(Received for publication, December 4, 1995; and in revised form, January 12, 1996)
From the
We have recently shown that a four-amino acid epitope (VTIL) on
the m2 muscarinic receptor (corresponding to Val,
Thr
, Ile
, and Leu
) is
essential for G
coupling specificity and G
activation (Liu, J., Conklin, B. R., Blin, N., Yun, J., and Wess,
J.(1995) Proc. Natl. Acad. Sci. U. S. A. 92,
11642-11646). Because this sequence element is thought to be
located at the junction between the third intracellular loop and the
sixth transmembrane helix (TM VI), we speculated that agonist binding
to the m2 receptor protein results in conformational changes that
enable the VTIL motif to interact with G
proteins. To
test the hypothesis that such structural changes might involve a
relative movement of TM VI toward the cytoplasm, we created a series of
mutant m2 muscarinic receptors in which one to four extra Ala residues
were inserted into TM VI immediately after Leu
. Based on
the geometry of an
-helix, such mutations are predicted to
``push'' the VTIL sequence away from the lipid bilayer.
Consistent with our working hypothesis, second messenger assays with
transfected COS-7 cells showed that all mutant m2 receptors containing
extra Ala residues C-terminal of Leu
could activate the
proper G proteins even in the absence of agonist. However, replacement
of the VTIL motif in such constitutively active m2 receptors with the
corresponding m3 muscarinic receptor sequence (AALS) or deletion of
Ala
from the wild type m2 receptor completely abolished G
protein coupling. Interestingly, introduction of extra Ala residues
C-terminal of the AALS motif in the m3 muscarinic receptor completely
abolished functional activity. Mutant m2 and m3 receptors that
contained extra Ala residues immediately N-terminal of the VTIL and
AALS motif, respectively, displayed wild type-like coupling properties.
Our data are consistent with a model in which agonist binding to the m2
muscarinic receptor leads to a relative movement of TM VI toward the
cytoplasm, thus enabling the adjacent VTIL sequence to interact with
the C terminus of G
subunits.
All members of the superfamily of G protein-coupled receptors
are predicted to share a similar molecular architecture consisting of
seven -helically arranged transmembrane domains (TM I-VII) (
)connected by three extracellular and three intracellular
loops (i1-i3) (Watson and Arkinstall, 1994). Binding of an
agonist to the receptor protein (which involves residues in the
extracellular receptor domains and/or the TM helices) is predicted to
cause conformational changes in the TM receptor core that are
propagated to the intracellular receptor surface where the interaction
with specific classes of G proteins is thought to occur (Dohlman et
al., 1991; Savarese and Fraser, 1992; Hedin et al., 1993;
Wess, 1993; Strader et al., 1994). The molecular nature of
these agonist-induced conformational changes remains unknown at
present.
We have used the m2 and m3 muscarinic acetylcholine
receptors as model systems to study the molecular basis of receptor/G
protein coupling selectivity and receptor-mediated G protein activation
(Wess, 1996). Whereas the m2 receptor is selectively linked to G
proteins of the G class (primary biochemical response:
inhibition of adenylyl cyclase), the m3 receptor is preferentially
coupled to G proteins of the G
family (primary
biochemical response: stimulation of phosphatidylinositol (PI)
hydrolysis via activation of phospholipase C
) (Peralta et
al., 1988; Parker et al., 1991; Berstein et al.,
1992; Offermanns et al., 1994).
In a recent study (Liu et al., 1995a), we identified a four-amino acid epitope on the
m2 muscarinic receptor (VTIL, corresponding to Val,
Thr
, Ile
, and Leu
; see Fig. 1) that is essential for G
coupling
specificity and G
activation. In agreement with this
notion, substitution of this structural motif into the wild type m3
muscarinic receptor resulted in a mutant receptor that gained the
ability to mediate inhibition of adenylyl cyclase (Liu et al.,
1995a). Moreover, coexpression studies with hybrid m2/m3 muscarinic
receptors and C-terminally modified mutant G protein
(G
) subunits suggested that the VTIL epitope
(corresponding sequence in the m3 receptor: AALS; see Fig. 1)
can functionally interact with the C-terminal five amino acids of
G
subunits of the G
family. Consistent with this
notion, only those mutant receptors that contained the VTIL motif were
able to activate mutant G
subunits in which the last
five amino acids of G
were replaced with the
corresponding
(qi5) or
(qo5)
sequence (Liu et al., 1995a).
