©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping of Single Amino Acid Residues Required for Selective Activation of G by the m3 Muscarinic Acetylcholine Receptor (*)

(Received for publication, May 2, 1995)

Nathalie Blin June Yun Jrgen Wess (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Each G protein-coupled receptor can interact only with a limited number of the many structurally similar G proteins expressed within a cell. This study was undertaken to identify single amino acids required for selectively coupling the m3 muscarinic acetylcholine receptor to G proteins of the G family. To this goal, distinct intracellular segments/amino acids of the m3 receptor were systematically substituted into the structurally closely related m2 muscarinic receptor, which couples to G proteins, not G proteins. The resultant mutant receptors were expressed in COS-7 cells and studied for their ability to induce agonist-dependent stimulation of phosphatidylinositol hydrolysis, a response known to be mediated by G proteins of the G class. Using this approach, we were able to identify four amino acids in the second intracellular loop and four amino acids at the C terminus of the third intracellular loop of the m3 muscarinic receptor that are essential for efficient G activation. We could demonstrate that these amino acids, together with a short segment at the N terminus of the third intracellular loop, fully account for the G protein coupling preference of the m3 muscarinic receptor. Taken together, our data strongly suggest that only a limited number of amino acids, located on different intracellular regions, are required to determine the functional profile of a given G protein-coupled receptor.


INTRODUCTION

A remarkable number of neurotransmitters, peptide hormones, neuromodulators, and autocrine and paracrine factors regulate cellular activity through binding to specific plasma membrane receptors coupled to heterotrimeric G proteins (Watson and Arkinstall, 1994). Characteristically, each member of this superfamily of G protein-coupled receptors can recognize and activate only a limited number of the many structurally closely related G proteins expressed within a cell (Dohlman et al., 1991; Savarese and Fraser, 1992; Hedin et al., 1993). Molecular genetic studies (Dohlman et al., 1991; Savarese and Fraser, 1992; Hedin et al., 1993; Strader et al., 1994) as well as experiments with short synthetic peptides that can mimic or inhibit receptor interactions with G proteins (Knig et al., 1989; Mnch et al., 1991; Okamoto and Nishimoto, 1992) have shown that multiple intracellular receptor domains including the second intracellular loop (i2), the N- and C-terminal portions of the third intracellular loop (i3), and the membrane-proximal portion of the C-terminal tail (i4) are involved in determining the specificity of receptor/G protein coupling. At present, very little is known about which specific amino acids contained within these receptor regions are of particular importance for proper G protein recognition. However, such information is essential to gain deeper insight into the molecular basis of receptor/G protein coupling selectivity.

The muscarinic acetylcholine receptors (m1-m5) have served as useful model systems to analyze the structural basis of receptor/G protein interactions (Wess, 1993). Based on their differential G protein coupling profiles, the muscarinic receptors can be divided into two major functional categories. The m1, m3, and m5 receptors preferentially couple to G proteins of the G family (Peralta et al., 1988; Bonner et al., 1988; Berstein et al., 1992; Offermanns et al., 1994), whereas the m2 and m4 receptors selectively activate G proteins of the G class (Peralta et al., 1988; Parker et al., 1991; Dell'Acqua et al., 1993; Offermanns et al., 1994). Studies with hybrid m2/m3 muscarinic receptors have shown that the N-terminal 16-21 amino acids of the i3 loop play an important role in determining the G protein coupling profile of a given muscarinic receptor subtype (Wess et al., 1989, 1990a; Lechleiter et al., 1990). Moreover, mutational analysis of the rat m3 muscarinic receptor (Blml et al., 1994a, 1994b) has shown that Tyr-254, located at the N terminus of the i3 loop, is essential for efficient stimulation of m3 receptor-mediated PI()hydrolysis, a response known to be mediated by G proteins of the G class (Smrcka et al., 1991; Berstein et al., 1992). This residue is conserved among the m1, m3, and m5 receptors, but is replaced with a different residue (Ser) in the m2 and m4 receptors.

