©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of the Relative Orientation of Transmembrane Helices I and VII in G Protein-coupled Receptors (*)

(Received for publication, June 2, 1995)

Jie Liu Torsten Schöneberg Michiel van Rhee Jürgen Wess (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Currently, detailed structural information about the arrangement of the seven transmembrane helices (TM I-VII) present in all G protein-coupled receptors is still lacking. We demonstrated previously that hybrid m2/m5 muscarinic acetylcholine receptors which contain m5 sequence in TM I and m2 sequence in TM VII were unable to bind significant amounts of muscarinic radioligands (Pittel, Z., and Wess, J.(1994) Mol. Pharmacol. 45, 61-64). By using immunocytochemical and enzyme-linked immunosorbent assay techniques, we show in the present study that these pharmacologically inactive mutant receptors are present (at high levels) on the surface of transfected COS-7 cells. Strikingly, all misfolded m2/m5 hybrid receptors could be pharmacologically rescued by introduction of a single point mutation into either TM I (m5Thr m2Ala) or TM VII (m2Thr m5His). All our experimental data are consistent with the notion that the two altered threonine residues face each other at the TM I/TM VII interface in the pharmacologically inactive m2/m5 hybrid receptors, thus interfering with proper helix-helix packing. Our data provide the first experimental evidence as to how TM I and TM VII are oriented relative to each other and also strongly suggest that the TM helices in G protein-coupled receptors are arranged in a counterclockwise fashion (as viewed from the extracellular membrane surface).


INTRODUCTION

The seven transmembrane helices (TM I-VII) (^1)predicted to be present in all G protein-coupled receptors (GPCRs) are thought to be sequentially arranged in a ringlike fashion, thus forming a very tightly packed TM receptor core (Baldwin, 1993, 1994; Schwartz, 1994). 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 (Strader et al., 1994; Baldwin, 1993, 1994; Schwartz, 1994). Moreover, ligand-induced conformational changes in the TM receptor core are thought to be intimately involved in receptor activation (Dohlman et al., 1991; Wess, 1993; Strader et al., 1994). Detailed structural information about the membrane-embedded portion of GPCRs is therefore essential for understanding how GPCRs function at a molecular level.

By using the atomic coordinates of the heptahelical bacterial membrane protein bacteriorhodopsin (Henderson et al., 1990) as a template, several three-dimensional models of the TM core of GPCRs have been proposed (Trumpp-Kallmeyer et al., 1992; Nordvall and Hacksell, 1993; Hibert et al., 1993). However, the usefulness of these models has been questioned by recent mutagenesis studies (Zoffmann et al., 1993; Blüml et al., 1994) and the availability of a low resolution (9 Å) electron density map of rhodopsin, a G protein-coupled photoreceptor (Schertler et al., 1993).

In the absence of high resolution structural data on any GPCR, we have chosen a mutagenesis approach to identify interhelical interactions in GPCRs. In a previous study (Pittel and Wess, 1994), we identified a series of hybrid m2/m5 muscarinic receptors (C1-C4; Fig. 1) that were unable to bind significant amounts of muscarinic radioligands. A common structural feature of these pharmacologically inactive mutant receptors was the presence of m5 receptor sequence in TM I and of m2 receptor sequence in TM VII. Interestingly, hybrid m2/m5 muscarinic receptors in which both TM I and TM VII contained m2 receptor sequence (such as C5 or C6; Fig. 1) regained the ability to bind muscarinic ligands normally, suggesting that molecular interactions between TM I and TM VII are required for proper GPCR folding (Pittel and Wess, 1994).


Figure 1: Structure of mutant muscarinic receptors. A, hybrid m2/m5 muscarinic receptors (C1-C6). Transfection of COS-7 cells with the underlined constructs (C1-C4) did not result in an appreciable number of [^3H]NMS or [^3H]QNB binding sites (Pittel and Wess, 1994). B, sites in C1-C4 that were targeted by site-directed mutagenesis. C, comparison of the TM I and TM VII amino acid sequences of the human m2 and m5 muscarinic receptors. #, positions at which the sequences of the m2 and m5 receptors differ. Residues marked with an arrow were targeted by site-directed mutagenesis. Numbers refer to amino acid positions within the human m2 and m5 muscarinic receptors (Bonner et al., 1987, 1988). The N termini of the TM helices were chosen as defined by Baldwin(1993).



Based on these results, this study was designed to identify specific amino acids on TM I and TM VII, which are responsible for the folding defect present in C1-C4 (Fig. 1). The identification of such residues should allow predictions as to how TM I and TM VII are oriented relative to each other. In addition, immunocytochemical and ELISA studies were carried out to examine the subcellular localization and the expression levels of various mutant receptors.

