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(Received for publication, January 23,
1995; and in revised form, May 31, 1995) From the
To test the hypothesis that G protein-coupled receptors consist
of multiple autonomous folding domains, the rat m3 muscarinic
acetylcholine receptor was ``split'' in all three
intracellular (i1-i3) and all three extracellular loops (o2-o4). The
six resulting polypeptide pairs (N G protein-coupled receptors (GPCRs)
Figure 1:
Structure of truncated m3 muscarinic
receptors. A, the rat m3 muscarinic receptor (Bonner et
al., 1987) was split (arrows) in all three intracellular
(i1-i3) and all three extracellular loops (o2-o4), resulting in six
pairs of fragmented receptors. B, structure of fragmented m3
muscarinic receptors. The positions of the seven transmembrane domains
(I-VII) are indicated. An HA-epitope tag was added to the N terminus of
the wild type receptor (m3-N-HA) and all fragments of the N-series
(N
We have recently
shown that muscarinic receptors (which are typical GPCRs) behave
structurally in a fashion analogous to two-subunit receptors (Maggio et al., 1993a, 1993b). When truncated m2 or m3 muscarinic
receptors (containing TM I-V) were coexpressed in COS-7 cells with
their corresponding C-terminal receptor segments (containing TM VI and
VII), functional muscarinic receptors were obtained. Similar findings
have also been described for ``split'' In this study, we have tested the hypothesis that GPCRs may consist
not only of two but of multiple autonomous folding units. To this goal,
the rat m3 muscarinic receptor (which was used as a model system) was
split in all three intracellular (i1-i3) and all three extracellular
loops (o2-o4). The six resulting polypeptide pairs
(N The immunological studies
showed that all N- and C-terminal receptor fragments examined (except
N
Binding data were
analyzed by nonlinear least squares curve-fitting procedures, using the
computer program LIGAND (saturation binding data; Munson and
Rodbard(1980)) or KALEIDAGRAPH (competition binding data; Synergy
Software), respectively.
Figure 2:
Immunocytochemical localization of
epitope-tagged wild type and truncated m3 muscarinic receptors. COS-7
cells were transfected with DNA constructs coding for m3-N-HA (a wild
type m3 receptor epitope-tagged at the N terminus; A and B), m3-C-HA (a wild type m3 receptor epitope-tagged at the C
terminus; C and D), and N
Figure 3:
Immunocytochemical localization of m3
muscarinic receptor fragments. COS-7 cells were transfected with DNA
constructs coding for N
Moreover, an HA-epitope was also added to the
C terminus of two of the N-terminally truncated receptor fragments,
C We next examined the
ability of an affinity-purified polyclonal antibody (prepared by W. F.
Simonds) To quantify the amount of C-terminally truncated
receptor fragments (N The m3-N-HA construct was expressed at different receptor
densities (B
Figure 4:
Relationship between m3 muscarinic
receptor (m3-N-HA) density and extinction determined in an indirect
cellular ELISA system. COS-7 cells were transfected in 100-mm dishes
with increasing amounts of m3-N-HA receptor DNA (0.125-4 µg,
supplemented with vector DNA to keep the amount of transfected plasmid
DNA constant at 4 µg). For ELISA measurements, cells were split
into 96-well plates about 24 h after transfections, and the remaining
cells were grown for saturation binding assays. ELISA and
[
Based on this
finding, ELISA experiments were carried out with nonpermeabilized
COS-cells individually transfected with receptor fragments of the N
series containing an HA-epitope at their N termini
(N
The results of the [
Figure 5:
Carbachol-induced PI hydrolysis mediated
by fragmented m3 muscarinic receptors coexpressed in COS-7 cells. The
following DNA constructs were used for transfection of COS-7 cells (see Fig. 1for structure of encoded receptor fragments): m3-N-HA (A), N
Figure 6:
Carbachol-induced PI hydrolysis mediated
by the N
To test the hypothesis that GPCRs consist of multiple
structural subunits (folding units), the rat m3 muscarinic receptor was
split in all three intracellular (i1-i3) and all three extracellular
loops (o2-o4), thus generating six polypeptide pairs
(N In contrast to all other
C-terminally truncated receptor fragments
(N We found that coexpression of
N To address the question of
which fraction of the N Interestingly,
immunocytochemical studies with two of the C-terminal receptor
fragments, C Functional
studies showed that the N In contrast to
N In contrast to
N Taken together, our data strongly suggest that
muscarinic receptors and, most likely, other GPCRs are composed of
multiple structural/functional subunits. These subunits appear to be
able to fold independently of each other in a fashion such that they
can interact with each other to form a functional receptor complex. The
findings described here obtained with a eucaryotic plasma membrane
protein are consistent with previous studies on a structurally related
bacterial membrane protein, bacteriorhodopsin, which, however, does not
couple to G proteins but functions as a light-driven proton pump (for a
review, see Popot and Engelman, 1990). While the present study was
carried out in an in vivo system, all studies on the
microassembly of bacteriorhodopsin were performed in vitro under conditions clearly different from those found in an intact
cell. It could be shown that bacteriorhodopsin can be functionally
reconstituted from individual receptor fragments resulting from
proteolytic cleavage of various loop regions (Liao et al.,
1983; Popot etal., 1987, Kahn and Engelman, 1992).
