From the Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Alanine-scanning mutagenesis has been applied to
residues 100-121 in transmembrane domain 3 of the M1
muscarinic acetylcholine receptor. This study complements a previous
investigation of the triad
Asp122-Arg123-Tyr124 (Lu, Z-L.,
Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234-241). The results
demonstrate the Models of the rhodopsin-like superfamily of G protein-coupled
receptors suggest that transmembrane domain 3 (TMD
3)1 forms the core of the
seven-membered helical bundle that traverses the phospholipid bilayer
(1, 2). Spin labeling and cross-linking studies on Cys-substituted
rhodopsin mutants have shown that a reorganization of the contacts
between TMD 3 and surrounding transmembrane domains probably
accompanies receptor activation (3-5). Substituted cysteine
accessibility mutagenesis of TMD 3 of the D2 dopamine receptor has supported its TMD 3 of the M1 muscarinic acetylcholine receptor (mAChR)
is constrained at its N terminus by the conserved disulfide-bonded residue Cys98 (7, 8). It carries the acetylcholine (ACh)
binding side chain of Asp105 (9, 10) and, near its C
terminus, Arg123, which is completely conserved in the
rhodopsin-like G protein-coupled receptors and is important in G
protein binding and activation (11, 12). In the M1 mAChR,
the highly conserved residues Asp122 and Tyr124
appear to make intramolecular contacts whose integrity is important for
receptor expression (13). Other residues within TMD 3 may also make
inter- or intramolecular contacts, and these may be important
determinants of receptor structure and activation.
Scanning mutagenesis can help to establish the secondary structure of
receptor domains (6) and to locate and define functional epitopes
within them (14). Replacement of individual amino acids by alanine,
which deletes the side chain beyond the The results show that different, topographically distinct, groups of
amino acids are responsible for the selective stabilization of the
ground and activated states of the receptor. We propose that ACh
binding causes TMD 3 to switch from one set of contacts to the other. A
preliminary account of some of these results has been given (16).
Experimental procedures were performed as described
previously (10, 12, 13).
Mutagenesis and Expression--
Briefly, residues 100-121 of
the rat M1 mAChR were individually mutated to Ala (or, if
they were Ala in the parent sequence, to Gly), using a polymerase chain
reaction method. After verification by dideoxy sequencing, mutant
receptors were subcloned into the pCD expression vector and expressed
transiently in COS-7 cells by electroporation using a Bio-Rad Gene
Pulser at 180 V and 960 microfarads with 10 µg of DNA/0.4-cm cuvette
(4 × 107 cells, 0.8 ml). Membrane preparations were
made after 72 h as described previously (13).
Binding and Functional Assays--
Binding of
(
Phosphoinositide (PI) turnover was assayed as described previously (12,
13). Briefly, transfected COS-7 cells were prelabeled with
[3H] inositol for 48 h, followed by washing and
preincubation with Krebs bicarbonate solution containing 10 mM LiCl for 30 min, and then ACh or other agonists were
added for 30 min. When the effects of antagonists on the basal
phosphoinositide response were to be studied, the antagonist was added
at different times up to 48 h before the performance of the PI
assay. In some instances, the antagonist was washed out (three changes
of Krebs bicarbonate solution without Li+ over a period of
about 15 min) before adding Li+ for 30 min to initiate the
PI assay.
Immunocytochemistry--
Expression of receptor protein was
assessed 72 h after transfection by incubation of fixed,
permeabilized cells with a 1:100 dilution of an immunoaffinity-purified
antiserum raised against the C-terminal 13 amino acids of the
M1 mAChR sequence (13). This was followed by incubation
with a 1:5000 dilution of an alkaline phosphatase-labeled second
antibody and visualization as described (13).