We recently demonstrated that substitution of Tyr-254 into a mutant m3 muscarinic receptor in which the N-terminal segment of the i3 loop was replaced with the corresponding m2 receptor sequence (a modification that eliminated m3 receptor-mediated PI hydrolysis) was able to confer on the resultant mutant receptor the ability to efficiently activate the PI pathway (Blml et al., 1994b). However, when m3Tyr-254 was substituted directly into the wild-type m2 receptor, the resulting m2(Ser-210 Tyr) mutant receptor (m2-Y), similar to the wild-type m2 receptor, failed to stimulate PI hydrolysis to a significant extent (Blml et al., 1994b). These findings indicated that residues located in other intracellular domains, besides the N-terminal portion of the i3 loop, must also play important roles in proper recognition of G proteins of the G class.

To identify such residues, we have employed a ``gain-of-function'' mutagenesis approach. Initially, distinct intracellular loops/segments of the m3 muscarinic receptor were substituted into the wild-type m2 receptor as well as into the m2-Y mutant receptor, and the resulting hybrid receptors were studied for their ability to mediate carbachol-induced PI hydrolysis. In a second step, the functional roles of individual amino acids were examined by site-directed mutagenesis.

Using this approach, we identified a series of single amino acids, located in the i2 domain and at the C terminus of the i3 loop of the m3 receptor, that play key roles in G activation. We demonstrate that these residues, together with the N-terminal portion of the i3 loop, fully account for the unique functional profile of the m3 muscarinic receptor.


EXPERIMENTAL PROCEDURES

Construction of Mutant Muscarinic Receptor Genes

All mutations were introduced into Hm2pCD, a mammalian expression plasmid coding for the human m2 muscarinic receptor (Bonner et al., 1987), by using standard polymerase chain reaction mutagenesis techniques (Higuchi, 1989). Initially, distinct intracellular sequences of the wild-type m2 receptor and the m2(Ser-210 Tyr) mutant receptor (m2-Y) were systematically replaced with the corresponding m3 receptor segments (see Table 1). The Rm3pCD expression plasmid (Bonner et al., 1987), which codes for the rat m3 muscarinic receptor, was used as a template for polymerase chain reactions. In addition, single, double, and multiple point mutations were introduced into different loop regions of the wild-type m2 receptor and m2-Y. The construction of m2-Ctail (see Table 1) has been described previously (Wess et al., 1990b). The identity of all mutant constructs and the correctness of all polymerase chain reaction-derived coding sequences were verified by dideoxy sequencing of the mutant plasmids (Sanger et al., 1977).



Transient Expression of Mutant Muscarinic Receptors

COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum 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 using a DEAE-dextran method (Cullen, 1987).

Radioligand Binding Assays

Transfected COS-7 cells were harvested 48 h after transfections. Binding assays were carried out with membrane homogenates prepared as described previously (Drje et al., 1991). Incubations were carried out for 3 h at 22 °C in a 25 mM sodium phosphate buffer (pH 7.4) containing 5 mM MgCl. In N-[H]methylscopolamine (NMS; 81.4 Ci/mmol; DuPont NEN) saturation binding experiments, six different radioligand concentrations (12.5-400 pM) were used. In carbachol competition binding experiments, 10 different carbachol concentrations (50 nM to 1 mM) were tested in the presence of 200 pM [H]NMS. Nonspecific binding was measured 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).

PI Hydrolysis Assays

About 24 h after transfections, cells were split into six-well dishes (0.75 10 cells/well) in culture medium supplemented with 3 µCi/ml myo-[H]inositol (20 Ci/mmol; ARC). After a 24-h labeling period, cells were preincubated for 30 min at 37 °C in 1 ml of Hanks' balanced salt solution containing 20 mM HEPES and 10 mM LiCl. Cells were then stimulated in the same buffer with increasing concentrations of carbachol (1 nM to 1 mM) for 1 h at 37 °C. After removal of the medium, the reaction was stopped by the addition of 0.3 ml of 0.1 N NaOH. Cell homogenates were collected after a 30-min incubation period at 37 °C and neutralized with 0.4 ml of 1 N acetic acid. The inositol monophosphate (IP) fraction was then isolated by anion-exchange chromatography as described previously (Berridge et al., 1983) and counted on a Pharmacia Biotech Inc. liquid scintillation counter. Concentration-response curves were analyzed using the computer program KALEIDAGRAPH.