We found that all misfolded m2/m5 hybrid receptors (C1-C4) could be pharmacologically rescued by introduction of a single point mutation into either TM I (m5Thr m2Ala) or TM VII (m2Thr423 m5His). Based on a recent model of the possible arrangement of TM I-VII in GPCRs (Baldwin, 1993, 1994), our data provide the first direct experimental evidence as to how TM I and VII are oriented relative to each other.


EXPERIMENTAL PROCEDURES

Construction of Mutant Muscarinic Receptors

Hybrid m2/m5 muscarinic receptors were created by employing standard polymerase chain reaction (PCR) mutagenesis techniques (Higuchi, 1989), using the human m2 (Hm2pcD; Bonner et al., 1987) and human m5 (Hm5pcDp1; Bonner et al., 1988) expression plasmids as templates. The construction of C1-C6 (Fig. 1) has been described previously (Wess et al., 1992; Pittel and Wess, 1994). C1-C6 are composed as follows (numbers refer to amino acid positions in the human m2 and m5 muscarinic receptors): C1, m5 1-162/m2 156-466; C2, m5 1-337/m2 301-466; C3 m5 1-445/m2 391-466; C4, m5 1-477/m2 423-466; C5, m2 1-69/m5 77-445/m2 391-466. C6, m2 1-46/m5 54-477/m2 423-466. Point mutations were introduced into C1-C4 by standard PCR mutagenesis approaches. In addition, the wild type m5 receptor and various mutant muscarinic receptors were epitope-tagged with a 9-amino acid sequence (YPYDVPDYA) (Kolodziej and Young, 1991) derived from the influenza virus hemagglutinin protein (HA-tag). The HA-tag was inserted after the methionine translation start codon. The identity of the various constructs and the correctness of all PCR-derived coding sequences were verified by restriction endonuclease analysis and by dideoxy sequencing of the mutant plasmids (Sanger et al., 1977).

Transient Expression of Mutant 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(2) incubator. For transfections, 2 10^6 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).

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 (Dörje 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(2). Saturation binding experiments were carried out with [^3H]N-methylscopolamine ([^3H]NMS, 81.4 Ci/mmol; DuPont NEN) and (-)-[^3H]N-quinuclidinyl benzilate ((-)-[^3H]QNB; 43.0 Ci/mmol; DuPont NEN). In these studies, six different concentrations of the radioligands (range: 12.5-400 pM) were used. In competition binding studies, membrane homogenates were incubated with 200 pM [^3H]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), using a Bio-Rad protein assay kit.

Binding data were analyzed by nonlinear least squares curve-fitting procedures, using the computer programs LIGAND (saturation binding data; Munson and Rodbard(1980)) or KALEIDAGRAPH (competition binding data; Synergy Software), respectively.

Stimulation of PI Hydrolysis

For PI assays, COS-7 cells were transfected in 100-mm dishes with 1 µg of receptor DNA as described above. About 24 h after transfections, cells were transferred into six-well plates (0.75 10^6 cells/well), and 3 µCi/ml myo-[^3H]inositol (20 Ci/mmol, American Radiolabeled Chemicals Inc.) was added to the growth medium. After a 24-h labeling period, cells were incubated for 1 h at 37 °C with the muscarinic agonist, carbachol (1 mM). Increases in intracellular inositol monophosphate (IP(1)) levels were determined as described (Berridge et al., 1983; Blin et al., 1995).

cAMP Assays

For cAMP assays, COS-7 cells were cotransfected in 100-mm dishes with 4 µg of muscarinic receptor DNA and 1 µg of an expression plasmid coding for the human V2 vasopressin receptor (V2pcD-PS; kindly provided by Dr. Allen Spiegel, NIDDK). Approximately 24 h after transfections, cells were transferred into 6-well plates (0.75 10^6 cells/well), and 2 µCi/ml [^3H]adenine (15 Ci/mmol, American Radiolabeled Chemicals Inc.) was added to the growth medium. After a 40-48-h labeling period, cells were preincubated in medium containing 20 mM Hepes and 1 mM 3-isobutyl-1-methylxanthine for 15 min, and then stimulated for 15 min at 37 °C with 0.5 nM of [Arg^8]vasopressin (AVP), in the absence or presence of the muscarinic agonist, carbachol (0.1 mM). The reaction was terminated by aspiration of the medium and addition of 1 ml of ice-cold 5% trichloroacetic acid containing 1 mM ATP and 1 mM cAMP. [^3H]cAMP was determined by anion-exchange chromatography as described (Salomon et al., 1974).