Materials--
( Data Analysis--
Binding curves were analyzed (10, 12, 13)
using SigmaPlot (SPSS Software Inc.). Ligand saturation curves were
fitted to a one-site model of binding to yield an affinity constant, KBin, and a total concentration of binding
sites, RT. Competition curves were fitted to the
Hill equation, or to one-site or two-site models of binding. Binding
constants were corrected for the Cheng-Prusoff shift, as necessary
(13). Unless otherwise stated, the agonist KBin
is taken to be the reciprocal of the corrected IC50 value. PI dose-response curves were fitted to a four-parameter logistic function, yielding a basal activity (PIbas), a maximum
response (PImax), an EC50 value, and a slope
factor. The slopes of the PI dose-response curves were, on average, not
significantly different from 1.0. The KAct value
is defined as 1/EC50. Emax is the
maximum receptor-induced signal, defined as
(PImax,mut
Agonist signaling efficacy values were calculated by fitting the ratio
([RG] + [ARG])/[GT] calculated from the
ternary complex model (17) to the dose-response data, where [RG] is
the concentration of the receptor-G protein binary complex, [ARG] is
the concentration of the agonist-receptor-G protein ternary complex,
and [GT] is the receptor-accessible
concentration of G protein. Unless the basal activity is raised, the
contribution of [RG] can be ignored. The calculation uses the agonist
binding constant, KBin, and an estimate of the
effective ratio of total receptor concentration to receptor-accessible
G protein, [RT]/[GT],
denoted RT. In performing these
calculations, we have used the value of 20 for
RT for the wild-type receptor
estimated in a previous study in which the effect of an irreversible
blocking agent on the ACh dose-response curve was studied (13). Values
of RT for the different mutants
were calculated from the expression of [3H]NMS binding
sites. For the L116A mutant, the calculated
RT value of 0.4 is consistent
with the experimentally determined Emax of 40%
of wild type (legend to Fig. 3).
The agonist signaling efficacy parameter is
KG·[GT] (denoted
KG), where KG
is the apparent affinity constant of the G protein for the ensemble of
agonist-receptor complexes. KG was
computed to reproduce the pEC50 of the PI dose-response curve.
For values of RT > 1, the efficacy
parameter, KG, was calculated as
follows.
3H-Labeled Antagonist Binding and Receptor
Expression--
Several Ala substitution mutations in the N-terminal
half of TMD 3 strongly reduced the affinity of the quaternary
antagonist NMS (Fig. 1a).
D105A and Y106A gave 100-fold reductions, measured by direct
radioligand saturation or by competition binding assays (10). 10-Fold
reductions followed mutations of Trp101,
Leu102, Asn110, and Val113. Large
decreases were also seen in the affinity of the tertiary antagonist QNB
(legend to Fig. 1), 6000-fold for D105A (measured by competition with
[3H]NMS), and 31-fold for Y106A. In contrast, mutations
in the C-terminal part of TMD 3 had little effect on NMS affinity. The
largest change was a 2-fold reduction, from I119A (Fig.
1a).
Ala substitutions in the C-terminal part of the domain had the largest
effects on the expression of [3H]NMS binding sites. The
L116A mutant was expressed at 2% of the wild-type level. Mutations of
Val113, Asn115, and Leu117 also
caused greater than 3-fold reductions (Fig. 1b). In the N-terminal region, only the mutation of Asp105 decreased
receptor expression, to 10% of the wild-type level. Unusually,
mutation of Asn110 somewhat enhanced receptor expression.
Alterations in the expression of the receptor protein were also
visualized by immunocytochemical staining of transfected cells using an
antibody directed against the C terminus of the receptor (Fig.
2). As noted previously, the apparent
variation in staining intensity was less than might be expected from
the variation in expression of binding sites, suggesting that,
particularly in the case of poorly expressing mutants such as D105A
(Fig. 2e), part of the receptor protein was misfolded or was
inappropriately localized (13).
Acetylcholine Binding--
Acetylcholine binding was assayed by
inhibition of the binding of [3H]NMS. ACh binding to the
wild-type receptor expressed in COS-7 cells gave a slightly flattened
isotherm (nH = 0.9) with a
KBin of 1.26 × 105
M
Three mutations located between the center and the C terminus of TMD 3 increased the affinity of ACh by between 4- and 30-fold. They were
V113A, L116A, and S120A.
ACh-stimulated Phosphoinositide Response--
In COS-7 cells
transfected with the wild-type M1 mAChR, ACh elicits a
robust phosphoinositide response, with an EC50 of 70 nM and a maximum stimulation of 3 times the basal activity
(13). Qualitatively, the effects of the Ala substitution mutations on the PI response were parallel to their effects on ACh binding. Mutations that decreased ACh affinity decreased the
KAct (defined as 1/EC50), while
mutations that increased ACh affinity increased the
KAct (Fig. 3b). However, S112A
decreased ACh potency without affecting its affinity.