Figure 1: Amino acid sequences of the m2 and m3 muscarinic receptors at the junction between the i3 loop and TM VI. Numbers refer to amino acid positions in the human m2 and the rat m3 muscarinic receptors, respectively (Bonner et al., 1987). Mutant muscarinic receptors were created by inserting one or more alanine residues at the indicated positions (arrows). The abbreviations for the amino acids residues are as follows: A, Ala; I, Ile; K, Lys; L, Leu; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val. Residues known to be important for proper G protein recognition are highlighted in black (Blin et al., 1995; Liu et al., 1995a).
Whereas Val and
Thr
are predicted to be located at the C terminus of the
i3 loop of the m2 muscarinic receptor, Ile
and
Leu
are thought to be contained within the N terminus of
TM VI (see Fig. 1; Bonner et al., 1987; Hulme et
al., 1990). Computational approaches suggest that the region at
the i3 loop/TM VI junction in muscarinic and other G protein-coupled
receptors is
-helically arranged (Strader et al., 1989).
Based on this notion, Val
, Thr
,
Ile
, and Leu
are predicted to be located on
one side of an
-helix and may thus form a contiguous hydrophobic
surface that can interact with the C terminus of G
subunits. We therefore hypothesized that this receptor surface
becomes available for interaction with G
subunits only in the
agonist-bound receptor conformation, resulting perhaps from an
agonist-induced rotation or movement toward the cytoplasm of the
N-terminal portion of TM VI and the adjacent loop sequence.
To test
this hypothesis, we speculated that such structural changes might be
mimicked (at least partially) by the insertion of one or more extra Ala
residues into the N-terminal segment of TM VI of the m2 muscarinic
receptor, immediately after Leu (see Fig. 1).
Consequently, a series of mutant m2 receptors containing one or more
additional Ala residues C-terminal of Leu
were created
and studied for their ability to mediate inhibition of adenylyl cyclase
and to functionally interact with C-terminally modified mutant
G
subunits such as qo5 or qi5. For comparison, the
functional effects of inserting one or two additional Ala residues
immediately N-terminal of Val
were also examined.
Moreover, the m3 muscarinic receptor was structurally modified in a
fashion analogous to that described for the m2 receptor to study the
effects of such mutations on the function of a G
-coupled
receptor.
Consistent with our working hypothesis, we show in this
study that mutant m2 muscarinic receptors containing one or more
additional Ala residues after Leu are able to activate G
proteins even in the absence of agonist. In contrast, similarly
modified mutant m3 muscarinic receptors are functionally completely
inactive, suggesting that the molecular mode of receptor-mediated G
protein activation may differ between the G
- and
G
-coupled muscarinic receptors.
The construction
of pcDNAI-based expression plasmids coding for wild type murine
G, wild type murine G
(Sullivan et al., 1986), and the various mutant G
subunits has been described previously (Conklin et al.,
1993).
The functional properties of all wild type and mutant m2 and m3 muscarinic receptors were examined after their transient expression in COS-7 cells. Mutant muscarinic receptors containing extra Ala residues were denoted as m2(x + yA) (or m3(x + yA)) where x indicates the position of the amino acid in the human m2 and rat m3 receptor (Bonner et al., 1987), respectively, after which y extra Ala residues were inserted (Fig. 1).
We first examined the ability of the m2(390
+ 1A) mutant receptor to functionally interact with G subunits in which the last five amino acids were replaced with
the corresponding
(qi5) or
(qo5)
sequences. Consistent with published results (Liu et al.,
1995a), the wild type m2 receptor, when coexpressed with qo5 or qi5 and
challenged with the muscarinic agonist carbachol (1 mM), was
able to induce a pronounced increase in phospholipase C activity
(4-7-fold increase in inositol phosphate levels above basal; Fig. 2). This effect was not observed upon coexpression of the
wild type m2 receptor with wild type G
(q(wt)) or a
mutant G
subunit (qs5) in which the last five amino
acids of q(wt) were replaced with the corresponding
sequence (Fig. 2; Liu et al., 1995a). As shown in Fig. 2, the m2(390 + 1A) mutant receptor displayed a G
protein coupling pattern very similar to that of the wild type m2
receptor (no or poor coupling to q(wt) and qs5 but efficient coupling
to qo5 and qi5). However, in contrast to the wild type receptor, m2(390
+ 1A) was able to activate qo5 and qi5 even in the absence of
agonist (3-4-fold stimulation in phospholipase C activity above
basal; Fig. 2). The addition of carbachol led to only a minor
increase (10-20%) in the magnitude of this response (Fig. 2). The agonist-independent increase in inositol phosphate
production seen with cells coexpressing m2(390 + 1A) and qo5 or
qi5 could be completely prevented by the addition of the muscarinic
antagonist, atropine (5 µM; Fig. 2).