RESULTS

All wild-type and mutant muscarinic receptors were transiently expressed in COS-7 cells and studied for their ability to mediate carbachol-induced stimulation of PI hydrolysis. In addition, the carbachol binding properties of all receptors were determined in [H]NMS competition binding studies. [H]NMS saturation binding studies showed that all mutant receptors were expressed at similar levels (B) compared with the wild-type m2 and m3 muscarinic receptors (see Tables II-V), thus allowing a meaningful comparison between the functional responses mediated by the wild-type and mutant receptors.

Role of Intracellular Loops/Segments of the m3 Muscarinic Receptor in GCoupling

Initially, distinct intracellular loops/segments of the m3 muscarinic receptor (i1, i2, Ni3 (corresponding to the first 21 amino acids of the i3 loop), Ci3 (corresponding to the last 30 amino acids of the i3 loop), and a region including the C-terminal i4 region (Ctail)) were substituted into the wild-type m2 muscarinic receptor or into the m2-Y mutant receptor containing an additional m2Ser-210 m3Tyr-254 point mutation at the N terminus of the i3 loop (Fig. 1A and Table 1). The pharmacological profile of the resultant mutant receptors is summarized in Fig. 1B and Table 2.


Figure 1: Stimulation of PI hydrolysis mediated by chimeric m2/m3 muscarinic receptors. A, shown is the structure of hybrid m2/m3 receptors (see also Table 1). B, COS-7 cells were transfected with the indicated wild-type (wt) and mutant muscarinic receptor constructs. Approximately 48 h later, cells were incubated with increasing concentrations of carbachol for 1 h at 37 °C, and the resultant increases in intracellular IP levels were determined as described under ``Experimental Procedures.'' Data are presented as percent increase in IP above basal levels in the absence of carbachol. The maximum response to wild-type m3 receptor stimulation (5-8-fold increase in IP) was set at 100%. Basal IP levels in cells expressing the wild-type m2 and m3 muscarinic receptors amounted to 2072 ± 357 and 2236 ± 296 cpm/sample, respectively. The basal IP levels observed with the various mutant receptor constructs were not significantly different from these values. E and carbachol EC values are summarized in Table 2. Similar to the wild-type m2 receptor, the m2-i1, m2-Y-i1, m2-Ctail, and m2-Y-Ctail mutant receptors were unable to stimulate PI hydrolysis to an appreciable extent (Table 2). Each curve is representative of three independent experiments, each carried out in duplicate.





Similar to the wild-type m2 muscarinic receptor, the m2-i1 and m2-Ctail mutant receptors as well as m2-Y-i1 and m2-Y-Ctail were unable to stimulate PI hydrolysis to a significant extent (Table 2), indicating that the i1 and i4 domains of the m3 muscarinic receptor are unlikely to play important roles in coupling to G. Consistent with previous findings (Wess et al., 1989, 1990a; Lechleiter et al., 1990), a mutant m2 receptor in which the N-terminal portion of the i3 loop was replaced with the corresponding m3 receptor sequence (resulting in m2-Ni3) gained the ability to activate the PI pathway ( Fig. 1and Table 2) with considerable efficacy. However, this response was characterized by an 15-fold reduction in carbachol potency, and the maximum accumulation of inositol phosphates was clearly smaller than that found with the wild-type m3 receptor (E 40% of wild-type m3).

Interestingly, the m2-i2 mutant receptor showed a functional profile very similar to that of m2-Ni3 ( Fig. 1and Table 2). The m2-Y-i2 mutant receptor (containing an additional point mutation at the N terminus of the i3 loop; see above) displayed a slightly higher E value (48% of wild-type m3) and an 3-fold increase in carbachol potency, which, however, may be primarily due to the ability of this mutant receptor to bind the agonist carbachol with very high affinity (Table 2).