The quantitative effects of the D105A, Y106A, and I119A mutations on
ACh potency in the PI response were much greater than their effects on
ACh binding. In particular, the D105A mutant completely failed to give
a PI response to ACh stimulation, and ACh potency was decreased by more
than 1000-fold by both the Y106A and I119A mutations. The basal PI
activities of most of the mutant receptors were between 92% (I119A)
and 112% of the wild-type basal, but that of the S120A mutant was
significantly raised (122 ± 6%; p < 0.01;
Basal = 0.07).
Computation of Agonist Signaling Efficacy--
Receptor-transducer
models (19), such as the ternary complex model (17), can be used to
quantitate the effects of mutations on signaling efficacy. Provided
that signal generation is directly proportional to the sum of the
concentrations of the RG binary and ARG ternary complexes, there exists
a quantitative relationship between the maximum response,
Emax (as a fraction of the maximum obtainable
signal); the KAct; the basal activity parameter,
Basal; and the agonist binding constant,
KBin (parameter definitions are given under
"Experimental Procedures"). When the ratio of receptor
concentration to accessible G protein concentration is greater than 1, the relationship is as follows.
A measure of agonist signaling efficacy, adjusted for differences in
receptor expression level, is provided by the affinity of the G protein
for the ensemble of agonist-receptor complexes, KG, multiplied by the concentration of
receptor-accessible G protein, [GT] (Refs. 13,
16, 18; see "Experimental Procedures"). The efficacy values
obtained for ACh activation of the TMD 3 mutants are illustrated in
Fig. 4b.
Deleting the side chains of Tyr106 and Ile119
caused reductions in the calculated signaling efficacy of ACh of
100-fold or greater. The D105A mutant had zero efficacy. There were
also 10-fold effects from Trp101, Ser109, and
Ser112. In contrast, the mutations of Leu116
and Ser120 caused 2.2- and 1.4-fold increases in the ACh
signaling efficacy, respectively.
GTP Shifts--
The effects of the nonhydrolyzable GTP analogue
GTP Pilocarpine Activation--
The mutants that showed increased ACh
affinity and efficacy were probed with the partial agonist pilocarpine.
Pilocarpine gave 78% of the maximum PI response elicited by ACh at the
wild-type receptor (Fig. 5). With respect
to the wild-type receptor, V113A and L116A gave approximately 10-fold
increases in affinity for pilocarpine, while S120A gave less than a
2-fold enhancement (Fig. 5). The potencies of pilocarpine in the PI
response were increased by all three mutations. At the V113A and S120A
mutants, pilocarpine became a full agonist. At the L116A mutant, its
maximum effect was maintained with respect to that of ACh, despite the
very low expression of the mutant receptor.
Calculation of the efficacy of pilocarpine gave a value equal to 1.4%
of the efficacy of ACh at the wild-type receptor. This was increased to
5% (3.6-fold) for the S120A and V113A mutants and to 30% (21-fold)
for the L116A mutant.
Tests for Constitutive Activity--
The mutants that showed
enhanced ACh and pilocarpine efficacies were examined for inverse
agonist effects. Preincubation of transfected cells with NMS for 24-48
h before the addition of LiCl and the isolation of phosphoinositides
reduced the basal activity of the S120A mutant (Basal = 7% of the maximum PI signal) to the wild-type basal level with an
IC50 of 10
Prolonged incubations of cells transfected with the L116A mutant with
NMS (Fig. 6a) or atropine increased the basal PI signal. To
see whether this effect might be partly mediated by an ability to
stabilize, and so up-regulate, the receptor, we examined the effect of
atropine on the level of expression of the L116A mutant by
immunocytochemistry and by [3H]NMS binding.