Taken together, these findings support the notion that the folding of
both procaryotic and eucaryotic membrane proteins occurs in two
consecutive steps (Popot and Engelman, 1990). In step I, the individual
transmembrane helices are established across the lipid bilayer. The
helices can then, in step II, interact with each other to form a
functional protein complex. It should be mentioned that truncated
versions of GPCRs have also been shown to be of clinical relevance.
Nonsense or frameshift mutations in the V2 vasopressin receptor gene,
for example, which lead to truncations of the receptor protein in
various intracellular and extracellular loops, can be the cause of
X-linked nephrogenic diabetes insipidus (Raymond, 1994). Moreover, a
nonsense mutation within the rhodopsin gene leading to a translational
stop site in the i3 loop has been shown to give rise to autosomal
recessive retinitis pigmentosa, a common hereditary form of retinal
degeneration (Rosenfeld et al., 1992). Based on the results
presented in this study, one may speculate that such clinically
relevant mutant receptors represent targets for novel therapeutic
strategies (e.g. peptide or gene therapy) designed to rescue
the function of these mutant proteins. In conclusion, we could
demonstrate under in vivo conditions that GPCRs are composed
of multiple structural subunits. Our findings should be of general
relevance for understanding the molecular mechanisms underlying
membrane insertion, assembly, and intracellular trafficking of
eucaryotic plasma membrane proteins.
Volume 270,
Number 30,
Issue of July 28, pp. 18000-18006, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C,
N
C, etc.) were coexpressed in COS-7
cells and studied for their ability to bind muscarinic ligands and to
activate G proteins. In addition, immunocytochemical and ELISA studies
were carried out to study the expression and subcellular localization
of the individual receptor fragments. Interestingly, all N- and
C-terminal receptor fragments studied (except N
, which
contained only the first transmembrane domain) were found to be
localized to the plasma membrane, even when expressed alone.
Coexpression of three of the six polypeptide pairs, generated by
splitting the m3 muscarinic receptor in the i2, o3, or i3 loop,
resulted in receptor complexes (N
C,
N
C, and
N
C, respectively), which were able to
bind muscarinic agonists and antagonists with high affinity. The
N
C and N
C polypeptide combinations, but not the
N
C complex, were also able to stimulate
carbachol-dependent phosphatidyl inositol hydrolysis to a similar
maximum extent as the wild type m3 muscarinic receptor. These findings
strongly suggest that G protein-coupled receptors are composed of
several independent folding units and may shed light on the molecular
mechanisms governing receptor assembly and membrane insertion.
()are
integral membrane proteins characterized by the presence of seven
transmembrane helices (TM I-VII) linked by three intracellular (i1-i3)
and three extracellular loops (o2-o4) (Fig. 1). The structural
elements in GPCRs involved in ligand binding and G protein recognition
have been mapped in considerable detail (Dohlman et al., 1991;
Savarese and Fraser, 1992; Strader et al., 1994). In contrast,
little is known about the molecular mechanisms governing proper
membrane insertion and assembly (folding) of GPCRs.