ELISA

To quantitate the amount of receptor protein present on the cell surface (even in the absence of ligand binding activity), an indirect cellular ELISA protocol was employed. COS-7 cells expressing epitope-tagged versions of wild type and mutant muscarinic receptors were transferred into 96-well plates (4-5 10^4 cells/well) approximately 24 h after transfections. About 48 h later, cells were fixed with 4% formaldehyde in phosphate-buffered saline (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-tag (12CA5, 20 µg/ml; Boehringer Mannheim). Plates were then washed and incubated with a 1:2,500 dilution of a peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) for 1 h at 37 °C. H(2)O(2) 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 H(2)SO(4) solution containing 0.05 M Na(2)SO(3), and the color development was measured bichromatically in a BioKinetics reader (EL 312, Bio Tek Instruments, Inc.) at 490 and 630 nm.

Immunofluorescence Microscopy

Immunofluorescence studies were carried out to examine the subcellular distribution of epitope-tagged versions of wild type and mutant muscarinic receptors. About 24 h after transfections, COS-7 cells were transferred into six-well plates (1-2 10^5 cells/well) containing sterilized glass coverslips. Approximately 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 monoclonal antibody 12CA5 (20 µg/ml in DMEM, 10% FCS). After washing off the excess of unbound 12CA5 antibody with PBS, cells were incubated for 1 h at 37 °C with a 1:100 dilution of a FITC-conjugated goat anti-mouse IgG antibody (Sigma). 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). Images were obtained using a confocal laser-scanning microscope (MRC-600, Bio-Rad).

Drugs

4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) was purchased from Research Biochemicals Inc. (Natick, MA). Acetylcholine chloride, carbamylcholine chloride (carbachol), and AVP were obtained through Sigma.


RESULTS

All wild type and mutant muscarinic receptor constructs were transiently expressed in COS-7 cells. As reported previously (Pittel and Wess, 1994), the C1-C4 hybrid receptors (Fig. 1) were unable to bind significant amounts of muscarinic radioligands such as [^3H]NMS or [^3H]QNB, whereas C5 and C6 (Fig. 1) could bind muscarinic ligands normally (Table 1). It is therefore likely that a conformational incompatibility exists between the m5 receptor sequence in TM I and the m2 receptor sequence in TM VII in C1-C4, leading to improperly folded receptor proteins.



Immunological Studies

To determine whether the pharmacologically inactive hybrid receptors were retained in an intracellular compartment or properly delivered to the cell surface, a 9-amino acid HA-tag was added to the N terminus of one of these mutant receptors, C3. For comparison, the wild type m5 muscarinic receptor was epitope-tagged in a similar fashion. The incorporation of the HA-tag had no significant effect on the ligand binding properties of the wild type m5 receptor or the virtual lack of ligand binding activity found with C3 (data not shown).

Immunocytochemical studies (confocal fluorescence microscopy) demonstrated that C3-HA showed a subcellular distribution similar to that found with the wild type m5 receptor. As shown in Fig. 2, non-permeabilized COS-7 cells expressing the C3-HA construct displayed a distinct staining of the plasma membrane in a fashion similar to the wild type m5 receptor.


Figure 2: Immunocytochemical localization of wild type and mutant muscarinic receptors. COS-7 cells were transfected with the following DNA constructs: vector (control, A), wild type m5 (B), C3 (C), C3(m5Thr m2Ala) (D), and C3(m2Thr m5His) (E). All receptors contained an HA-tag at their N terminus. Immunofluorescence studies were carried out with non-permeabilized COS-7 cells grown on glass coverslips, as described under ``Experimental Procedures.'' Cells were treated with a monoclonal antibody directed against the HA-tag (12CA5) and then incubated with a FITC-linked goat anti-mouse IgG secondary antibody. Fluorescence images were obtained with a confocal laser scanning microscope (MRC-600, Bio-Rad). Each picture is representative of three independent experiments.



To quantitate the amount of C3-HA present on the cell surface, an indirect cellular ELISA was employed (for details, see ``Experimental Procedures''). For ELISA measurements, non-permeabilized COS-7 cells transiently expressing C3-HA or the epitope-tagged version of the wild type m5 muscarinic receptor were first incubated with a monoclonal antibody directed against the HA-tag (12CA5), followed by addition of a peroxidase-conjugated secondary antibody and the photometric determination of peroxidase activity. Control experiments with non-permeabilized COS-7 cells expressing an m3 muscarinic receptor containing an HA-tag at its C terminus resulted in optical density readings that were similarly low as those found with the nontagged versions of the m3 and m5 muscarinic receptors (data not shown). This finding, together with the observation that the C-terminally tagged m3 muscarinic receptor could be easily detected in permeabilized cells, demonstrates that the employed ELISA procedure does not interfere with the intactness of the plasma membrane barrier.