A 48-h incubation of the cells with atropine increased the accumulation
of the L116A protein (Fig. 2d) but not the wild-type receptor (Fig. 2c). Measurement of [3H]NMS
saturation curves after atropine pretreatment of the cells showed no
effect on the wild-type receptor, but the expression of the L116A
mutant was increased by up to 50-fold, close to the wild-type level,
without changing the NMS affinity (Fig. 6b) or the ACh
binding properties (data not shown). Preincubation with atropine
(10
Atropine pretreatment of the transfected cells, followed by washout,
before measurement of the PI response increased the basal activity of
the L116A mutant to a level equivalent to 70-120% of the maximum
signal obtained in cells transfected with the wild-type receptor. The
enhanced basal activity was partially inhibited by the addition of
atropine, with an IC50 of 10 One category of residues is those whose mutation had minimal
effect, defined as less than a 3-fold change in the level of receptor
expression, in NMS and ACh affinities and less than a 6-fold change in
ACh functional potency. Five of these (Leu100,
Ala103, Leu104, Val107, and
Ala108) occur in the N-terminal half of TMD 3, and three
(Met114, Leu118, and Phe121) occur
in the C-terminal half. In a helical wheel plot, they are clustered in
one sector (Fig. 7a), strongly
supporting an -helical secondary structure of the domain and
suggest its orientation with respect to the other transmembrane
domains. The C-terminal part of the helix appears to be largely buried
within the receptor structure. On its surface, there is a patch of
three residues, Val113, Leu116, and
Ser120, which may form intramolecular contacts that help to
stabilize the inactive ground state of the receptor. Mutagenic
disruption of these increased agonist affinity and signaling efficacy.
In two cases (L116A and S120A), this led to constitutive activation of
the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of
transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the
activated state. The binding of acetylcholine may destabilize the first
set of contacts while favoring the formation of the second.
INTRODUCTION
Top
Abstract
Introduction
References
-helical character (6).
-carbon (in this study, Ala
residues themselves were replaced by Gly), is normally well tolerated
and allows the functions of individual side chains to be probed and
analyzed (15). We have applied this systematic approach to residues
100-121 of TMD 3 of the M1 mAChR.
EXPERIMENTAL PROCEDURES
)-N-[3H]methylscopolamine
([3H]NMS), and (
)-[3H]quinuclidinyl
benzilate ([3H]QNB) to membrane preparations was measured
at 30 °C in a buffer containing 20 mM Na-HEPES, 100 mM NaCl and 1 mM MgCl2 using an incubation time of 60 min to 3 h, as described previously (10). The
binding of ACh and other unlabeled ligands was measured by inhibition
of the binding of the tritiated antagonist. For the study of guanine
nucleotide shifts, MgCl2 was replaced by 2 mM MnCl2 (12).
)-N-[3H]methylscopolamine
(85 Ci/mmol), (
)-[3H]quinuclidinyl benzilate (50 Ci/mmol), and myo-D-[3H]inositol (80 Ci/mmol)
were obtained from Amersham Pharmacia Biotech. Guanosine
5'-O-(3-thiotriphosphate) tetralithium salt, was from
Boehringer Mannheim.
PIbas,wt)/(PImax,wt
PIbas,wt). For two constitutively active
mutants, the basal activity parameter, denoted Basal, was
calculated relative to the wild-type receptor as
(PIbas,mut
PIbas,wt)/(PImax,mut
PIbas,wt). With these two exceptions,
Basal = 0.
or
(Eq. 1)
These equations are extensions of those derived by Whaley
et al. (18). Other details are given by Hulme and Lu (16). When RT was <1, the efficacy
was calculated from a fit of the ternary complex model to the
dose-response data, as described previously (13).
(Eq. 2)
RESULTS
View larger version (13K):
[in a new window]
Fig. 1.
Effects of mutations of Leu100
through Phe121 on the binding of
[3H]NMS. Results are the mean of three or more
independent experiments. a, effects on the affinity. Results
are expressed as the log of the ratio of the affinity of NMS
(designated KNMS) for the mutant to the affinity
for the wild-type receptor ± S.E. The affinity of NMS for the
wild-type receptor was 1.0 ± 0.05 × 1010
M 1. The effects on the binding of
[3H]QNB were measured for selected mutants. The affinity
of [3H]QNB for the wild-type was 3.6 ± 0.03 × 1010 M
1. The logs of the affinity
ratios were as follows:
1.2 ± 0.05 (W101A);
0.8 ± 0.05 (L102A);
3.83 ± 0.07 (D105A);
1.49 ± 0.05 (Y106A)
0.0 ± 0.01 (S109A);
0.75 ± 0.08 (N110A). b,
effects on the expression. Results are expressed as the log of the
ratio of expression (designated RT) of the
mutant to the expression of the wild-type receptor in a parallel
transfection ± S.E. Expression of the wild-type receptor varied
from 0.5 to 1.2 pmol/mg of protein in different transfections.
View larger version (129K):
[in a new window]
Fig. 2.