-N
) (see ``Experimental
Procedures''). N
contains only the first 21 amino
acids of the i3 loop of the rat m3 muscarinic receptor
(Arg
-Thr
). A 115-amino acid segment of the
i3 loop (Glu
-Asn
) is contained in neither
N
nor C
but was deleted during the
construction of the two gene fragments. The construction and precise
amino acid composition of the various receptor fragments are described
under ``Experimental Procedures'' (Table 1).
2-adrenergic
receptors coexpressed in Xenopus oocytes (Kobilka et
al., 1988). Based on these findings, we proposed that GPCRs are
composed of at least two independent folding domains, one containing TM
I-V and the other TM VI and VII (Maggio et al., 1993a, 1993b). C,
N
C, etc.; Fig. 1) were
coexpressed in COS-7 cells and studied for their ability to bind
muscarinic ligands and to mediate agonist-dependent stimulation of
phosphatidyl inositol (PI) hydrolysis (through interaction with G
proteins of the G
/G family). In addition,
individual receptor fragments were epitope-tagged at their N and C
termini, respectively, to allow their subcellular localization to be
studied by confocal immunofluorescence microscopy and a newly
developed, indirect cellular ELISA system.
; Fig. 1) were inserted into the plasma membrane,
even when expressed alone. The results of the coexpression experiments
strongly suggest that muscarinic receptors and, most likely, other
GPCRs are composed of multiple structural subunits.
DNA Constructs
All mutations were introduced
into Rm3pcD, a mammalian expression vector containing the entire coding
sequence of the rat m3 muscarinic receptor (Bonner et al.,
1987), using standard polymerase chain reaction (PCR) mutagenesis
techniques (Higuchi, 1989). To allow the detection of receptor protein
in immunological assays, a stretch of nucleotides coding for a
nine-amino acid epitope (YPYDVPDYA) (Kolodziej and Young, 1991) derived
from the influenza virus hemagglutinin protein (HA-epitope) was
inserted after the initiating Met codon (yielding Rm3pcD-N-HA) or
before the translation stop codon of Rm3pcD (yielding Rm3pcD-C-HA),
respectively. For the construction of truncated m3 receptors of the
``N series'' (N-N
; Fig. 1, Table 1), stop codons (TGA) were inserted at the appropriate
positions in Rm3pcD-N-HA. The newly created stop codons were linked to
a unique SstI site in the 3`-untranslated region of
Rm3pcD-N-HA. For the construction of truncated m3 receptors of the
``C series'' (C
-C
; Fig. 1, Table 1), PCR primers were designed to contain a PstI
site at their 5`-ends (to link the various PCR fragments to the PstI site at position 3154 of the pcD vector), followed by six
bases of 5`-untranslated sequence adjacent to the ATG translation
initiation codon in Rm3pcD (GTCACA) and an in-frame ATG start codon.
For immunological studies, C
and C
were
epitope-tagged at their C termini by subcloning a 1.31-kilobase pair
Rm3pcD-C-HA NheI-SstI restriction fragment containing
the HA-epitope into these mutant constructs. The identity of the
various constructs and the correctness of all PCR-derived sequences
were verified by restriction endonuclease analysis and by dideoxy
sequencing of the mutant plasmids.