Consistent with the microscopic studies described above, the ELISA experiments showed that C3-HA was properly expressed on the cell surface. As illustrated in Fig. 3, the optical density readings found with C3-HA were only 25-30% lower than those found with the epitope-tagged version of the wild type m5 muscarinic receptor. Taken together, the immunological studies suggest that C3 (and, most likely, other pharmacologically inactive m2/m5 hybrid receptors such as C1, C2, and C4) are stably incorporated into the plasma membrane. The virtual lack of ligand binding activity observed with C1-C4 thus appears to be due to a specific folding defect.


Figure 3: ELISA determination of the amount of wild type and mutant muscarinic receptors expressed on the cell surface. COS-7 cells were transfected with the following constructs all of which (except A, which was control, wild type m5, non-tagged) contained an HA-tag at their N terminus: wild type m5 (B), C3 (C), C3(m5Thr m2Ala) (D), and C3(m2Thr m5His) (E). ELISA measurements were carried out with non-permeabilized COS-7 cells in 96-well plates as described under ``Experimental Procedures.'' Optical density values can be considered a direct measure of the amount of receptor protein present on the cell surface. Data are presented as means ± S.D. of three independent experiments, each performed in triplicate.



Pharmacological Rescue of C1-C4

It seemed reasonable to assume that the amino acids responsible for the structural defect present in C1-C4 are located at the interface between TM I and VII. To identify these residues, the TM I and TM VII sequences (derived from the m5 and the m2 receptor, respectively) of C1-C4 were initially depicted in a helical wheel model assuming an anticlockwise connectivity of the TM helices (as viewed from the extracellular surface of the membrane; Fig. 4A). The positions of the TM helices shown in Fig. 4is based on the recently published ``Baldwin model'' (Baldwin, 1993, 1994), which is compatible with a low resolution electron density map of rhodopsin (Schertler et al., 1993). However, the model proposed in Fig. 4differs from the Baldwin projection in that TM VII has been rotated by approximately 30° (counterclockwise as viewed from the extracellular membrane surface). This orientation would allow m2Tyr (TM VII), which has been shown to be critically involved in the recognition of muscarinic ligands (Wess et al., 1991; Matsui et al., 1995) to project into the central ligand cavity formed by TM III-VII (Dohlman et al., 1991; Wess, 1993; Strader et al., 1994; Schwartz, 1994). Consistent with this notion, residues located at the homologous position in other GPCRs are also known to be important for ligand binding (Baldwin, 1994; Schwartz, 1994).


Figure 4: Helical wheel model of the possible orientation of TM I and VII in muscarinic receptors. A, anticlockwise (proposed) connectivity of TM I-VII, as viewed the extracellular surface of the membrane. The TM I (m5) and TM VII (m2) sequences of the pharmacologically inactive m2/m5 hybrid receptors, C1-C4 (Fig. 1), are shown. Amino acids that differ between the m2 and m5 muscarinic receptors are highlighted (blackbars). The model shown here is consistent with a low resolution electron density map of rhodopsin (Schertler et al., 1993; Baldwin, 1993). It differs from the Baldwin projection (Baldwin, 1993, 1994) in that TM VII has been rotated by about 30° (counterclockwise) to allow Tyr (underlined) to project into the ligand binding cavity formed by TM III-VII. Site-directed mutagenesis data obtained with different muscarinic receptor subtypes suggest that this tyrosine residue is critically involved in ligand recognition (Wess et al., 1991; Matsui et al., 1995). Residues marked with an arrow were targeted by site-directed mutagenesis. The size of the circles next to the individual amino acids reflects their relative depth in the plasma membrane. B, clockwise (``incompatible'') connectivity of TM I-VII (as viewed from the extracellular surface of the membrane), based on the assumptions made in A. The predicted positions of m5Thr (TM I) and m2Thr (TM VII) in C1-C4 are highlighted. Positions at which the TM I and TM VII sequences of the m2 and m5 muscarinic receptors differ are marked with an asterisk.