Immunocytochemical visualization of
expression of mutants using an anti-C-terminal antibody. The
magnification is × 100. a, wild type; b,
L116A; c, wild type, after 48-h pretreatment of the
transfected cells with atropine (10 6 M);
d, L116A after 48-h pretreatment with atropine
(10
6 M); e, D105A; f,
nontransfected control.
1 (10). Mutations that diminished NMS
affinity caused reductions in the affinity of ACh (Fig.
3a). Mutation of
Ser109, which was without effect on NMS binding, gave a
10-fold reduction in the affinity of ACh.
View larger version (15K):
[in a new window]
Fig. 3.
Effects of mutation of Leu100
through Phe121 on the binding affinity and signaling
potency of acetylcholine. Results are the mean of three or more
independent experiments. a, effects on the affinity of ACh.
Results are expressed as the log of the ratio of the affinity
(designated KACh) of ACh for the mutant to that
for the wild-type receptor ± S.E. KACh for
the wild-type receptor was 1.26 ± 0.1 × 105
M 1. The Hill coefficient of the wild-type
binding curve was 0.89 ± 0.02. and was unaltered except for D105A
(1.0 ± 0.1), Y106A (0.97 ± 0.01), S109A (0.96 ± 0.03), N110A (0.95 ± 0.02), L116A (0.62 ± 0.08), I119A
(0.95 ± 0.04), and S120A (0.71 ± 0.07). b,
effects on the potency of ACh. Results are expressed as the log of the
ratio of 1/EC 50 (designated KAct)
of ACh in the PI response elicited by the mutant to that elicited by
the wild-type receptor ± S.E. The KAct for
ACh at the wild-type receptor was 1.26 ± 0.1 × 107 M
1, and the ratio of the
maximum ACh-stimulated signal to the basal signal was 3.3 ± 0.1. The D105A mutant was inactive in the PI assay. The basal activities of
the mutant receptors were between 92 and 112% of the wild-type basal,
except for S120A, which was 122 ± 6% of wild-type basal. The
Emax values were 100% of the wild-type, except
for D105A (0%), Y106A (70 ± 4%), S112A (81 ± 7%), N115A
(67 ± 5%), L116A (40 ± 2%), and I119A (31 ± 7%).
For the present set of mutants, as well as R123A, R123K (12), and
D105E (10), this relationship is reasonably well obeyed (Fig.
4a). In only one case, N115A,
was the deviation of the measured from the predicted
Emax greater than 20% of the total signal, suggesting a reduction in the signaling activity of the ternary complex
in this case.
(Eq. 3)
View larger version (12K):
[in a new window]
Fig. 4.
Analysis of ACh-stimulated PI signal using
the ternary complex model. a, relationship between the
measured and the predicted Emax values. The
predicted Emax was calculated as shown in
Equation 3. Emax values are expressed relative
to that of the wild-type receptor. b, log of the efficacy of
ACh at the mutant relative to the efficacy at the wild-type receptor.
The method of calculation is described under "Experimental
Procedures." The values of the efficacy parameter calculated for the
wild-type receptor, L116A, and S120A were 6.4, 13.8, and 8.8, respectively. The D105A mutant had zero efficacy.
S (10
5 M) were investigated on the
mutants that showed enhanced ACh affinity and efficacy. Binding studies
were performed in the presence of 2 mM Mn2+
ions to promote the stability of the agonist-receptor G protein complex
(12). Mn2+ increased the affinity of ACh for the wild-type
receptor by 1.5-fold relative to the standard
Mg2+-containing buffer, but no significant GTP
S shift
resulted. The V113A, and L116A mutants also showed no GTP shift.
Mn2+ enhanced the affinity of ACh for the S120A mutant by
3-fold; in this case, the binding data could be described by a two-site model of binding (pKdL = 5.74 ± 0.01 (60%
of total sites); pKdH = 7.33 ± 0.06 (40%
of total sites)). GTP
S gave a significant decrease in the ACh
affinity for this mutant. Analysis showed a 3.5-fold decrease in the
high affinity binding constant (pKdH = 6.79 ± 0.11; p < 0.05) without an effect on the low
affinity binding constant. The partial nature of this effect indicates that only a fraction of the high affinity sites were affected.
View larger version (12K):
[in a new window]
Fig. 5.
Pilocarpine binding and stimulation of the
wild-type, V113A, L116A, and S120A mutants. Results are expressed
as mean ± S.E. of three independent experiments.
