©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Plasma Membrane Localization and Functional Rescue of Truncated Forms of a G Protein-coupled Receptor (*)

(Received for publication, January 23, 1995; and in revised form, May 31, 1995)

Torsten Schneberg Jie Liu Jrgen Wess (§)

From the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (NC, NC, 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 (NC, NC, and NC, respectively), which were able to bind muscarinic agonists and antagonists with high affinity. The NC and NC polypeptide combinations, but not the NC 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.


INTRODUCTION

G protein-coupled receptors (GPCRs)()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.


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-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).





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'' 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).

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 (NC, NC, 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.

The immunological studies showed that all N- and C-terminal receptor fragments examined (except N; 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.


EXPERIMENTAL PROCEDURES

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).

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.

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).


RESULTS

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).


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 (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.




Figure 3: Immunocytochemical localization of m3 muscarinic receptor fragments. COS-7 cells were transfected with DNA constructs coding for N (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.



Moreover, an HA-epitope was also added to the C terminus of two of the N-terminally truncated receptor fragments, C 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.

We next examined the ability of an affinity-purified polyclonal antibody (prepared by W. F. Simonds)()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.

To quantify the amount of C-terminally truncated receptor fragments (N-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.

The m3-N-HA construct was expressed at different receptor densities (B, 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.


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 [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.



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-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, NC, or NC (Table 3). No specific [H]NMS binding activity was found after coexpression of the polypeptide pairs NC, NC, or NC, even at very high [H]NMS concentrations (up to 4 nM).



The results of the [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, NC, or NC 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 NC 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 NC and NC 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, NC, NC, and NC) (Fig. 5). In contrast, the NC and NC 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 NC 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 NC 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 NC complex displayed a carbachol affinity similar to that of the wild type receptor (Table 3).


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), NC (B), NC (C), NC (D), NC (E), NC (F), NC (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, NC, or NC 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.






Figure 6: Carbachol-induced PI hydrolysis mediated by the NCo3 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 NC () 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.




DISCUSSION

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 (NC, NC, 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.

In contrast to all other C-terminally truncated receptor fragments (N-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.

We found that coexpression of N, 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 NC and NC 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).

To address the question of which fraction of the NC, NC, and NC 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.

Interestingly, immunocytochemical studies with two of the C-terminal receptor fragments, C 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 NC, NC, and NC polypeptide complexes can be assembled both in the plasma membrane and in the endoplasmic reticulum network.

Functional studies showed that the NC and NC 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 NC 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 NC 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).

In contrast to NC and NC, the NC 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.

In contrast to NC, NC, and NC, coexpression of the NC, NC, and NC polypeptide combinations did not result in significant ligand binding activity. The lack of functional activity found with NC 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 NC and NC polypeptide combinations to produce functional receptors remains unknown at present.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: National Inst. of Diabetes and Digestive and Kidney Diseases, Laboratory of Bioorganic Chemistry, Bldg. 8A, Rm. B1A-09, Bethesda, MD 20892. Tel.: 301-402-4745; Fax: 301-402-4182.

The abbreviations used are: GPCR, G protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; IP, 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.

W. F. Simonds, unpublished results.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Berridge, M. J., Dawson, M. C., Downes, C. P., Heslop, J. P., and Irvine, R. F.(1983) Biochem. J. 212, 473-482 [Medline] [Order article via Infotrieve]
  2. Bonner, T. I., Buckley, N. J., Young, A. C., and Brann, M. R.(1987)Science 237, 527-532 [Medline] [Order article via Infotrieve]
  3. Bradford, M. M. (1976)Anal. Biochem.72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  4. Cheng, Y., and Prusoff, W. H.(1973)Biochem. Pharmacol. 22, 3099-3108 [CrossRef][Medline] [Order article via Infotrieve]
  5. Cullen, B. R. (1987)Methods Enzymol.152,684-704 [Medline] [Order article via Infotrieve]
  6. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J.(1991)Annu. Rev. Biochem. 60, 653-688 [CrossRef][Medline] [Order article via Infotrieve]
  7. Drje, F., Wess, J., Lambrecht, G., Tacke, R., Mutschler, E., and Brann, M. R. (1991)J. Pharmacol. Exp. Ther. 256, 727-733 [Abstract]
  8. Hartmann, E., Rapoport, T. A., and Lodish, H. F.(1989)Proc. Natl. Acad. Sci. U. S. A. 86, 5786-5790 [Abstract]
  9. Higuchi, R. (1989) in PCR Technology (Erlich, H. A., ed) pp. 61-70, Stockton Press, New York
  10. Kahn, T. W., and Engelman, D. M.(1992)Biochemistry 31, 6144-6151 [Medline] [Order article via Infotrieve]
  11. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1988)Science 240, 1310-1316 [Medline] [Order article via Infotrieve]
  12. Kolodziej, P. A., and Young, R. A.,(1991)Methods Enzymol. 194, 508-519 [Medline] [Order article via Infotrieve]
  13. Lewis, M. J., and Pelham, H. R. B.(1992)Cell 68, 353-364 [Medline] [Order article via Infotrieve]
  14. Liao, M.-J., London, E., and Khorana, H. G.(1983)J. Biol. Chem. 258, 9949-9955 [Abstract/Free Full Text]
  15. Maggio, R., Vogel, Z., and Wess, J.(1993a)FEBS Lett. 319, 195-200 [CrossRef][Medline] [Order article via Infotrieve]
  16. Maggio, R., Vogel, Z., and Wess, J.(1993b)Proc. Natl. Acad. Sci. U. S. A. 90, 3103-3107 [Abstract]
  17. Moro, O., Lameh, J., Hgger, P., and Sadee, W.(1993) J. Biol. Chem. 268, 22273-22276 [Abstract/Free Full Text]
  18. Munson, P. J., and Rodbard, D.(1980)Anal. Biochem. 107, 220-239 [Medline] [Order article via Infotrieve]
  19. Popot, J.-L., and Engelman, D. M.(1990)Biochemistry 29, 4031-4037 [Medline] [Order article via Infotrieve]
  20. Popot, J.-L., Gerchman, S.-E., and Engelman, D. M.(1987)J. Mol. Biol. 198, 655-676 [Medline] [Order article via Infotrieve]
  21. Raymond, J. R. (1994)Am. J. Physiol.266,F163-F174
  22. Rosenfeld, P. J., Cowley, G. S., McGee, T. L., Sandberg, M. A., Berson, E. L., and Dryja, T. P.(1992)Nature Genet. 1, 85-91 [Medline] [Order article via Infotrieve]
  23. Savarese, T. M., and Fraser, C. M.(1992)Biochem. J. 283, 1-19 [Medline] [Order article via Infotrieve]
  24. Strader, C. D., Fong, T. M., Tota, M. R., Underwood, D., and Dixon, R. A. F.(1994) Annu. Rev. Biochem. 63, 101-132 [CrossRef][Medline] [Order article via Infotrieve]
  25. Wess, J.(1993) Trends Pharmacol. Sci.14,308-313 [CrossRef][Medline] [Order article via Infotrieve]
  26. Wong, S. K.-F., Parker, E. M., and Ross, E. M.(1990)J. Biol. Chem. 265,6219-6224 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.