Transient Expression of Mutant Muscarinic
Receptors
COS-7 cells were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum
(FCS) at 37 °C in a humidified 5% CO
incubator. For
transfections, 2 10
cells were seeded into 100-mm
dishes. About 24 h later, cells were transfected with 4 µg of
plasmid DNA/dish by a DEAE-dextran method (Cullen, 1987).Membrane Preparation and Radioligand Binding
Assays
For radioligand binding studies, transfected COS-7 cells
were harvested 48-72 h after transfections. Binding assays were
carried out with membrane homogenates prepared from transfected COS-7
cells essentially as described (Drje et
al., 1991). Samples were incubated for 3 h at 22 °C in a 1-ml
volume. Incubation buffer consisted of 25 mM sodium phosphate
(pH 7.4) containing 5 mM MgCl. In N-[
H]methylscopolamine
([H]NMS, 81.4 Ci/mmol; DuPont NEN) saturation
binding experiments, six different concentrations of the radioligand
(range: 6.25-200 pM) were used. In competition binding
studies, membrane homogenates were incubated with 200 pM [H]NMS and 10 different concentrations of
cold inhibitor. Nonspecific binding was defined as binding in the
presence of 1 µM atropine. Protein concentrations were
determined by the method of Bradford (1976).Stimulation of PI Hydrolysis
Transfected COS-7
cells were transferred into six-well plates (about 0.75
10
cells/well) about 24 h after transfections, and 3
µCi/ml of [myo-H]inositol (20
Ci/mmol, American Radiolabeled Chemicals Inc.) was added to the growth
medium. After a 24 h-labeling period, cells were washed once with 2 ml
of phosphate-buffered saline (PBS) and then incubated for 20 min (room
temperature) with 0.5 ml of Hank's balanced salt solution
containing 20 mM HEPES and 10 mM LiCl. Following the
addition of different concentrations of carbachol, cells were incubated
for 1 h at 37 °C. After this time, the assay medium was removed,
and the reaction was stopped by adding 0.75 ml of 0.1 N NaOH,
followed by a 10-min incubation at 37 °C. The alkaline solution was
then neutralized by adding 0.3 ml of 0.2 M formic acid, and
the inositol monophosphate (IP) fraction was isolated by
anion exchange chromatography as described (Berridge et al.,
1983) and counted on an LKB liquid scintillation counter.ELISA
An indirect cellular ELISA protocol was
developed to quantify the amount of epitope-tagged receptor fragments
present in the plasma membrane. One day after transfections, COS-7
cells were transferred into 96-well plates (4-5 10
cells/well). About 48 h later, cells were fixed with 4%
formaldehyde in PBS for 30 min at room temperature. After washing with
PBS and blocking with DMEM containing 10% FCS, cells were incubated for
2 h at 37 °C with a monoclonal antibody directed against the
HA-epitope tag (12CA5, Boehringer Mannheim; 10 µg/ml in DMEM, 10%
FCS). Plates were then washed and incubated with a 1:2,500 dilution (in
DMEM, 10% FCS) of a peroxidase-conjugated goat anti-mouse IgG antibody
(Sigma) for 1 h at 37 °C. HO and o-phenylenediamine (2.5 mM each in 0.1 M phosphate-citrate buffer, pH 5.0) were then added to serve as
substrate and chromogen, respectively. The enzymatic reaction (carried
out at room temperature) was stopped after 30 min with 1 M HSO solution containing 0.05 M NaSO, and the color development was
measured bichromatically in the BioKinetics reader (EL 312, Bio Tek
Instruments, Inc., Winooski, VT) at 490 and 630 nm.Immunofluorescence Microscopy
One day after
transfections, COS-7 cells were transferred into six-well plates
(1-2 10
cells/well) containing sterilized
glass coverslips. About 48 h later, cells were fixed with 4%
formaldehyde in PBS for 30 min at room temperature. After rinsing with
PBS, unspecific binding was blocked with DMEM containing 10% FCS. Cells
were then incubated for 2 h at 37 °C with the monoclonal antibody
12CA5 (10 µg/ml in DMEM, 10% FCS) or an affinity-purified
polyclonal antibody (rabbit) raised against a peptide corresponding to
the C-terminal 10 amino acids of the rat m3 muscarinic receptor
(FHKRVPEQAL) (kindly provided by W. F. Simonds, NIH). After washing off
the excess of unbound primary antibody with PBS, cells were incubated
for 1 h at 37 °C with a 1:100 or 1:200 dilution of a fluorescein
isothiocyanate-conjugated goat anti-mouse or goat anti-rabbit IgG
antibody (Sigma), respectively. The unbound secondary antibody was
removed by washing with PBS, and coverslips were mounted on microscope
slides using a glycerol/PBS mixture (1:1, v/v). To permeabilize the
cell membranes, cells were treated with 0.5% Triton X-100 in PBS for 10
min at room temperature (Lewis and Pelham, 1992). Images were obtained
using a confocal laser-scanning microscope (MRC-600, Bio-Rad).Drugs
Acetylcholine chloride and carbamylcholine
chloride (carbachol) were obtained through Sigma.