Emax is expressed relative to the value obtained
for ACh in the same cells.
8 M (Fig.
6a). There was no detectable
effect of NMS on the basal activity of the wild-type receptor, but in
some experiments a reduction of about 3% in the V113A Basal
parameter was seen (not shown).
View larger version (14K):
[in a new window]
Fig. 6.
Analysis of S120A and L116A mutants for
constitutive activity. Results are representative experiments that
were repeated three times with essentially the same results.
a, effect of pretreatment of transfected cells with NMS on
the PI response. Different concentrations of NMS were added to the
transfected cells for 48 h before measurement of the PI response.
, S120A;
, L116A. b, effect of pretreatment with
atropine on the the binding of [3H]NMS to L116A and
wild-type receptors. Atropine (10
6 M) was
added to the transfected cells for 48 h before harvesting and the
preparation of membranes.
, wild type;
, wild type plus atropine;
, L116A;
, L116A plus atropine. c, effect of
pretreatment with atropine on the PI response of L116A and wild-type
receptors.
and
, wild type;
and
, L116A. Open
symbols, pretreated with 10
6 M
atropine for 48 h followed by washout; closed
symbols, not pretreated. Full lines,
challenged with carbachol; dotted lines,
challenged with atropine.
4 M) also doubled the expression of the
D105A mutant (data not shown).
8 M
(Fig. 6c). The PI signal was stimulated by 40% by the
addition of carbachol (Fig. 6c) or ACh (not shown). The
Basal parameter was equivalent to 70% of the maximum signal
in these cells. In contrast, pretreatment with atropine had no effect
on the PI response of the wild-type receptor.
DISCUSSION
-helical conformation for TMD 3. Cys substitution
mutants of the equivalent positions in the D2 receptor
failed to react with a polar sulfhydryl reagent (6). In both receptor
types, these residues probably face toward the lipid bilayer. A helical
net representation (Fig. 7b) shows that the sector of TMD 3 in which mutations can be made without major effect narrows toward the cytoplasmic end of the helix, in agreement with models based on the
structure of rhodopsin, which suggest that this part of the helix is
mostly surrounded by the other TMDs (1).
View larger version (17K):
[in a new window]
Fig. 7.
Structural plots of residues in
transmembrane domain 3. a, helical wheel plot.
Filled symbols show the locations of the null
mutations. b, helical net representation of the changes
produced by the mutations. The radii of the circles indicate the
changes in expression level,
log(RT,mut/RT,wt).
Changes in expression of less than 3-fold are shown as
circles of unit radius. The filled
arrows represent the change in the ground state binding
constant of ACh, log(KACh,mut/KACh,wt). The
open arrows represent the change in the stability
of the ternary complex relative to the the binary complex,
log(
K·KG,mut)/(
K·KG,wt). The
right pointing arrows indicate a
decrease, and left pointing arrows
indicate an increase in these parameters. Details of the method of
calculation are in Ref. 16. The dotted arrow
denotes decreased catalytic activity of the ternary complex. *, a
10-fold decrease in NMS affinity; **, a 100-fold decrease in NMS
affinity. Residues whose mutation increased both ACh affinity and
signaling efficacy are colored red. Residues whose mutation
decreased signaling efficacy are colored dark
blue. Those whose mutation only decreased ACh affinity are
colored light blue. Asp122
(D122) and Tyr124 (Y124), for which
mutations decrease expression levels and in several cases increase ACh
affinity, are colored pink; expression levels are for the
D122A-d loop and Y124C mutants (13).
The residues whose mutation lowered receptor expression levels have the opposite distribution. They are concentrated in the C-terminal half of TMD3 (Fig. 7b) and are spread over 220° of arc. We have argued that the side chains of such residues make intramolecular contacts that contribute to the stability of the transmembrane structure of the ground state of the receptor (13). Their removal reduces the free energy of the folded state with respect to inactive intermediates on the folding pathway. Besides Asp122 and Tyr124 (13), we have now found a third position, Leu116, at which a side chain deletion strongly reduced the level of expression. Three other mutations in the C-terminal part of the helix also had significant effects. Only one such sensitive residue occurs in the N-terminal half of the helix. Interestingly, it is the binding site aspartate itself. The distribution of these mutants suggests that the side chains of residues in the C-terminal half of TMD 3 make more frequent intramolecular contacts than those in the N-terminal half. The free energy difference between the folded and unfolded states was restored by incubation of the expressing cells with a high affinity antagonist. This counteracted the effect of the L116A mutation on expression and doubled the expression of the D105A mutant.
