich ELISA assay are shown in Fig.
3. Cell lysates prepared from COS-7 cells
that were transfected with the m3-trunk polypeptide alone gave only a
very weak ELISA signal (optical density (OD) measured at 490 nm:
0.018 ± 0.002), which was similarly low as the signal found with
vector-transfected cells (0.014 ± 0.002). However, lysates
prepared from cells cotransfected with m3-trunk and m3-tail resulted in
a 9.5-fold increase in OD readings (above background), indicative of
fragment association (Fig. 3). The ELISA signal observed in the
m3-trunk/m3-tail coexpression experiments amounted to approximately
70% of the signal found with lysates prepared from cells expressing
the m3'(wt) receptor.
Correlation of ELISA Signals with Receptor/Receptor Fragment
Expression Levels--
We next wanted to verify that the magnitude of
the ELISA signals observed in the coexpression experiments directly
correlated with the amount of expressed receptor polypeptides. Toward
this goal, COS-7 cells were cotransfected with increasing amounts of m3-trunk and m3-tail DNA (0.25-4 µg for each fragment). Transfection mixtures were supplemented with vector DNA (pcD-PS) to keep the amount
of transfected plasmid DNA constant at 8 µg. Analogous experiments
were carried out with the m3'(wt) receptor.
Initially, protein expression was studied via Western blotting analysis
(Fig. 4). For these studies, the m3-trunk
and m3-tail fragments were detected with the 12CA5 anti-HA monoclonal
and the anti-C-m3 polyclonal antibodies, respectively (expression of
the m3'(wt) receptor was probed with both antibodies). All polypeptides
yielded immunoreactive bands of the expected molecular mass (Fig. 4).
To quantitate the intensities of these bands, blots were subjected to
scanning densitometry. The results of these measurements are summarized
in Fig. 4, in which protein expression levels are expressed in
arbitrary units. Fig. 4 illustrates that the expression levels of
m3-trunk and m3-tail as well as of m3'(wt) increased in an almost
linear fashion with the amount of transfected receptor/polypeptide
DNA.
Similarly, OD values determined via sandwich ELISA strictly correlated
with the amount of transfected receptor/fragment DNA, as found with
m3'(wt)-expressing cells as well as cells cotransfected with m3-trunk
and m3-tail (Fig. 5A).
Likewise, radioligand binding studies showed that the number of
detectable [3H]NMS binding sites
(Bmax) increased with the amount of transfected receptor/polypeptide DNA (Fig. 5B). Taken together, these
results demonstrate that the employed sandwich ELISA allows
measurements that correlate well with the amount of expressed
receptors/receptor fragments.
Specificity of Fragment Association--
To study the specificity
of the interaction between the m3-trunk and m3-tail fragments, an
additional series of ELISA experiments were conducted. Initially, COS-7
cells were cotransfected with the m3-tail fragment and m3-trunk,
V2-trunk, or GnRH-trunk. V2-trunk and GnRH-trunk are fragments derived
from the human V2 vasopressin (16) and GnRH receptors (17),
respectively, and are structurally homologous to m3-trunk. Like
m3-trunk, these two polypeptides also carried an HA epitope tag at
their N terminus. For this first set of experiments, ELISA plates
coated with the anti-C-m3 antibody were used. As shown in Fig. 3,
coexpression of m3-tail with V2-trunk or GnRH-trunk led to marked
reductions in OD readings (OD = 0.049 ± 0.002 and 0.043 ± 0.003, respectively) in the sandwich ELISA, as compared with cells
coexpressing m3-trunk and m3-tail. Interestingly, when the muscarinic
agonist carbachol (1 mM) was added to lysates prepared from
cells cotransfected with m3-trunk and m3-tail, a small (about 10%) but
significant (p < 0.01) increase in ELISA signals was
observed (Fig. 3). This effect was not observed in the presence of the
vasopressin receptor agonist, arginine-vasopressin (AVP; 10 µM). Similarly, addition of carbachol or AVP to lysates prepared from cells coexpressing m3-tail and V2-trunk (or GnRH-trunk) did not lead to a significant increase in OD readings (Fig. 3).