4-Diphenylacetoxy-N-methylpiperidine methiodide was purchased
from Research Biochemicals Inc. (Natick, MA).
Immunological Studies
The rat m3 muscarinic
receptor was split in all three intracellular (i1-i3) and all three
extracellular loops (o2-o4) by PCR-based mutagenesis techniques (Fig. 1, Table 1). To study the expression and subcellular
localization of the resulting individual receptor fragments, a
nine-amino acid HA-epitope tag (Kolodziej and Young, 1991) was added to
the N terminus of all C-terminally truncated receptors (N series,
N-N
; Fig. 1). Immunocytochemical
studies (confocal fluorescence microcopy) demonstrated that all
polypeptides of this series (except N
) showed a
subcellular distribution similar to that of the wild type m3 muscarinic
receptor containing an HA-tag at its N terminus (m3-N-HA).
Nonpermeabilized COS-7 cells expressing N
, N
,
N
, N
, or N
displayed a distinct
staining of the plasma membrane indistinguishable from that seen with
m3-N-HA (shown for N
in Fig. 2). In contrast,
N
was not found in the plasma membrane (no signal in
nonpermeabilized cells), but it was retained in the cytoplasm
(endoplasmic reticulum/Golgi complex) as visualized with permeabilized
cells (Fig. 3).
(a truncated
m3 receptor epitope-tagged at the N terminus; E and F). Immunofluorescence studies were carried out with
transfected cells grown on glass coverslips as described under
``Experimental Procedures.'' Cells were treated with a
monoclonal antibody directed against the HA-epitope tag and then
incubated with a fluorescein isothiocyanate-linked goat anti-mouse IgG
antibody. Immunofluorescence experiments were carried out with
nonpermeabilized (A, C, and E) and
permeabilized cells (B, D, and F).
Fluorescence images were obtained with a confocal laser scanning
microscope (MRC-600, Bio-Rad). Each picture is representative
of three independent experiments.
(a C-terminally truncated m3
muscarinic receptor containing only the first TM domain; Fig. 1) (A and B) and C
(an N-terminally
truncated m3 muscarinic receptor containing TM VI and VII; Fig. 1) (C and D). N
contained an
HA-epitope tag at its N terminus, whereas C
was used in
its nontagged form. Immunofluorescence studies with nonpermeabilized (A and C) and permeabilized cells (B and D) were carried out as described under ``Experimental
Procedures.'' For the detection of C
, an
affinity-purified polyclonal antibody directed against the C-terminal
10 amino acids of the rat m3 muscarinic receptor (FHKRVPEQAL) was
employed. Each picture is representative of two or three
independent experiments.
and C
. Immunocytochemical studies with
permeabilized COS-7 cells transfected with the epitope-tagged versions
of C
or C
showed that the two polypeptides
could be detected in the endoplasmic reticulum/Golgi complex, similar
to the wild type m3 receptor containing an HA-epitope at its C terminus
(m3-C-HA; Fig. 2). However, it could not be determined with
certainty whether or not these two fragments were also incorporated
into the plasma membrane. Coexpression of C
and C
(HA-tagged) with their corresponding N-terminal receptor
fragments, N
and N
, respectively (see below),
also did not result in a clear staining of the cell surface (studied in
permeabilized COS-7 cells). This finding indicated that the sensitivity
of the employed immunofluorescence procedure was too low to
unambiguously detect the plasma membrane localization of the HA-tagged
versions of C
and C
.
()raised against a peptide (FHKRVPEQAL)
corresponding to the C-terminal 10 amino acids of the rat m3 muscarinic
receptor to detect C and C
(nontagged) on the
surface of transfected COS-7 cells. In this case, both C-terminal
polypeptides could be clearly localized to the plasma membrane (shown
for C
in Fig. 3D). The intensity of cell
surface staining was not further increased when C
and
C
were coexpressed with N
and N
,
respectively.