At three of the six positions in the C-terminal half of the domain
(Val113, Leu116, and, as reported in Ref. 13,
Asp122), mutations that decreased expression levels
increased ACh affinity; in two cases (Val113 and
Leu116), signaling efficacy and/or basal activity was also
increased. Mutations of acidic residues homologous to
Asp122 in rhodopsin (20, 21), the 1 receptor (22), and
the
2 receptor (23) increase basal activity, but we have not
observed this for mutations of Asp122 in the M1
mAChR, even after antagonist preincubation to increase the expression
levels.2 In contrast, S120A
behaved like a classical constitutively active mutant, showing raised
basal activity, increased agonist affinity, enhanced efficacy of a
partial agonist, and an increased GTP shift (24, 25). Together,
Val113, Leu116, and Ser120 form a
compact patch of residues located on the inward facing surface of the
cytoplasmic half of TMD 3 (Fig. 7b).
In the extended ternary complex model of agonist-receptor-G protein
interaction, a conformational equilibrium is postulated to exist
between preexisting ground and active states of the receptor, governed
by an isomerization constant, K (24). Agonists bind to the ground state
conformation with low affinity, Ka, but to the activated state
with a higher affinity, ·Ka. In the simplest case, the
G-protein binds selectively to the activated conformation. The extended
ternary complex model offers three routes to mutational effects on
efficacy: a change in the isomerization constant, K; a change in the
agonist cooperativity,
; or a change in the affinity of the G
protein for the activated receptor, KG.
A selective increase in K provides the most economical explanation for
the simultaneously increased basal activity, agonist affinity, and
signaling efficacy of the group of constitutively active mutants (24),
although it is not possible entirely to exclude an increase in
KG, combined with a simultaneous increase in Ka or
in the cooperativity factor . A priori, an increase in
KG seems unlikely to result from a side chain deletion.
Detailed calculations based on the assumption of a changed
isomerization constant (described in Ref. 16) have suggested a value of
about 2 × 104 for K for the wild-type
M1 receptor and increases of about 2- and 40-fold in this
parameter for the S120A and L116A mutants. Particularly in the case of
L116A, this may reflect a selective destabilization of the ground state
of the receptor, because the expression of the mutant was decreased by
approximately the same ratio. These changes are enough to account for
the observed increases in the ACh binding affinity, as well as in the
ACh signaling efficacy, without an increase in the ground state binding
constant, Ka (16). However, in the case of the L116A mutant,
Ka for pilocarpine may be increased. The pharmacology of this
mutant would repay further investigation, particularly as it retains some signaling activity even in the presence of NMS and atropine (Fig.
6, a and c). This may imply a partial uncoupling
of the ligand binding site from the G protein binding site.
For V113A, there is a 30% increase in the signaling efficacy of ACh (16), and a 3-fold increase for pilocarpine. However, the 10-fold increases observed in ACh and pilocarpine binding affinity and a significant decrease in antagonist affinity imply an additional perturbation of the ground state conformation of the receptor by this mutation.
The residues whose mutation decreased the signaling efficacy of ACh form a strip extending the full length of TMD 3 (Fig. 7b). In the outer, N-terminal, part of the helix, they lie on the opposite face to the sector of null residues. In the inner, C-terminal, part, they are displaced by one residue (100° of arc) from the group of residues whose mutations enhanced signaling.
The mutation of Trp101, Asp105, Tyr106, and Ser109 strongly reduced ACh signaling efficacy. Mutation of these residues also reduced the ground state binding of ACh and, with the exception of Ser109, decreased the affinities of NMS and QNB. Mutation of the two more peripheral residues, Leu102 and Asn110, inhibited ground state binding of ACh but had little effect on receptor activation. Our studies suggest that Tyr106 contributes almost as much free energy as Asp105 to the binding of NMS. This may indicate an amino-aromatic interaction (26), since mutation of the equivalent Tyr to Phe in the M3 mAChR had little effect on the binding of NMS, although it reduced the affinity of ACh (27). In contrast, Asp105 plays a much larger part in the binding of the tertiary amine QNB, consistent with the formation of a charge-reinforced hydrogen bond. In the case of Trp101, the aromaticity of the side chain has been reported to be important for both NMS and ACh binding (28). In general, the data support the conjecture of Hibert et al. that aromatic as well as polar interactions are important for the binding of muscarinic ligands (29).