In contrast to m2-Ni3 and m2-i2, the m2-Ci3 mutant receptor, in which the C-terminal 30 amino acids of the i3 loop of the m2 receptor were replaced with the corresponding m3 receptor sequence, completely lacked the ability to stimulate agonist-dependent phospholipase C activity. However, introduction of the additional m2Ser-210 m3Tyr-254 point mutation into m2-Ci3 resulted in a mutant receptor (m2-Y-Ci3) that was able to stimulate PI hydrolysis in a fashion similar to m2-Ni3 and m2-i2 ( Fig. 1and Table 2).

Four Residues in the i2 Loop of the m3 Muscarinic Receptor Are Critical for Recognition of G

Based on the observation that the m2-i2 and m2-Y-i2 mutant receptors gained the ability to stimulate PI hydrolysis with considerable efficacy, we next examined which specific amino acids within the i2 loop of the m3 muscarinic receptor are responsible for this effect. This region of the m3 receptor contains four amino acids (Ser-168 (S), Arg-171 (R), Arg-176 (R), and Arg-183 (R)) that are conserved among all G-coupled muscarinic receptors (m1, m3, and m5), but are replaced with different residues (Cys, Lys, Pro, and Met, respectively) in the m2 and m4 receptors (Fig. 2A). As shown in Fig. 2B and Table 3, substitution of these four residues into the wild-type m2 receptor and into m2-Y (resulting in m2-i2(SRRR) and m2-Y-i2(SRRR), respectively) quantitatively mimicked the PI response mediated by m2-i2 and m2-Y-i2, respectively.


Figure 2: Four amino acids in the i2 loop of the m3 muscarinic receptor play key roles in receptor-mediated PI hydrolysis. A, shown is a comparison of the i2 loop sequences of the m1-m5 muscarinic receptors (rat = human). *, positions at which all five receptors have identical residues; #, positions at which the m1, m3, and m5 receptors have identical residues, which differ from those present in the m2 and m4 receptors. Numbers refer to amino acid positions in the human m2 and rat m3 muscarinic receptors, respectively (Bonner et al., 1987). The entire i2 loop of the m3 receptor or the boxed m3 receptor residues were substituted into the wild-type m2 or the m2-Y mutant receptor, either individually or in combination (see Tables I and III). B, COS-7 cells expressing the indicated wild-type and mutant muscarinic receptor constructs were incubated with increasing concentrations of carbachol, and the resultant increases in intracellular IP levels were determined as described under ``Experimental Procedures'' (for additional experimental details, see legend to Fig. 1B). E and carbachol EC values are summarized in Table 3. Each curve is representative of three independent experiments, each carried out in duplicate.





To further explore which of these residues is (are) of particular importance for G activation, triple, double, and single point mutants were created and functionally analyzed (Table 3). The m2-i2(RRR) mutant receptor and its m2-Y analog (m2-Y-i2(RRR)) were considerably less active than m2-i2(SRRR) and m2-Y-i2(SRRR), respectively (50% reduction in E and 8-fold decrease in carbachol potency), indicating that m3Ser-168 plays an important role in G activation. Moreover, all mutant receptors lacking m3Arg-176 (R) were unable to stimulate PI hydrolysis to an appreciable extent, indicating that this residue also plays a key role in coupling to G. Consistent with this notion, the m2-i2(R) mutant receptor containing a single m2Pro-132 m3Arg-176 point mutation was able to (modestly) stimulate PI hydrolysis in a fashion similar to m2-i2(RRR) (E 20-30% of wild-type m3). In contrast, all other single point mutations that were introduced into the i2 loop of the wild-type m2 receptor (or m2-Y) yielded mutant receptors that were unable to stimulate PI hydrolysis to a significant extent (Table 3).

Four Residues at the C Terminus of the i3 Loop of the m3 Muscarinic Receptor Are Critical for Efficient GActivation