-N
) present in the
plasma membrane, an indirect cellular ELISA protocol was developed (for
details, see ``Experimental Procedures''). The usefulness of
this assay system was initially demonstrated by studying
nonpermeabilized COS-7 cells transfected with m3-N-HA (a wild type m3
receptor containing an HA-epitope at its N terminus). Control
experiments with nonpermeabilized cells transfected with m3-C-HA (a
wild type m3 receptor containing an HA-epitope at its C terminus)
resulted in optical density readings similarly low as those found with
cells expressing the nontagged version of the wild type m3 receptor
(data not shown), demonstrating the intactness of the plasma membrane
barrier.
, fmol/mg membrane protein; studied
in [H]NMS saturation binding studies) by stepwise
reduction of the amount of transfected plasmid DNA (Fig. 4). In
parallel, ELISA experiments were carried out with nonpermeabilized
COS-7 cells derived from the same batch of cells used for the B measurements. Fig. 4shows that the
optical density values observed in the ELISA studies were directly
proportional to receptor densities (B)
determined in the radioligand binding studies.
H]NMS saturation binding studies were carried
out as described under ``Experimental Procedures.'' The curve shown is representative of two independent experiments,
each carried out in duplicate (binding assays) or triplicate (ELISA),
respectively. OD, optical density.
-N
; Fig. 1). Consistent with the
microscopic studies described above, the optical density readings found
with N
were not significantly different from background
values determined with COS-7 cells expressing the nontagged version of
the wild type m3 muscarinic receptor (Table 2). In contrast,
transfection of COS-7 cells with all other C-terminally truncated m3
receptor fragments (N
, N
, N
,
N
, and N
) yielded optical density readings
that were significantly higher than the background values (Table 2). Assuming a linear relationship between optical density
readings and protein amount present in the plasma membrane (Fig. 4), these polypeptides are predicted to be expressed at
about 2-10-fold lower levels than the wild type receptor
(m3-N-HA).
Ligand Binding Studies
COS-7 cells individually
expressing the various m3 muscarinic receptor fragments were unable to
specifically bind the muscarinic antagonist,
[H]NMS. In contrast, a significant number of
specific [H]NMS binding sites (44-122
fmol/mg) could be observed after coexpression of
NC, N
C,
or N
C (Table 3). No specific
[
H]NMS binding activity was found after
coexpression of the polypeptide pairs
NC, N
C,
or N
C, even at very high
[
H]NMS concentrations (up to 4 nM).
H]NMS saturation and
competition binding studies are summarized in Table 3. B values (determined in
[H]NMS saturation binding assays) after
coexpression of NC,
N
C, or N
C amounted to about 5-20% of the corresponding wild type
receptor value when equal amounts of plasmid DNA (4 µg) were used
for transfections. The N
C receptor
complex displayed agonist (acetylcholine, carbachol) and antagonist
(NMS, 4-diphenylacetoxy-N-methylpiperidine methiodide) binding
affinities similar to those of the wild type receptor (Table 3).
The N
C and N
C polypeptide complexes showed 2.5-8-fold and 4-20-fold
reduced affinities, respectively, for all ligands examined.
Stimulation of PI Hydrolysis
To study whether the
various m3 receptor fragments (fragment pairs) were capable of
activating G proteins, their ability to mediate carbachol-induced
stimulation of PI hydrolysis was examined in transfected COS-7 cells.
No functional activity was observed with cells expressing the various
polypeptides individually (data not shown). Likewise, only residual PI
activity was observed with four of the six fragment pairs examined
(NC,
N
C, N
C,
and N
C) (Fig. 5). In contrast,
the N
C and N
C receptor complexes were able to stimulate the production of
inositol phosphates to a similar maximum extent as the wild type m3
muscarinic receptor (Fig. 5, Table 4). The PI response
mediated by N
C was characterized by a
5-6-fold reduction in carbachol potency (Fig. 6, Table 4), consistent with the 6-fold decrease in carbachol
affinity observed with this polypeptide combination in the radioligand
binding studies (Table 3). Surprisingly, carbachol was able to
stimulate PI hydrolysis in cells transfected with
N
C with about 60-fold increased potency
as compared with cells expressing similar levels of the wild type m3
receptor (m3-N-HA) (Fig. 6, Table 4). In competition
binding studies, however, the N
C complex displayed a carbachol affinity similar to that of the
wild type receptor (Table 3).