To exclude the possibility that the pronounced reduction in ELISA
signals observed with cells coexpressing m3-tail and V2-trunk (or
GnRH-trunk) was due to poor expression of V2-trunk (as compared with
m3-trunk) and to further examine the receptor specificity of fragment
association, we carried out a second set of sandwich ELISA studies
(Fig. 6). In this case, COS-7 cells were
cotransfected with V2-tail (a human V2 vasopressin receptor fragment
that is structurally homologous to m3-tail; Ref. 16) and V2-trunk,
m3-trunk, or GnRH-trunk. For these studies, plates were coated with a
rabbit polyclonal antibody directed against the C-terminal segment of the human V2 receptor (anti-C-V2; Ref. 16) (all other steps were
identical to those outlined in Fig. 2). As shown in Fig. 6,
coexpression of V2-tail with V2-trunk resulted in robust ELISA signals
(OD = 0.310 ± 0.008), which were about 10-fold greater than
those determined with cells transfected with V2-trunk or vector DNA
alone. In contrast, ELISA signals were drastically reduced when lysates
prepared from cells cotransfected with V2-tail and m3-trunk or
GnRH-trunk were analyzed (OD = 0.056 ± 0.002 and 0.053 ± 0.003, respectively). Addition of the agonists AVP (10 µM) or carbachol (1 mM) had no significant
effect on these residual responses. However, addition of AVP (10 µM) to lysates prepared from cells coexpressing V2-tail
and V2-trunk led to a small (about 10%) but significant
(p < 0.01) increase in OD readings (Fig. 6). In
contrast, OD readings determined with V2-trunk/V2-tail-cotransfected cells remained unaffected upon addition of the muscarinic agonist, carbachol (1 mM) (Fig. 6).
Importance of Conserved Proline Residues for m3 Muscarinic Receptor
Assembly--
It is generally believed (though not proven directly
experimentally) that some of the residues that are highly conserved
among GPCRs are important for proper receptor assembly. We therefore speculated that mutational modification of these residues should interfere with the efficient association between N- and C-terminal receptor fragments, as probed via sandwich ELISA.
To test this hypothesis, we initially focused our attention on a set of
three proline residues that are present not only in the m3 muscarinic
receptor but in almost all GPCRs of the rhodopsin family (4, 5). As
shown in Fig. 1, these proline residues are located in TM V
(Pro242), TM VI (Pro505), and TM VII
(Pro540). We first created three m3 receptor fragments in
which these proline residues were individually replaced with alanine,
resulting in P242A-m3-trunk, P505A-m3-tail, and P540A-m3-tail. We then
coexpressed these mutant fragments with m3-tail (P242A-m3-trunk) or
m3-trunk (P505A-m3-tail and P540A-m3-tail) and monitored fragment
association via ELISA. As outlined above, lysates prepared from COS-7
cells coexpressing the m3-trunk and m3-tail polypeptides resulted in a
robust increase in OD readings (9.5-fold above values determined for
cells expressing m3-trunk alone; Fig. 7).
In contrast, ELISA signals were found to be drastically reduced when
one of the cotransfected receptor fragments contained a Pro
Radioligand binding studies yielded results that were in good agreement
with the ELISA data. Fig. 8 shows that
the number of detectable [3H]NMS binding sites
(Bmax) was strongly reduced (by 3.3-8.7-fold, as compared with m3-trunk/m3-tail-expressing cells) in coexpression experiments that included fragments containing a Pro
In order to exclude the possibility that the reduced signals seen in
the coexpression experiments that included m3 receptor fragments
containing Pro Effect of Ligands on the Assembly of m3 Receptor Fragments
Containing Pro Effect of Other Point Mutations on the Assembly of N- and
C-terminal m3 Receptor Fragments--
To examine the specificity of
the detrimental effects of the different Pro
To examine the effect of the different mutations on m3 receptor
assembly, sandwich ELISA measurements were carried out using lysates
prepared from cells coexpressing a mutant m3-trunk or m3-tail
polypeptide and the corresponding non-mutated N- or C-terminal m3
receptor fragment. These studies showed that the majority of the
mutations had little or no effect on proper fragment assembly (Fig.