As discussed above, a mutant m2 receptor (m2-Y-Ci3) containing 30 amino acids of m3 receptor sequence at the C terminus of the i3 loop and the additional m2Ser-210 m3Tyr-254 point mutation gained the ability to stimulate phospholipase C activity with considerable efficiency ( Fig. 1and Table 2). To identify single amino acids responsible for this effect, we initially focused on four m3 receptor residues located at the junction between the i3 loop and transmembrane domain VI (Ala-488 (A), Ala-489 (A), Leu-492 (L), and Ser-493 (S)) (Fig. 3A). These amino acids are present in all G-coupled muscarinic receptors, but are replaced with Val, Thr, Ile, and Leu/Phe, respectively, in the m2 and m4 receptors (Fig. 3A). Substitution of these four m3 receptor residues into m2-Y resulted in a mutant receptor (m2-Y-Ci3(AALS)) that gained the ability to stimulate the production of inositol phosphates in a fashion very similar to m2-Y-Ci3 (Fig. 3B and Table 4). However, as observed with the m2-Y-Ci3 mutant receptor (Fig. 1B), this response was completely abolished in the absence of the m2Ser-210 m3Tyr-254 point mutation (m2-Ci3(AALS)) (Table 4). Substitution of the m3Ala-488-Ala-489 amino acid pair (AA) into m2-Y partially mimicked the effect of the quadruple point mutation (Fig. 3B and Table 4). On the other hand, the m2-Y-Ci3(LS) mutant receptor (containing the m3Leu-492-Ser-493 amino acid pair) was completely inactive (Table 4). Similarly, all mutant receptors in which Ala-488, Ala-489, Leu-492, and Ser-493 were individually substituted into m2-Y were unable to stimulate PI hydrolysis to a significant extent (Table 4).


Figure 3: Four amino acids at the C terminus of the i3 loop of the m3 muscarinic receptor are of critical importance for receptor-mediated PI hydrolysis. A, amino acid sequences of the C-terminal portion of the i3 loop of the m1-m5 muscarinic receptors (except for m3 (rat), human sequences are shown). *, positions at which all five receptors have identical residues; #, positions at which the m1, m3, and m5 receptors have identical residues, which differ from those present in the m2 and m4 receptors. Numbers refer to amino acid positions in the human m2 and rat m3 muscarinic receptors, respectively (Bonner et al., 1987). Gaps were introduced to allow for maximum sequence identity. The C-terminal segment of the i3 loop of the m3 receptor (Lys-464-Ser-493) or the boxed m3 receptor residues were substituted into the wild-type m2 or the m2-Y mutant receptor, either individually or in combination (see Table 1and Table 4). TM, transmembrane domain. B, COS-7 cells expressing the indicated wild-type and mutant muscarinic receptor constructs were incubated with increasing concentrations of carbachol, and the resultant increases in intracellular IP levels were determined as described under ``Experimental Procedures'' (for additional experimental details, see legend to Fig. 1B). E and carbachol EC values are given in Table 4. Each curve is representative of three independent experiments, each carried out in duplicate.





Multiple Substitutions Confer on the m2 Muscarinic Receptor m3-like PI Activity

As outlined above, mutant m2 receptors in which specific amino acids in the i2 loop or in the N- and C-terminal segments of the i3 domain were replaced with the corresponding m3 receptor residues gained the ability to stimulate the production of inositol phosphates. However, none of these mutant receptors was able to stimulate PI hydrolysis with the same efficacy (E) and efficiency (carbachol EC) as the wild-type m3 receptor. To test whether the functional effects of the various m2 m3 substitutions were additive, a series of ``combination mutants'' were created and functionally analyzed ( Fig. 4and Table 5).


Figure 4: Stimulation of PI hydrolysis mediated by mutant m2 muscarinic receptors containing substitutions in multiple intracellular receptor regions. COS-7 cells were transfected with the indicated wild-type and mutant muscarinic receptor constructs. The various substitutions are defined in Table 1(see also Figs. 1A-3A). Increases in intracellular IP levels were determined as described under ``Experimental Procedures'' (for additional experimental details, see legend to Fig. 1B). E and carbachol EC values are listed in Table 5. Each curve is representative of three independent experiments, each carried out in duplicate.





