(Received for publication, May 2, 1995)
From the
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 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 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
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
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
[
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
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 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 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
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
To further explore which of these residues is (are) of
particular importance for G
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
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
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 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 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 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
Figure 5:
Residues in the m3 muscarinic receptor
that play key roles in receptor-mediated activation of
G
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
Secondary structure analysis of muscarinic and
other G protein-coupled receptors suggests that the region at the i3
loop/transmembrane domain VI junction is
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
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 Although this study provides additional evidence
for the importance of m3Tyr-254 for proper G 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. 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.
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.
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.
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.
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.
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 G
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 Coupling
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.
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.
. 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).
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).
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.
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.
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 G
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 Activation
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).
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).
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.
(
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).
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).
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.
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).
. 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).
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).
-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).
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.
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
G
activation. 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.
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.
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.
, inositol monophosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.