C (B),
N
C (C),
N
C (D),
N
C (E),
N
C (F),
N
C (G). Similar to the wild
type receptor (m3-N-HA), all truncated receptors of the N series
(N
-N
) contained an HA-epitope tag at their N
termini. In the coexpression experiments, 2 µg of each plasmid (per
100-mm plate) were used. m3-N-HA was expressed at levels (B
) similar to those found after coexpression of
NC, N
C,
or N
C by reducing the amount of
transfected m3-N-HA plasmid DNA to 0.4 µg (supplemented with 3.6
µg of vector DNA) (see Table 3). m3-N-HA gave a similar
maximum PI response as the nontagged version of the wild type m3
receptor (data not shown). Transfected COS-7 cells were incubated in
six-well plates for 1 h at 37 °C with 1 mM carbachol, and
the resulting increases in intracellular IP
levels were
determined as described under ``Experimental Procedures.''
Data are presented as percentage increase in IP above basal
levels in the absence of carbachol. Basal IP levels for
m3-N-HA amounted to 2610 ± 797 cpm/well. The basal IP levels observed in the coexpression experiments were not
significantly different from this value. Data are given as means
± S.E. of a single experiment performed in triplicate; two
additional experiments gave similar
results.
Co3 and NC polypeptide complexes. COS-7 cells were transfected in 100-mm
dishes with epitope-tagged wild type m3 receptor DNA (0.4 µg of
m3-N-HA supplemented with 3.6 µg of vector DNA) (
) and
mixtures (2 µg of each plasmid) of N
C (
) or NC (
). m3-N-HA
stimulated the generation of inositol phosphates in a fashion similar
to the nontagged version of the wild type m3 receptor (data not shown).
PI assays were carried out in six-well plates as described under
``Experimental Procedures.'' Data are presented as percentage
increase in IP above basal levels in the absence of
carbachol. Basal IP levels for m3-N-HA amounted to 2236
± 415 cpm/well. The basal IP levels observed in the
coexpression experiments were not significantly different from this
value. Each curve is representative of three independent
experiments, each carried out in duplicate.
C,
N
C, etc.). Initially, COS-7 cells were
transfected with the individual receptor fragments to study their
expression and subcellular localization. Interestingly,
immunocytochemical and ELISA studies showed that all C-terminally
truncated receptors except N
were present in the plasma
membrane. This finding demonstrates that proper intracellular
trafficking and plasma membrane insertion of GPCRs does not require the
presence of the full-length receptor protein. In fact, even rather
short polypeptides such as N
and N
, which
contain only the first two and three TM regions, respectively, are
properly targeted to the plasma membrane.
-N
), the N
polypeptide, which
contains only the first TM domain, could only be detected
intracellularly but not on the cell surface. It has been suggested that
proper membrane insertion/orientation of eucaryotic plasma membrane
proteins critically depends on the character of the 15 amino acids N-
and C-terminal of TM I (Hartmann et al., 1989). N
contains only the first six amino acids of the i1 loop and
therefore may not contain the complete structural information required
for proper plasma membrane insertion.
, N
, and N
with their
corresponding C-terminal receptor segments (C
,
C
, and C
, respectively) resulted in a
considerable number (44-122 fmol/mg) of specific
[
H]NMS binding sites. This finding suggests that
a covalent connection between TM III and TM IV, TM IV and TM V, and TM
V and TM VI, respectively, is not essential for the formation of the
ligand binding site. However, whereas the NC receptor complex displayed wild type-like ligand binding
affinities, the N
C and
N
C polypeptide complexes showed
2.5-20-fold reduced binding affinities for all muscarinic
agonists and antagonists examined. This observation suggests that the
i2 and o3 loops may exert indirect conformational effects on the proper
arrangement of the transmembrane receptor core (formed by TM I-VII)
where the binding of small ligands such as acetylcholine is thought to
occur (Dohlman et al., 1991; Savarese and Fraser, 1992; Wess,
1993; Strader et al., 1994).