10). All mutant fragments (with the exception of Y529F- and Y533F-m3-tail, which led to 32% and 45% reductions in ELISA signals, respectively) gave OD readings that were similar to those found with
lysates prepared from m3-trunk/m3-tail-expressing cells. Moreover,
addition of the agonist, carbachol (1 mM), or the
antagonist, NMS (0.1 µM), led to small increases in
ELISA signals (by approximately 10%; p < 0.01) in all
coexpression experiments (again with the exception of Y529F-
and Y533F-m3-tail) (Fig. 10).
Studies with split GPCRs have shown that GPCRs can be assembled
from two or more coexpressed receptor fragments (11-19). In this
study, we have examined the association between two m3 muscarinic receptor fragments, referred to as m3-trunk and m3-tail (Fig. 1), as a
model system. Consistent with previous studies (12, 14), coexpression
of these two polypeptides in COS-7 cells resulted in the
"reconstitution" of a significant number of high affinity binding
sites for the muscarinic radioligand, [3H]NMS.
To examine the interaction between the m3-trunk and m3-tail fragments
in a more direct fashion, we employed a sandwich ELISA using three
different antibodies. The first antibody, a rabbit polyclonal antibody
referred to as anti-C-m3, was used for the coating of microtiter plates
and allowed the capture of the m3-tail fragment from cell lysates
prepared from cotransfected COS-7 cells. The second antibody was a
mouse monoclonal antibody (12CA5) that was able to detect the HA
epitope tag that had been added to the N terminus of the m3-trunk
polypeptide. Addition of the third antibody, an anti-mouse IgG antibody
linked to horseradish peroxidase, allowed the detection of the m3-trunk
polypeptide by a simple enzymatic reaction.
Cell lysates prepared from cells transfected with the m3-trunk
polypeptide or vector DNA alone gave only residual ELISA signals (OD
measured at 490 nm). However, a robust increase (9.5-fold) in the
magnitude of ELISA signals was observed when cells coexpressing m3-trunk and m3-tail were analyzed (Fig. 3). These observations indicate that the employed sandwich ELISA provides a sensitive and
convenient experimental system to study the association between the
m3-trunk and m3-tail fragments.
To further investigate the potential usefulness of the sandwich ELISA
for studying mechanisms of GPCR assembly, we next examined whether the
strengths of the ELISA signals correlated with actual amounts of
coexpressed receptor fragments. For these studies, COS-7 cells were
transfected with increasing amounts of m3-trunk and m3-tail DNA,
followed by the preparation of cell lysates and Western blotting
analysis to monitor polypeptide expression levels. These studies showed
that increasing the amount of transfected fragment DNA led to almost
linear increases in the expression levels of the m3-trunk and
m3-tail fragments (Fig. 4).
Sandwich ELISA measurements showed that the observed increases in
polypeptide expression levels led to gradual increases in the number of
detectable m3-trunk/m3-tail complexes (Fig. 5A). A similar
correlation was also found in [3H]NMS saturation binding
studies (Fig. 5B). Taken together, these findings indicate
that the sandwich ELISA employed here provides a highly sensitive
method to monitor the efficiency of fragment (GPCR) assembly.
Another question that we addressed in a set of initial experiments was
related to the receptor specificity of fragment assembly. For these
studies, COS-7 cells were cotransfected with the m3-tail polypeptide
and two different N-terminal GPCR fragments referred to as V2-trunk and
GnRH-trunk. These latter two fragments were derived from the human V2
vasopressin (16) and GnRH receptors (17), respectively, and are
structurally homologous to m3-trunk (note that all "trunk
fragments" contained an N-terminal HA tag). ELISA measurements showed
that the m3-tail polypeptide was unable to interact with V2-trunk and
GnRH-trunk in an efficient manner (Fig. 6). To exclude the possibility
that the low ELISA signals seen in these experiments was due to reduced
expression levels of V2-trunk (or GnRH-trunk), measurements were
also carried out with cells coexpressing V2-trunk and V2-tail (a
fragment that is structurally homologous to m3-tail; Ref. 16). For
these experiments, plates were coated with a rabbit polyclonal antibody
(anti-C-V2; Ref. 16) directed against the C terminus of the V2
receptor. These studies demonstrated that V2-trunk and V2-tail were
able to interact with each other with high efficiency. In contrast, ELISA signals were very weak when cells cotransfected with
V2-tail and m3-trunk or GnRH-trunk were analyzed. In sum, these
findings convincingly show that efficient association of receptor
fragments is highly receptor-specific.