Substitution of the AALS motif (located at the C terminus of the i3 loop of the m3 receptor) (Fig. 3A) into m2-i2(SRRR) or m2-Y-i2(SRRR) did not lead to a further increase in PI activity. In contrast, substitution of the Ni3 region of the m3 receptor (Arg-252-Thr-272) into m2-i2(SRRR) or m2-Ci3(AALS) resulted in a pronounced increase in E (70-90% of wild-type m3) and carbachol potency (EC = 0.3-0.6 µM). Interestingly, the m2-i2(SRRR)-Ni3-Ci3(AALS) mutant receptor containing substitutions in three different intracellular regions gained the ability to stimulate PI hydrolysis with a carbachol potency similar to that found with the wild-type m3 receptor (0.1-0.2 µM). Surprisingly, this mutant receptor stimulated the production of inositol phosphates to an even greater maximum extent (E = 160% of wild-type m3) than the wild-type m3 receptor (Table 5).


DISCUSSION

A large body of evidence suggests that the specificity of receptor/G protein interactions is determined by multiple intracellular receptor domains (Dohlman et al., 1991; Savarese and Fraser, 1992; Hedin et al., 1993; Strader et al., 1994). To gain deeper insight into the molecular mechanisms governing receptor/G protein coupling selectivity, specific amino acids that are of particular importance for proper G protein recognition need to be identified. Toward this goal, we have used the m2 and m3 muscarinic receptors, which are selectively coupled to G proteins of the G and G families, respectively (Peralta et al., 1988; Parker et al., 1991; Offermanns et al., 1994), as model systems. In contrast to many previous studies that attempted to eliminate G protein coupling by deletion or substitution of distinct receptor domains/amino acids, we have employed a gain-of-function mutagenesis approach. Specifically, distinct intracellular m3 receptor domains/amino acids were substituted into the corresponding regions/sites of the wild-type m2 and m2-Y mutant receptors (containing an additional m2Ser-210 m3Tyr-254 point mutation) (Blml et al., 1994a, 1994b), and the resulting mutant receptors were assayed for their ability to gain coupling to PI hydrolysis, a response known to be due to G-mediated activation of phospholipase C- (Smrcka et al., 1991; Berstein et al., 1992).

Consistent with several previous studies (Wess et al., 1989, 1990a; Lechleiter et al., 1990), substitution of the first 21 amino acids of the i3 loop of the m3 receptor into the wild-type m2 receptor yielded a mutant receptor (m2-Ni3) that, in contrast to the wild-type m2 receptor, could stimulate PI hydrolysis to a significant extent. However, in keeping with earlier findings (Wess et al., 1990a), this effect was clearly weaker than the corresponding wild-type m3 response (Fig. 1B), indicating that the N-terminal segment of the i3 loop is not the sole structural element responsible for efficiently coupling the m3 muscarinic receptor to G proteins of the G family. This conclusion is also in agreement with several previous studies using chimeric adrenergic (Cotecchia et al., 1990, 1992; Liggett et al., 1991) and chimeric muscarinic/adrenergic (Wong et al., 1990) receptors.

Interestingly, we could show that a mutant m2 muscarinic receptor in which the i2 loop was replaced with the corresponding m3 receptor sequence (resulting in m2-i2) was able to stimulate PI hydrolysis in a fashion similar to m2-Ni3 (Fig. 1B). This result suggests that the i2 loop contributes to proper recognition of G to an extent similar to that of the N-terminal segment of the i3 loop. Consistent with this notion, replacement of the i2 loop in the m1 muscarinic receptor with the corresponding -adrenergic receptor sequence resulted in a mutant receptor that was clearly less efficient in activating the PI signaling pathway, as indicated by a 10-fold reduction in carbachol potency (Wong et al., 1990). Moreover, we could demonstrate that four amino acids present in the i2 loop of the m3 receptor (Ser-168, Arg-171, Arg-176, and Arg-183) fully account for the ability of this receptor region to support preferential coupling to G (Fig. 2B and 5). Systematic substitution of these residues into the wild-type m2 receptor (or into m2-Y), either individually or in combination, showed that the presence of all four residues is required for optimum G recognition. Interestingly, Arg-176 seems to be of particular functional importance since only those ``i2 loop mutants'' that contained this residue (including the m2(Pro-132 m3Arg-176) single point mutant) were able to stimulate PI hydrolysis to an appreciable extent (Table 3). Interestingly, in the m2 and m4 muscarinic receptors, Arg-176 is replaced with a Pro residue, an amino acid that frequently causes drastic changes in protein secondary structure. It should be noted that the i2 loop also contains several residues that are conserved among most (or many) G protein-coupled receptors (corresponding to Asp-164, Arg-165, Pro-172, and Leu-173 in the rat m3 muscarinic receptor) (Fig. 5). Loss-of-function mutagenesis studies suggest that these residues are generally required for efficient G protein activation (O'Dowd et al., 1988; Fraser et al., 1989; Moro et al., 1993b; Zhu et al., 1994).