These findings underline the primary role of Asp105, supported by Tyr106 in ACh binding and signal transduction (9, 10, 27). Notably, Asp105 is the only residue in this set whose mutation completely abolished signaling. However, Asp105 appears to have a complex function. It may contribute to the stability of the receptor's ground state structure, as well as being vital for ligand contact. This may imply that its side chain also forms an intramolecular interaction, as appears to be the case for Glu113 in rhodopsin (30).
The tightly delimited strip of residues whose mutation strongly diminished ACh signaling efficacy extends to the cytoplasmic end of the helix. At this point, it becomes continuous with residues homologous to the functionally conserved residues recently identified by random saturation mutagenesis in the second intracellular loop of the M5 mAChR (31), which provides a cytoplasmic extension of TMD 3.
Mutation of Ser112 and particularly Ile119 as well as Arg123 (12) had large effects on ACh signaling efficacy. However, these mutations did not strongly affect receptor expression levels, suggesting that these residues make limited contributions to the stability of the ground state.
The mutation S112A caused a small but significant enhancement of NMS affinity, while I119A decreased NMS as well as ACh affinity. We suggest that the side chains of these two residues make weak intramolecular contacts in the ground state and that these become stronger in the active conformation of the receptor. An interdomain contact has also been postulated for the residue homologous to Ile119 in the GnRH receptor (32). If this is true, the main effect of the deletion of these side chains should be to decrease the isomerization constant, K. However, it is not possible to exclude the possibility of a more direct role for them in the formation of the G protein binding pocket, leading to effects on KG; the basal activity of the I119A mutant is decreased,2 showing that there are reductions in the product K·KG, which governs basal activity. Arg123 also has the potential for direct binding to the G protein as well as for intrareceptor contacts (12). With the possible exception of Asn115, there is no evidence that these residues influence the catalytic steps within the ARG ternary complex that occur subsequent to its formation.
In summary, scanning mutagenesis has revealed strong functional
differentiation of the surface of TMD 3 of the M1 mAChR. It has demonstrated the -helical secondary structure of the domain and
suggested its orientation with respect to the other transmembrane elements and the lipid bilayer. The results suggest that the C-terminal half of the helix is deeply buried within the receptor structure. On
the buried surface, we propose that a distinct group of amino acid
side-chains make intramolecular bonds that selectively stabilize the
ground state of the receptor. Mutagenic disruption of these characteristically decreases receptor expression and increases agonist
affinity and signal transduction. Running parallel to the helix axis
and spanning the whole transmembrane region, a topologically distinct
strip of residues on one face of TMD 3 forms intermolecular
(ACh-receptor, receptor-G protein) or intramolecular bonds that are
necessary for the stability or activity of the ARG ternary complex.
Mutation of these decreases signal transduction. The binding of ACh may
disrupt the first set of contacts while promoting the second. This may
trigger the rotational movement of TMD 3 deduced from spectroscopic
studies of rhodopsin (4) and the
2 receptor (23), and
could mobilize the cytoplasmic ends of the surrounding TMDs. We propose
that TMD 3 and its cytoplasmic extension (31) may act as an
intramolecular rotary switch in receptor activation. Several of the key
amino acids, particularly Ser112, Leu116,
Ile119, Ser120, Asp122,
Arg123, and Tyr124, are very highly conserved
in the G protein-coupled receptor superfamily (1), suggesting the
potential generality of such a mechanism.
![]() |
FOOTNOTES |
---|
* This work was supported by the Medical Research Council, United Kingdom, and by a Travelling Fellowship from the Wellcome Trust (to Z-L. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44181-959-3666;
Fax: 44181-906-4477; E-mail: e-hulme{at}nimr.mrc.ac.uk.
2 Z-L. Lu, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TMD, transmembrane
domain;
ACh, acetylcholine;
mAChR, muscarinic acetylcholine receptor;
NMS, ()-N-methyl scopolamine;
QNB, (
)-3-quinuclidinyl
benzilate;
PI, phosphoinositide;
GTP
S, guanosine
5'-3-O-(thio)triphosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|