Currently, little is known about the structural mechanisms required to
maintain GPCRs in a functionally competent fold. We therefore decided
to use the sandwich ELISA outlined above to identify amino acids in the
m3 muscarinic receptor that are critical for proper receptor assembly.
Initially, we focused our attention on a set of three conserved proline
residues (Pro242, Pro505, and
Pro540) that are present not only in the m3 muscarinic
receptor but in almost all GPCRs of the rhodopsin family (4, 5). A
previous study (22) had shown that individual replacement of these
prolines with alanine residues in the full-length m3 receptor resulted in a pronounced reduction in specific radioligand binding sites (Bmax). Based on these results, we speculated
that the three conserved proline residues might be required for the
proper assembly of the transmembrane receptor core.
To test this hypothesis, mutant m3-tail or m3-trunk fragments were
created in which the three conserved prolines were individually substituted with alanine residues. Cell lysates prepared from cells
cotransfected with a mutant form of m3-trunk and the non-mutated m3-tail polypeptide (or vice versa) were then analyzed via
sandwich ELISA. These studies showed that each of the three Pro Based on the unique structure and geometry of the proline side chain,
proline residues are predicted to introduce "kinks" into TM
helices, resulting in an altered direction and/or orientation of the
helical backbones (29-31). It is therefore likely that the mutated
proline residues (Pro242, Pro505, and
Pro540) do not directly participate in establishing
interhelical contact sites but that their mutational modification
results in impaired helix-helix interactions due to indirect structural effects.
To examine the specificity of the detrimental effects of the different
Pro In another set of experiments, we examined the effect of different
classes of muscarinic ligands (agonists, antagonists, or allosteric
ligands) on the efficiency of association between the m3-trunk and
m3-tail polypeptides. Sandwich ELISA measurements using cells
cotransfected with m3-trunk and m3-tail showed that all ligands
investigated (except TMA, which left ELISA signals virtually
unaffected) led to small but significant increase in OD readings (by
approximately 10%). This effect was considerably more pronounced
(increase in specific ELISA signals by approximately 300-500%) when
one of the cotransfected fragments carried a Pro Interestingly, allosteric ligands such as alcuronium (33, 34),
gallamine (35, 36), and eburnamonine (28) promoted m3 receptor fragment
assembly in a fashion similar to classical muscarinic agonists and
antagonists. Such ligands can modulate the binding of classical
muscarinic agents to the primary muscarinic binding site (located
within the TM helical bundle) via interaction with a secondary
(allosteric) binding site (37). This allosteric site is predicted to be
located "extracellularly" of the primary (classical) ligand
recognition site and is thought to involve residues located on the
second and third extracellular loops and exofacial portions of adjacent
TM helices (38-40). Our observation that all studied allosteric
ligands were able to facilitate the interaction between the m3-trunk
and m3-tail polypeptides, especially when fragment association was
impaired due to Pro It should be noted that the partial muscarinic agonist TMA, a rather
small positively charged ammonium compound, failed to promote the
interaction between the m3-trunk and m3-tail polypeptides, even in the
presence of the different Pro In conclusion, our results provide novel insight into the structural
basis of GPCR assembly. Given the fact that all GPCRs share a similar
molecular architecture, the experimental approach outlined here should
be generally applicable.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Ala mutations.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
)-eburnamonine (Sigma), gallamine triethiodide
(Sigma), (
)-N-methylscopolamine bromide (NMS; Sigma),
(
)-quinuclidinyl benzilate (QNB; Research Biochemicals Inc.), and
tetramethylammonium chloride (Sigma).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Transmembrane topology of the rat m3
muscarinic receptor. The structures of the m3-trunk and m3-tail
fragments (which were generated by "splitting" the rat m3
muscarinic receptor within the i3 loop; Refs. 12 and 14) are indicated.
Residues that were targeted by site-directed mutagenesis are
boxed. The three Pro residues (Pro242,
Pro505, and Pro540) that are highly conserved
among GPCRs of the rhodopsin family are highlighted in
black. An HA epitope tag (underlined) was present
at the N terminus of the full-length m3 receptor and the m3-trunk
polypeptide. The m3'(wt) receptor (which served as a control because it
could be detected easily via immunoblotting; see text for details)
lacked the central portion of the i3 loop (amino acids
Ala274-Lys469) (25). A rabbit polyclonal
antibody (referred to as anti-C-m3) was raised against the indicated
C-terminal receptor sequence. Numbers refer to amino acid
positions in the rat m3 muscarinic receptor sequence (41).