Figure 5: Residues in the m3 muscarinic receptor that play key roles in receptor-mediated activation of G. The intracellular regions (except for the central portion of the i3 loop and a 25-amino acid segment of the i4 domain) and the endofacial portions of transmembrane domains I-VII of the rat m3 muscarinic receptor are shown. As demonstrated in this study, the highlighted amino acids (aa) are required for efficient activation of G. Moreover, our data suggest that optimum activation of G proteins requires one or more additional residues located in the N-terminal portion of the i3 loop (dotted sequence).



In contrast to the m2-Ni3 and m2-i2 hybrid receptors, a mutant m2 receptor in which the C-terminal 30 amino acids of the i3 loop were replaced with the corresponding m3 receptor sequence was unable to stimulate PI hydrolysis to an appreciable extent. However, introduction of the additional m2Ser-210 m3Tyr-254 point mutation into this hybrid construct yielded a mutant receptor that was capable of activating the PI pathway with the same efficiency as m2-i2 and m2-Ni3 (Fig. 1B). This finding, besides providing additional evidence for the key role of m3Tyr-254 in G activation (Blml et al., 1994a, 1994b), is consistent with the notion that Tyr-254 and distinct residues within the C-terminal segment of the i3 loop of the m3 receptor form a common recognition site for G proteins. Moreover, systematic mutational analysis showed that four m3 receptor residues (Ala-488, Ala-489, Leu-492, and Ser-493) quantitatively account for the contribution of the C-terminal segment of the i3 loop to proper recognition of G proteins (Figs. 3B and 5). Single amino acid substitution showed that all four residues are required for efficient G activation. Interestingly, substitution of Ala-488-Ala-489 (but not of Leu-492-Ser-493) into m2-Y resulted in a mutant receptor that could induce a small but significant PI response (E 30% of wild-type m3) (Fig. 3B), suggesting that these two Ala residues play key roles in m3 receptor-mediated G recognition. In keeping with this finding, mutant m3 muscarinic receptors in which Ala-489 was replaced with Lys or Glu (substitutions that render various other G protein-coupled receptors constitutively active) (Kjelsberg et al., 1992; Parma et al., 1993) resulted in a drastic reduction of receptor-mediated PI hydrolysis.()Similar findings were obtained when this Ala residue was structurally modified in the m5 muscarinic receptor (Burstein et al., 1995). Interestingly, the C-terminal portion of the i3 loop of many G protein-coupled receptors contains a series of charged residues (corresponding to Lys-384-Lys-387 in the rat m3 muscarinic receptor) (Fig. 5), some of which, as shown by loss-of-function mutagenesis studies, appear to be generally important for G protein coupling (Kunkel and Peralta, 1993; Hgger et al., 1995).

Secondary structure analysis of muscarinic and other G protein-coupled receptors suggests that the region at the i3 loop/transmembrane domain VI junction is -helically arranged (Strader et al., 1989). Based on this notion, Ala-488, Ala-489, Leu-492, and Ser-493 (m3 receptor sequence) are predicted to be located on one side of an -helical domain (Fig. 6). Similarly, mutagenesis studies suggest that the N-terminal portion of the i3 loop also forms an amphiphilic -helix and that the noncharged (hydrophobic) side of this helical segment is intimately involved in G protein recognition and activation (Cheung et al., 1992; Blml et al., 1994c). As already hinted at above, it is therefore likely that these two patches of primarily hydrophobic residues (located at the N and C termini of the i3 loop, respectively) lie adjacent to each other in the three-dimensional receptor structure, thus forming a common binding surface for specific classes of G proteins. This notion is strongly supported by the observation that the Ala-488-Ala-489-Leu-492-Ser-493 motif did not lead to an increase in phospholipase C activity when introduced into the wild-type m2 receptor or into m2-i2(SRRR), but drastically improved the efficiency of receptor-mediated PI hydrolysis when substituted into m2-Ni3 or m2-Y ( Table 4and Table 5).