In Fig. 4A, all TM I and TM VII residues that differ among the m2 and m5 muscarinic receptors are highlighted (blackbars). Strikingly, if this model is correct, there are only two residues at the TM I/TM VII interface that are not identical between the two receptors (TM I, m5Thr/m2Ala; and TM VII, m2Thr/m5His) (Fig. 1C and 4A). Virtually all other nonconserved residues are predicted to face the lipid bilayer. Characteristically, in all pharmacological inactive hybrid m2/m5 receptors (C1-C4), a TM I threonine residue (m5Thr) faces a TM VII threonine residue (m2Thr). We therefore speculated that a novel interaction (which does not occur in the wild type receptors) formed between these two polar residues (e.g. involving hydrogen bond interactions) might interfere with proper helix-helix packing. If this is correct, it should be possible to ``pharmacologically rescue'' C1-C4 by single point mutations such that m5Thr (TM I) faces its ``natural partner'', m5His (TM VII), or, vice versa, that m2Thr (TM VII) is located adjacent to m2Ala (TM I). To test this hypothesis, the corresponding mutations (m5Thr m2Ala and m2Thr m5His) were introduced into TM I and VII of C1-C4. The ligand binding properties of the resultant mutant receptors are summarized in Table 1. Whereas C1-C4 were unable to bind significant amounts of [^3H]NMS (highest concentration tested: 5 nM), mutational modification of m5Thr or m2Thr resulted in the appearance a considerable number of specific [^3H]NMS binding sites. Similar results were obtained with a structurally different muscarinic radioligand, (-)-[^3H]QNB (data not shown). All mutant receptors in which either m5Thr or m2Thr was modified were able to bind muscarinic antagonists (NMS, 4-DAMP) and agonists (acetylcholine, carbachol) with affinities similar to those found with the two wild type receptors (Table 1).

Characteristically, in the case of each pharmacologically inactive hybrid receptor (C1-C4), the two point mutations had a similar effect on the number of ``recovered'' [^3H]NMS binding sites (B(max); Table 1). The C1- and C2-derived mutant receptors yielded B(max) values (approximately 150 fmol/mg) that were clearly lower than the corresponding wild type receptor levels. In contrast, C3(m5Thr m2Ala) and C3(m2Thr m5His) as well as the corresponding C4-based mutant receptors yielded B(max) values (418-556 fmol/mg) that almost reached the corresponding wild type m2 receptor levels. Either of the two point mutations therefore fully mimicked the effect of replacing long N-terminal segments in C3 and C4 (including TM I and adjacent extramembranous regions) with the corresponding m2 receptor sequences (C5, C6; Fig. 1, Table 1). Immunocytochemical and ELISA studies with epitope-tagged versions of C3(m5Thr m2Ala) and C3(m2Thr m5His) showed that the two mutant receptors were present on the cell surface at levels similar to those found with C3 ( Fig. 2and Fig. 3).

For control purposes, two additional point mutations (m5Thr m2Phe and m5Val m2Ser) were introduced into one of the pharmacologically inactive hybrid receptors, C4, either alone or in combination (Fig. 1). These two sites were targeted based on the observation that they are located in the N-terminal segment of TM I and that the m2 and m5 receptor residues present at these positions clearly differ in their physicochemical properties (hydrophobic versus hydrophilic; Fig. 1C). Consistent with the predicted localization of m5Thr and m5Val on the outer surface of TM I (facing the lipid bilayer; Fig. 4), expression of the three resultant mutant receptors did not result in the appearance of a significant number of [^3H]NMS binding sites (B(max) < 25 fmol/mg).

To further explore the molecular mechanisms by which introduction of the m5Thr m2Ala and m2Thr m5His point mutations into C1-C4 can restore proper receptor folding, four additional mutant receptors were created and pharmacologically characterized. The results of these studies are summarized in Table 2and Table 3. Introduction of an additional m5Thr m2Ala point mutation into C4(m2Thr m5His) did not interfere with the ability of this mutant receptor to properly bind the muscarinic antagonist, [^3H]NMS. Interestingly, C4 could be pharmacologically rescued not only by replacement of m2Thr with histidine (see above) but also by replacement with glutamate and asparagine, which are present at the corresponding position in the m1 and m3 muscarinic receptors, respectively (Table 2). In contrast, exchange of m2Thr with alanine did not restore ligand binding activity to C4.