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Fig. 2.
Sandwich ELISA strategy for studying the
association between N- and C-terminal m3 muscarinic receptor
fragments. The ELISA protocol used for investigating interactions
between the m3-trunk and m3-tail polypeptides (for structures, see Fig.
1) consisted of the following major steps (see text for experimental
details). 1) 96-well-plates were coated with a rabbit polyclonal
antibody (anti-C-m3) directed against the C-terminal segment of the rat
m3 muscarinic receptor (highlighted in black). 2) Membrane
lysates prepared from COS-7 cells cotransfected with m3-trunk and
m3-tail were added to the coated plates. The lysates had been
preincubated with a monoclonal antibody directed against the N-terminal
HA epitope tag (hatched) and a secondary anti-mouse IgG
antibody, linked to horseradish peroxidase (POD). 3)
Peroxidase activity was determined by a simple color reaction following
the addition of substrate (H2O2) and chromogen
(o-phenylenediamine) (for further details, see
"Experimental Procedures").
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Fig. 3.
Assembly of m3 muscarinic receptor fragments
studied via ELISA: signal specificity and agonist effects. COS-7
cells were cotransfected with m3-tail and m3-trunk, V2-trunk (a V2
vasopressin receptor fragment that is structurally homologous to
m3-trunk; Ref. 16), or GnRH-trunk (a GnRH receptor fragment that is
structurally homologous to m3-trunk; Ref. 17) (4 µg of
DNA/plate/plasmid). Sandwich ELISA measurements were carried out as
described under "Experimental Procedures," using 96-well plates
coated with the anti-C-m3 polyclonal antibody (see Figs. 1 and 2).
Assays were carried out in the presence or absence of the agonists
carbachol (CBC, 1 mM) or AVP (10 µM). Data are presented as means ± S.E. of three or
four independent experiments, each performed in quadruplicate. *,
p < 0.01 (paired t test).
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Fig. 4.
Correlation between m3 receptor fragment
expression levels and amount of transfected plasmid DNA as studied via
Western blot analysis. COS-7 cells were transfected with
increasing amounts of m3'(wt) (0.25-4 µg) (filled
circles) or cotransfected with m3-trunk and m3-tail DNA
(0.25-4 µg for each fragment) (open circles).
Transfection mixtures were supplemented with vector DNA to keep the
amount of transfected plasmid DNA constant at 4 µg (m3'(wt)) or 8 µg (m3-trunk/m3-tail), respectively. Immunoblotting experiments were
carried out as described under "Experimental Procedures." Membrane
proteins were separated by 10% (A) or 15% (B)
SDS-PAGE, followed by Western blotting and scanning densitometry of
immunoreactive bands (see insets). A, the
full-length receptor (m3'(wt); filled circles)
and the m3-trunk fragment (open circles) were
visualized by using the 12CA5 anti-HA monoclonal antibody.
B, the m3-tail polypeptide (open
circles) and the m3'(wt) receptor (filled
circles) were detected with the anti-C-m3 polyclonal
antibody. The estimated molecular masses of the immunoreactive bands
were (in kDa): m3'(wt) (48-52), m3-trunk (26-28), and m3-tail
(18-20). The presented data are taken from one representative
experiment; two additional experiments gave similar results.
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Fig. 5.
Assembly of m3 receptor fragments studied via
sandwich ELISA and radioligand binding studies. COS-7 cells were
transfected with increasing amounts of m3'(wt) (filled
circles) or cotransfected with m3-trunk and m3-tail DNA
(open circles), as described in the legend to
Fig. 4. A, sandwich ELISA measurements were carried out as
described under "Experimental Procedures" (see also Fig. 2). OD
readings were taken at 490 nm. Data are presented as means (S.E. < 5%
for all data points) of three independent experiments, each performed
in quadruplicate. B, Bmax values were
determined in [3H]NMS saturation binding studies as
described under "Experimental Procedures." Data are presented as
means (S.E. < 5% for all data points) of three or four independent
experiments, each performed in triplicate.