Figure 6: Helical wheel representation of the C-terminal portion of the i3 loop of the m3 muscarinic receptor (Lys-486-Leu-496). The four m3 receptor residues that play key roles in m3 receptor-mediated G activation (highlighted in black) are predicted to be located on one side of an amphiphilic -helix (see ``Discussion''). The direction of view is from the N to the C terminus.



Functional analysis of hybrid m2/m3 muscarinic receptors containing multiple substitutions in various intracellular receptor domains showed that specific residues in the i2 loop and in the membrane-proximal portions of the i3 loop of the m3 receptor act in a concerted fashion to stimulate G proteins of the G family. We found that introduction of Ser-168, Arg-171, Arg-176, and Arg-183 (derived from the i2 loop of the m3 receptor) and of Ala-488, Ala-489, Leu-492, and Ser-493 (derived from the C terminus of the i3 loop of the m3 receptor) into m2-Ni3 resulted in a hybrid receptor that was able to stimulate PI hydrolysis with a similarly high carbachol potency as the wild-type m3 receptor ( Fig. 4and Table 5), indicating that these eight amino acids, together with the N-terminal portion of the i3 loop, quantitatively account for the efficiency of m3 receptor-mediated Gactivation. Surprisingly, this mutant receptor gained the ability to stimulate the production of inositol phosphates to an even greater maximum extent (E = 160% of wild-type m3) than the wild-type m3 receptor. A possible explanation for this phenomenon is that the central portion of the i3 loop of the m3 receptor contains structural elements (e.g. potential sites of receptor phosphorylation that are not contained in the mutant receptor (Moro et al., 1993a; Tobin and Nahorski, 1993)) that exert a negative regulatory effect on receptor/G protein coupling.

Although this study provides additional evidence for the importance of m3Tyr-254 for proper G recognition (see above) (Blml et al., 1994a, 1994b), our data also clearly indicate that this residue is not the only amino acid within the N-terminal portion of the i3 loop of the m3 receptor contributing to G recognition and activation. This notion is primarily based on the observation that the m2-Ni3, but not the m2-Y, mutant receptor (containing the m2Ser-210 m3Tyr-254 single point mutation) was able to stimulate the PI pathway to an appreciable extent. Moreover, all m2-Y-derived combination mutants were consistently less active than the corresponding m2-Ni3 analogs ( Fig. 4and Table 5). The specific residues within the N-terminal segment of the i3 loop of the m3 muscarinic receptor that, besides Tyr-254, also contribute to proper G recognition remain to be identified.

In summary, we have identified, in unprecedented molecular detail, the structural elements allowing the m3 muscarinic receptor to selectively couple to G proteins of the G family. We showed that four amino acids in the i2 loop and four amino acids at the C terminus of the i3 domain of the m3 muscarinic receptor are required for efficient activation of G proteins. These residues, together with the N-terminal segment of the i3 loop, quantitatively account for the G protein coupling preference of the m3 receptor (Fig. 5). Consistent with this finding, all eight amino acids are also present in the m1 and m5 muscarinic receptors (which, like the m3 receptor, are also selectively linked to G), but are absent in the m2 and m4 receptors, which selectively activate G proteins of the G class. Sequence analysis shows that these residues, except for m3Ala-489, are not well conserved among other classes of receptors that preferentially couple to G proteins. It should be interesting to examine whether the amino acids present at the corresponding positions in other G-coupled receptors play similar roles in selective G recognition as described here for the m3 muscarinic receptor. Our results strongly suggest that, as a general rule, the distinct functional profile of a given G protein-coupled receptor is determined by a rather limited number of amino acids present on multiple intracellular receptor domains.


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

The abbreviations used are: PI, phosphatidylinositol; NMS, N-methylscopolamine; IP, inositol monophosphate.

J. Wess, Z. Vogel, and S. Gutkind, unpublished results.


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