Functional Assays

To demonstrate that the pharmacologically ``rescued'' mutant receptors also displayed proper functional activity, PI assays were carried out with transfected COS-7 cells. Whereas the wild type m5 muscarinic receptor mediated a pronounced increase in the accumulation of inositol phosphates following carbachol stimulation, C1-C4 displayed only residual functional activity (shown for C4 in Fig. 5). Consistent with the results of the ligand binding studies, introduction of the m5Thr m2Ala and m2Thr m5His point mutations into C2-C4 resulted in mutant receptors capable of stimulating PI hydrolysis to a similar maximum extent as the wild type m5 receptor (shown for C4 in Fig. 5). The functional properties of C4(m2Thr Glu), C4(m2Thr Asn), and C4(m2Thr Ala) also paralleled the results of the ligand binding studies (Fig. 5, Table 2). The two C1-based mutant receptors, C1(m5Thr m2Ala) and C1(m2Thr m5His), were unable to stimulate PI hydrolysis to a significant extent (data not shown). This finding is in agreement with the previous observation (Wess et al., 1989) that the presence of m2 receptor sequence in the N-terminal segment of the third intracellular loop abolishes muscarinic receptor-mediated PI hydrolysis.


Figure 5: Carbachol-induced stimulation of PI hydrolysis mediated by wild type m5 and mutant m2/m5 muscarinic receptors. Transfected COS-7 cells transiently expressing the indicated receptors were incubated in six-well plates for 1 h at 37 °C with 1 mM carbachol, and the resulting increases in intracellular IP(1) levels were determined as described (Berridge et al., 1983; Blin et al., 1995). Data are presented as percent increase in IP(1) above basal levels in the absence of carbachol. Basal IP(1) levels for the wild type m5 receptor amounted to 4630 ± 290 cpm/well. The basal IP(1) levels observed with the various mutant receptors were similar to this value. Data are given as means ± S.E. of a single experiment performed in triplicate; one additional experiment gave similar results.



However, cAMP assays with COS-7 cells coexpressing the V2 vasopressin receptor and C1(m5Thr m2Ala) or C1(m2Thr m5His) showed that these two mutant receptors gained the ability to inhibit AVP-stimulated cAMP production (7-9-fold stimulation above basal levels in the absence of carbachol) by 20 ± 4% (n = 2; stimulation with 0.1 mM carbachol). Under the same experimental conditions, the wild type m2 muscarinic receptor mediated a reduction of AVP-stimulated cAMP levels by 30 ± 4%, whereas C1 was completely inactive (n = 2).


DISCUSSION

In the absence of detailed structural information on any GPCR, we (Pittel and Wess, 1994) and others (Suryanarayana et al., 1992; Zhou et al., 1994; Rao et al., 1994) have recently employed mutagenesis approaches to identify molecular interactions between the seven TM helices predicted to be present in all GPCRs. In a previous study (Pittel and Wess, 1994), we found that hybrid m2/m5 muscarinic receptors (C1-C4; Fig. 1) containing m5 receptor sequence in TM I and m2 receptor sequence in TM VII were unable to bind significant amounts of muscarinic radioligands. However, immunocytochemical and ELISA experiments demonstrated (this study) that the folding defect present in C1-C4 does not affect proper targeting of these mutant receptors to the cell surface (shown for C3).

As reported previously (Pittel and Wess, 1994), substitution of N-terminal m2 receptor sequences (including TM I) into misfolded hybrid m2/m5 muscarinic receptors such as C3 or C4 (Fig. 1) could rescue ligand binding activity (Table 1), suggesting that TM I and VII are located in close proximity to each other and that specific molecular interactions between these two helices are required for proper receptor folding. A similar conclusion was reached in a study examining the pharmacological properties of a series of hybrid alpha(2)/beta(2)-adrenergic receptors (Suryanarayana et al., 1992). Although both studies (Suryanarayana et al., 1992; Pittel and Wess, 1994) suggest that TM I and TM VII are located next to each other in the TM receptor core, they do not allow predictions as to how these two TM helices are oriented relative to each other.

To address this question, we assumed that the residues responsible for the misfolding of the pharmacologically inactive m2/m5 hybrid receptors (C1-C4) are likely to be located at the TM I/TM VII interface. To identify these amino acids, we initially displayed the TM I and VII sequences of C1-C4 in a helical wheel model (Fig. 4), based on the recently published Baldwin projection (Baldwin, 1993, 1994). The Baldwin model proposes a possible arrangement of TM I-VII in GPCRs, which is consistent with a low resolution electron density map of rhodopsin (Schertler et al., 1993). However, our model (Fig. 4) differs from the Baldwin projection in that TM VII has been rotated by approximately 30° (counterclockwise as viewed from the extracellular side of the membrane) to take into account data from several recent mutagenesis studies (Wess, 1993; Baldwin, 1994; Schwartz, 1994). This arrangement would allow a TM VII tyrosine residue (corresponding to m2Tyr in Fig. 4) known to be critically involved in the binding of muscarinic ligands (Wess et al., 1991; Matsui et al., 1995) to project into the ligand binding cavity formed by TM III-VII. Moreover, residues located at the homologous positions in many other GPCRs have also been shown to be essential for high affinity ligand binding (Baldwin, 1994; Schwartz, 1994). The model depicted in Fig. 4is consistent with the general assumption that the (mostly lipophilic) residues, which differ among functionally closely related GPCRs (in this case, subtypes of muscarinic receptors), are located on the lipid-facing side of the TM helices (Baldwin, 1993).