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Fig. 6.
Assembly of V2 vasopressin receptor fragments
studied via ELISA: signal specificity and agonist effects. COS-7
cells were cotransfected with V2-tail (a V2 vasopressin receptor
fragment that is structurally homologous to m3-tail; Ref. 16) and
V2-trunk, m3-trunk, or GnRH-trunk. Sandwich ELISA measurements were
carried out as described under "Experimental Procedures." In this
set of experiments, ELISA plates were coated with a rabbit polyclonal
antibody (anti-C-V2) directed against the C-terminal segment of the V2
receptor (16). Assays were carried out in the presence or absence of
the agonists AVP (10 µM) or carbachol (CBC, 1 mM). Data are presented as means ± S.E. of three or
four independent experiments, each performed in quadruplicate. *,
p < 0.01 (paired t test).
Ala
point mutation. As shown in Fig. 7, the remaining responses were only
about 2-fold greater than background values.
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Fig. 7.
Interaction of coexpressed N- and C-terminal
m3 receptor fragments carrying Pro Ala point mutations studied via
ELISA. COS-7 cells were transfected with the indicated plasmids (4 µg of DNA/plate/plasmid). Sandwich ELISA measurements were carried
out as described under "Experimental Procedures," using 96-well
plates coated with the anti-C-m3 polyclonal antibody. Assays were
carried out in the presence or absence of the full agonist carbachol (1 mM), the partial agonist TMA (10 mM), the
antagonists NMS (0.1 µM) and QNB (0.1 µM),
and the allosteric ligands alcuronium (0.1 mM), gallamine
(10 µM), and eburnamonine (0.1 mM). Data are
presented as means ± S.E. of three or four independent
experiments, each performed in quadruplicate. *, p < 0.01 (paired t test).
Ala mutation. These remaining [3H]NMS binding sites were characterized
by KD values similar to those obtained with cells
cotransfected with m3-trunk and m3-tail (m3-trunk/m3-tail, 130 ± 11 pM; m3-trunk/P505A-m3-tail, 171 ± 16 pM; m3-trunk/P540A-m3-tail, 148 ± 13 pM;
P242A-m3-trunk/m3-tail, 205 ± 23 pM).
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Fig. 8.
[3H]NMS binding studies using
cells coexpressing m3 receptor fragments carrying Pro Ala point
mutations. COS-7 cells were cotransfected with the indicated
m3-trunk and m3-tail fragments (4 µg of DNA/plate/plasmid).
Bmax values were determined in
[3H]NMS saturation binding studies as described under
"Experimental Procedures." Data are presented as means ± S.E.
of three independent experiments, each performed in triplicate.
Ala point mutations were due to lowered expression
of the mutant polypeptides, expression of the different mutant m3
receptor fragments was monitored via Western blotting analysis.
Polypeptides were visualized by immunoblotting using either the 12CA5
anti-HA monoclonal antibody (m3-trunk fragments) or the anti-C-m3
polyclonal antibody (m3-tail fragments). As shown in Fig.
9, all polypeptides yielded
immunoreactive bands of the expected molecular mass. Moreover, the
receptor fragments containing the indicated Pro
Ala mutations were
found to be expressed at similar levels as the corresponding
non-mutated fragments (Fig. 9). Scanning densitometry of the
immunoreactive bands revealed no significant differences in the
expression levels of the mutated versus the non-mutated
receptor polypeptides (data not shown).
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Fig. 9.
Expression of m3 receptor fragments carrying
Pro Ala point mutations studied via Western blotting. COS-7
cells were transfected with the following plasmids (4 µg each):
1 (m3'(wt)), 2 (m3-trunk + m3-tail), 3 (m3-trunk + P540A-m3-tail), 4 (m3-trunk + P505A-m3-tail),
5 (P242A-m3-trunk + m3-tail), 6 (m3-tail), and
7 (empty vector; pcD-PS). Membrane extracts were prepared
about 70 h later and subjected to 10% (A) or 15%
(B) SDS-PAGE. Polypeptides were visualized via
immunoblotting using the 12CA5 anti-HA monoclonal antibody
(A) or the anti-C-m3 polyclonal antibody (B) (the
receptor epitopes recognized by the two antibodies are indicated in
Fig. 1). Protein molecular size standards (in kDa) are indicated. Two
additional experiments gave similar results.