C,
N
C, and N
C fragments is actually associated with each other, we carried out
[
H]NMS binding studies with intact COS cells
coexpressing these polypeptide pairs. The B values obtained in these experiments (data not shown) were
similar to those obtained by the use of crude membrane homogenates (Table 3). Since [H]NMS is a permanently
charged ligand that does not penetrate the plasma membrane, the B values given in Table 3can be
considered a direct measure of the amount of functionally assembled
polypeptide complex present on the cell surface. On the other hand, the
ELISA experiments provide information about the total amount of the
N-terminal receptor fragments (free and complexed) present in the
plasma membrane. A comparison of the ELISA data (optical density values
can be converted into B values by using the
standard curve given in Fig. 4) and the number of
``recovered'' [H]NMS binding sites
therefore allows to estimate which fraction of N,
N
, and N
is complexed with C
,
C
, and C
, respectively. Based on these
considerations, it can be estimated that
60% of the N
and N
polypeptides and
15% of N
(present on the cell surface) exist in a complex with their
corresponding C-terminal receptor fragments.
and C
, demonstrated that these
polypeptides were delivered to the cell surface, even when expressed
alone. The use of a high affinity antibody directed against the C
terminus of the rat m3 muscarinic receptor clearly showed that both
fragments were localized to the plasma membrane. The immunological
studies thus demonstrated that both N- and C-terminal m3 muscarinic
receptor fragments can be independently targeted to the cell surface.
It is therefore likely that the N
C,
N
C, and N
C polypeptide complexes can be assembled both in the plasma
membrane and in the endoplasmic reticulum network.
C and
N
C receptor complexes were able to
stimulate PI hydrolysis to a similar maximum extent as the wild type m3
muscarinic receptor. Remarkably, the PI response mediated by
N
C was characterized by an
approximately 60-fold increase in carbachol potency (compared with the
wild type receptor). Since this polypeptide complex was shown to bind
carbachol with wild type-like affinity, this observation suggests that
the N
C complex can activate G proteins
with increased efficiency. Interestingly, a 115-amino acid segment of
the i3 loop of the rat m3 muscarinic receptor
(Glu
-Asn
) was not included in either
N
or C
but was deleted during the
construction of the two gene fragments. The possibility therefore
exists that this segment of the i3 loop contains structural elements (e.g. potential phosphorylation sites important for receptor
desensitization) that exert a negative regulatory effect on receptor-G
protein coupling. The correctness of this hypothesis will be studied in
coexpression experiments using modified versions of N
including this portion of the i3 loop
(Glu
-Asn
).
C and
N
C, the N
C peptide complex, despite its ability to bind muscarinic ligands
with relatively high affinity, was unable to stimulate PI hydrolysis to
a significant extent. Consistent with previous findings that the i2
loop of GPCRs plays an important role in proper G protein recognition
and activation (Wong et al., 1990; Dohlman et al.,
1991; Savarese and Fraser, 1992; Moro et al., 1993), this
observation suggests that the intactness of the i2 loop is essential
for efficient receptor-G protein coupling.
C, N
C,
and N
C, coexpression of the
N
C, N
C,
and N
C polypeptide combinations did not
result in significant ligand binding activity. The lack of functional
activity found with N
C can be explained
by the observation that N
is not inserted into the plasma
membrane (see above). On the other hand, the inability of the
N
C and N
C polypeptide combinations to produce functional receptors remains
unknown at present.
, inositol monophosphate; NMS, N-methylscopolamine; PBS, phosphate-buffered saline; PCR,
polymerase chain reaction; PI, phosphatidylinositol; TM I-VII, the
seven transmembrane domains of G protein-coupled receptors.
We thank June Yun for excellent technical assistance
and William F. Simonds (NIH) for providing us with an affinity-purified
polyclonal antibody raised against the C terminus of the rat m3
muscarinic receptor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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