The proposed arrangement of TM I and VII (Fig. 4) predicted that there are only two amino acids located at the TM I/TM VII interface (TM I, m5Thr/m2Ala; and TM VII, m2Thr423/m5His, respectively) that differ between the m2 and m5 muscarinic receptors. Characteristically, all pharmacologically inactive m2/m5 hybrid receptors (C1-C4) contain a threonine residue at these two positions. We therefore speculated that a novel interaction (which may involve hydrogen bonds) between these two residues interferes with proper helix-helix packing, ultimately resulting in misfolded receptor proteins unable to bind muscarinic ligands. Since both residues are predicted to be located about 1-2 helical turns away from the membrane surface (Fig. 1C), such contact appears theoretically possible.

To test this hypothesis, single m5Thr m2Ala (TM I) and m2Thr m5His (TM VII) point mutations were introduced into C1-C4, thus creating mutant receptors in which the amino acid configuration at the TM I/TM VII interface mimicked that found in the wild type m2 and m5 muscarinic receptors, respectively. We found, consistent with our working hypothesis, that mutant receptors in which either of the two threonine residues (m5Thr or m2Thr) was structurally modified gained the ability to bind significant amounts of muscarinic radioligands. All resulting mutant receptors were able to bind muscarinic agonists and antagonists with wild type affinities and, with the exception of the two C1-based mutant receptors, gained the ability to mediate a pronounced stimulation of PI hydrolysis. The observed lack of functional PI activity seen with the C1-based mutant receptors is consistent with the previous observation (Wess et al., 1989) that the presence of m2 receptor sequence in the N-terminal segment of the third intracellular loop abolishes muscarinic receptor-mediated PI hydrolysis.

However, in contrast to C1, both C1(m5Thr m2Ala) and C1(m2Thr m5His) gained the ability to inhibit the increase in cAMP levels mediated by stimulation of the cotransfected V2 vasopressin receptor. The degree of inhibition observed with these two mutant receptors (20 ± 4%) was somewhat smaller than that found with the wild type m2 receptor (30 ± 4%), probably due to the fact that the presence of m2 receptor sequence in the third intracellular loop and in the C-terminal ``tail'' is not sufficient to allow optimum coupling to G(i) (Wess et al., 1990; Liggett et al., 1991).

Immunocytochemical and ELISA studies showed that the m5Thr m2Ala (TM I) and m2Thr m5His (TM VII) point mutations did not lead to an increase in the amount of receptor protein expressed on the cell surface, suggesting that either substitution can specifically ``cure'' a conformational defect present in C1-C4. In contrast, exchange of residues predicted to be located on the lipid-facing side of TM I (m5Thr m2Phe and m5Val m2Ser) did not restore ligand binding activity.

Most strikingly, the C3- and C4-derived m5Thr m2Ala and m2Thr m5His mutant receptors yielded B(max) values that closely approached those found with the wild type m2 muscarinic receptor. In contrast, the analogous C1- and C2-based mutant receptors displayed about 4-6-fold lower B(max) values than the two wild type receptors, indicating that additional conformational incompatibilities, perhaps involving intracellular receptor domains, exist in C1 and C2 and interfere with optimum receptor folding and/or stability. This notion is consistent with the previous observation that an m3 muscarinic receptor in which the third intracellular loop was replaced with the corresponding m2 receptor sequence was expressed at 5-11-fold lower levels than the two wild type receptors (Wess et al., 1989).

Characteristically, in each case (C1-C4), replacement of either of the two critical threonine residues ((m5Thr or m2Thr) had a quantitatively similar effect on the number of ``recovered'' [^3H]NMS binding sites (Table 1). This result would be expected if the virtual lack of ligand binding activity found with C1-C4 is due to a direct (conformationally unfavorable) interaction between these two residues. Most of the residues thought to be located at the TM I/TM VII interface have a polar character (Fig. 4), suggesting that a complex network of hydrogen bond interactions may be involved in the proper positioning of these two TM helices. All our experimental data are therefore consistent with the notion that, in C1-C4, the formation of a novel hydrogen bridge between m5Thr (TM I) and m2Thr (TM VII) interferes with the proper formation of this hydrophilic network.