Ala Mutations--
We next wanted to examine
whether ligands were able to promote fragment association in
coexpression experiments that included m3 receptor fragments containing
Pro
Ala point mutations. For these studies, four different types of
ligands were used: the full agonist carbachol (1 mM), the
partial agonist tetramethylammonium (TMA, 10 mM), the
antagonists NMS (0.1 µM) and QNB (0.1 µM),
and the allosteric ligands alcuronium (0.1 mM), gallamine
(10 µM), and eburnamonine (0.1 mM; Ref. 28).
When added to lysates prepared from cells cotransfected with m3-trunk
and m3-tail, all ligands, except TMA, gave small (approximately 10%)
but significant (p < 0.01) increases in OD readings in
the sandwich ELISA (Fig. 7). Interestingly, this
ligand-dependent increase in ELISA signals was greatly
enhanced when one of the two cotransfected m3 receptor fragments
contained a Pro
Ala point mutation (P242A, P505A, or P540A). In
this case, all ligands (except TMA) led to OD readings that, when
corrected for background "noise" (determined with
m3-trunk-expressing cells), were increased by about 300-500%, as
compared with the values determined in the absence of ligands (Fig.
7).
Ala point mutations on
proper fragment assembly, we next introduced a variety of other point
mutations into either m3-trunk or m3-tail. Subsequently, we measured
the ability of the resulting mutant polypeptides to associate with
m3-tail (in the case of mutant m3-trunk fragments) or m3-trunk (in the
case of mutant m3-tail fragments) via sandwich ELISA. Three different types of mutations were included in this analysis, based on their effects on the function of the full-length m3 receptor. Two of the
mutations (I253Y/Y254I and 493+1A; for mutant receptor structures, see
legends to Figs. 1 and 10) have been
shown to completely disrupt m3 receptor/G protein coupling while having
little effect on ligand binding (23, 24). The second group comprises
mutations that are known to reduce agonist binding affinities but still
allow receptor/G protein coupling (Y148F, T231A, T234A, Y506F, Y529F, and Y533F; Refs. 20 and 21). Finally, two additional point mutations
(T502A and T537A) were included (for control purposes) that have
virtually no effect on the ligand binding and functional properties of
the full-length m3 receptor (20).
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Fig. 10.
Interaction of coexpressed mutant m3
receptor fragments studied via ELISA. COS-7 cells were
cotransfected with non-mutated m3-trunk and m3-tail fragments
(A, control) or the indicated mutant versions of m3-tail and
m3-trunk (B-D) (4 µg of DNA/plate/plasmid). Sandwich
ELISA measurements were carried out as described under "Experimental
Procedures" (using 96-well plates coated with the anti-C-m3
polyclonal antibody), either in the presence or absence of the agonist
carbachol (1 mM) or the antagonist NMS (0.1 µM). Three types of mutations were studied: B,
mutations known to disrupt receptor/G protein interactions in the
full-length m3 receptor (in I253Y/Y254I-m3-trunk, the positions of
Ile253 and Tyr254 were reversed (23); in
(493+1A)-m3-tail, an extra alanine residue was inserted directly after
Ser493 (24)); C, point mutations that reduce
agonist binding affinities in the full-length m3 receptor (20, 21);
D, point mutations that have no effect on the ligand binding
and functional properties of the full-length receptor (20). Data are
presented as means ± S.E. of three or four independent
experiments, each carried out in quadruplicate. *, p < 0.01 (paired t test).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Ala
mutations led to pronounced reductions in ELISA signals (by 6-19-fold
as compared with cells cotransfected with non-mutated m3-trunk and m3-tail fragments) (Fig. 7). This decrease in the magnitude of ELISA
signals was accompanied by strong reductions in the number of
detectable radioligand binding sites (Bmax)
(Fig. 8). Western analysis indicated that the polypeptides containing
the different Pro
Ala point mutations were expressed at similar
levels as the non-mutated m3-trunk and m3-tail fragments (Fig. 9).
Taken together, these results provide the first direct evidence that the three conserved proline residues play key roles in m3 muscarinic receptor assembly.
Ala point mutations on the interaction between m3-trunk and
m3-tail, we subsequently screened a larger number of m3 receptor point
mutations for their ability to interfere with proper fragment assembly.
These mutations were chosen based on their known effects on the
function of the full-length m3 receptor (20-24). As expected,
mutations that had no effect on the ligand binding and G
protein-coupling properties of the wild type receptor (T502A and T537A)
also did not interfere with the proper assembly between m3-trunk and
m3-tail fragments (as studied via ELISA; Fig. 10). Similar results were
obtained when fragments were used for coexpression studies that
contained mutations known to selectively disrupt receptor/G protein
coupling (I253Y/Y254I and 493+1A; for details regarding the structure
of these mutant receptors, see Fig. 1 and Refs. 23 and 24). This latter
observation excludes the possibility that fragment assembly requires
association of the fragment complex with G proteins. Finally, we
examined the effects of point mutations known to selectively reduce the
binding affinities of the neurotransmitter acetylcholine and other
muscarinic agonists (Y148F, T231A, T234A, Y506F, Y529F, and Y533F;
Refs. 20 and 21). ELISA measurements showed that these mutations (except for Y529F and Y533F, which caused 32% and 45% reductions in
OD readings, respectively), had little or no effect on the efficiency
of assembly between the m3-trunk and m3-tail polypeptides (Fig. 10),
indicating that these mutations do not interfere with the stable
assembly of the TM receptor core. This observation is consistent with
the view that these residues are predicted to project into the interior
of the TM helical bundle (where they can interact with agonist ligands)
and are not engaged in direct helix-helix interactions (4, 5, 20, 21).
In sum, these studies strongly support the notion that the ability of
the P242A, P505A, and P540A substitutions to interfere with m3
muscarinic receptor assembly is highly specific for these mutations.
More generally, our data indicate that the sandwich ELISA employed here
can be used to assess whether or not a specific residue plays a
critical role in proper receptor assembly.
Ala mutation (see
above) that were shown to interfere with efficient fragment assembly
(Fig. 7). A likely explanation for this observation is that muscarinic
ligands can act as anchors to stabilize fragment association by
contacting residues that are located on both the m3-trunk and m3-tail
fragments and that this stabilizing effect becomes more pronounced when
receptor assembly is impaired due to "destabilizing" mutations.
This view is consistent with a great number of mutagenesis studies
suggesting that classical muscarinic agonists and antagonists bind to
their target receptors by contacting many different amino acids located
on different TM domains (primarily TM III, V, VI, and VII; see Ref. 32
for a review).
Ala mutations, provides more direct evidence
for the notion that multiple extracellular epitopes are involved in the
recognition of this class of compounds.
Ala point mutations. Mutagenesis
studies (21) suggest that TMA primarily interacts with a conserved TM
III aspartate residue on the m3 receptor protein (Asp147 in
m3-trunk; Fig. 1) and that TMA binding does not critically depend on
residues located on TM VI and VII (which are contained in m3-tail).
This observation provides an explanation for the inability of TMA to
stabilize the interaction between the m3-trunk and m3-tail fragments.
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ACKNOWLEDGEMENTS |
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We thank Dr. Torsten Schöneberg for advice and for providing the GnRH-trunk expression construct and Dr. Paul Goldsmith for the kind gift of the anti-C-V2 polyclonal antibody.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Laboratory of
Bioorganic Chemistry, NIDDK, Bldg. 8A, Rm. B1A-05, Bethesda, MD 20892. Tel.: 301-402-3589; Fax: 301-402-4182; E-mail:
jwess{at}helix.nih.gov.
The abbreviations used are: GPCR, G protein-coupled receptor; AVP, arginine-vasopressin; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; GnRH, gonadotropin-releasing hormone; HA, hemagglutinin; i1-i3, the three intracellular loops of GPCRs; NMS, N-methylscopolamine; o1-o4, the four extracellular domains of GPCRs; OD, optical density; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.05% Tween 20; PBS-T-BSA, PBS-T containing 1% BSA; QNB, quinuclidinyl benzilate; TM, transmembrane; TM I-VII, the seven transmembrane domains of GPCRs; TMA, tetramethylammonium; wt, wild type.
2 F.-Y. Zeng and J. Wess, unpublished observations.
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REFERENCES |
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