Such an interaction cannot occur in the wild type m2 muscarinic receptor where m5Thr is replaced with an alanine residue (m2Ala). Likewise, introduction of an additional m5Thr m2Ala point mutation into C4(m2Thr m5His) did not interfere with proper receptor folding ( Table 2and Table 3), indicating that a hydrogen bond between m5Thr (TM I) and m5His (TM VII) is not required for the formation of a functional receptor protein.

To further explore by which mechanism the m2Thr m5His point mutation can rescue the function of C1-C4, m2Thr (in C4) was replaced with glutamate and asparagine (which are present at the corresponding positions in the m1 an m3 muscarinic receptors, respectively) as well as with alanine. Whereas the glutamate and asparagine substitutions, similar to the histidine substitution, yielded fully functional receptors, exchange of m2Thr with alanine was unable to correct the folding deficit present in C4. These data suggest that a hydrophilic residue at position m5His (TM VII) is required for proper receptor folding, perhaps by stabilizing TM VII via formation of an intrahelical hydrogen bond interaction with one of the conserved polar residues located on the same side of TM VII (Fig. 4A). This stabilizing interaction cannot occur in the inactive hybrid receptors (C1-C4) where m2Thr is predicted to be engaged in a hydrogen bridge with m5Thr on TM I. In contrast to threonine, other polar amino acids such as histidine, glutamate, and asparagine, due to the presence of two heteroatoms in their side chains, can form multiple hydrogen bridges (including the proposed stabilizing interaction within TM VII). This may explain why replacement of m2Thr (in C4) with the latter three residues can restore proper receptor folding.

In addition to providing experimental evidence for the relative orientation of TM I and TM VII, our findings also strongly suggest that the TM helices in GPCRs are arranged in a counterclockwise fashion (as viewed from the extracellular membrane surface), an arrangement that has been commonly assumed but one for which no strong experimental evidence has been available (Baldwin, 1993, 1994; Schwartz, 1994). In the case of a clockwise connectivity of TM I-VII in C1-C4, m2Thr on TM VII would be predicted to be located adjacent to TM VI (Fig. 4B) rather than TM I. However, such an orientation is absolutely inconsistent with our experimental data.

Interestingly, a possible interhelical contact site has recently also been identified in the gonadotropin-releasing hormone receptor (Zhou et al., 1994). In this receptor, two usually highly conserved aspartate (TM II) and asparagine (TM VII) residues are replaced with asparagine (Asn) and aspartate (Asp), respectively. Whereas an Asn Asp mutant receptor completely lacked pharmacological activity, an Asn Asp/Asp Asn double-mutant receptor regained the ability to bind ligands with high affinity, suggesting that these two residues are located next to each other within the TM helical bundle (Zhou et al., 1994). Moreover, valuable structural information about interhelical interactions in GPCRs has also been obtained in biophysical and mutational studies of constitutively active forms of rhodopsin (Robinson et al., 1992; Rao et al., 1994). It could be demonstrated that the TM VII Lys residue (Lys), which acts as the retinal attachment site is located in close proximity to both a TM III glutamate residue (Glu, the natural counterion for Lys) and a TM II glycine residue (Gly).

Very recently, mutational analysis of the tachykinin NK-1 receptor showed that replacement of two residues located at the top of TM V and VI (Glu and Tyr, respectively) with histidine enabled Zn ions to act as an antagonist on this receptor (Elling et al., 1995). It has therefore been proposed that the artificial creation of metal ion binding sites in GPCRs may represent a novel approach to probe helix-helix interactions (Elling et al., 1995).

In conclusion, by using a ``gain-of-function'' mutagenesis approach we could provide direct experimental evidence as to how TM I and VII are oriented relative to each other. Moreover, our data strongly support the concept that the TM helices in GPCRs are arranged in a counterclockwise fashion (as viewed from the extracellular membrane surface).


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: Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bldg. 8A, Rm. B1A-09, Bethesda, MD 20892. Tel.: 301-402-4745; Fax: 301-402-4182.

(^1)
The abbreviations used are: TM I-VII, the seven transmembrane domains of G protein-coupled receptors; AVP, [Arg^8]vasopressin; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GPCR, G protein-coupled receptor; HA-tag, a 9-amino acid epitope derived from the influenza virus hemagglutinin protein; IP(1), inositol monophosphate; NMS, N-methylscopolamine; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PI, phosphatidylinositol; QNB, quinuclidinyl benzilate.


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