Figure 2:
Stimulation of PI hydrolysis mediated by
the wild type m2 and the m2(390 + 1A) mutant muscarinic receptor
after coexpression with mutant G subunits. A,
COS-7 cells were cotransfected with expression plasmids coding for the
wild type m2 (top) or the m2(390 + 1A) mutant receptor (bottom) and the indicated G
subunits. In qo5, qi5, and
qs5, the last five amino acids of wild type G
(q(wt); EYNLV) were replaced with the corresponding sequences derived
from
(GCGLY),
(DCGLF), or
(QYELL),
respectively. About 48 h after transfections, cells were incubated for
1 h (at 37 °C) in the presence of the agonist carbachol (1
mM) or the antagonist atropine (5 µM) or in the
absence of drugs (basal). The resulting increases in
intracellular inositol monophosphate levels were determined as
described under ``Experimental Procedures.'' The data are
expressed as means ± S.E. and are representative of a single
experiment carried out in triplicate; one or two additional experiments
gave similar results. B, immunoblot analysis showing similar
levels of expression for wild type and mutant G
subunits in COS-7 cells cotransfected with the wild type m2
receptor. G
subunits were detected by Western blotting using the
12CA5 antibody as described under ``Experimental Procedures''
(Wedegaertner et al., 1993).
Consistent
with the results of the PI assays, the m2(390 + 1A) mutant
receptor also gained the ability to inhibit AVP-stimulated cAMP
production (when coexpressed with the wild type V2 vasopressin receptor
and wild type G) in an agonist-independent fashion
(20-25% inhibition; Fig. 3). The addition of carbachol
(0.1 mM) did not lead to a significant further increase in the
magnitude of this response. In contrast, efficient inhibition of
adenylyl cyclase activity (40-45%) by the wild type m2 receptor
was observed only in the presence of carbachol (Fig. 3).
Figure 3:
Inhibition of adenylyl cyclase by wild
type and mutant m2 muscarinic receptors. The structures of the various
m2 insertion mutants are given in Fig. 1. COS-7 cells
cotransfected with muscarinic receptor DNA and plasmids coding for the
V2 vasopressin receptor and wild type G were studied
for their ability to mediate carbachol-induced (0.1 mM)
inhibition of AVP-stimulated cAMP levels (Liu et al., 1995a).
Basal cAMP levels (no drug added) were not significantly different
between cells expressing the wild type or the various mutant receptors
(wild type m2 receptor: 1780 ± 220 cpm/well). The data are
expressed as the percentage of inhibition of maximum cAMP production
induced by 0.5 nM AVP (100%; 18 ± 3-fold above basal
levels), determined in the presence of pertussis toxin (500 ng/ml). The
data are given as means ± S.E. of triplicate determinations in a
single experiment; a separate experiment gave similar
results.
Figure 4:
Stimulation of PI hydrolysis mediated by
wild type and mutant m2 muscarinic receptors coexpressed with wild type
G (q(wt)) or the mutant G
subunit
(qo5). The structures of the various m2 insertion mutants are given in Fig. 1. In the m2(390 + 1A/VTIL->AALS) and m2(390 +
2A/VTIL->AALS) mutant receptors, Val
,
Thr
, Ile
, and Leu
(m2
receptor sequence) were replaced with the corresponding m3 receptor
residues (Ala
, Ala
, Leu
, and
Ser
; Fig. 1). In m2(
A391), Ala
was deleted from the wild type m2 receptor. qo5 is a mutant
G
subunit in which the C-terminal five amino acids of q(wt) were
replaced with the corresponding
sequence. COS-7 cells
coexpressing individual mutant receptors and q(wt) or qo5 were
incubated for 1 h (at 37 °C) in the absence or the presence of the
agonist carbachol (1 mM). The resulting increases in
intracellular inositol monophosphate levels were determined as
described under ``Experimental Procedures.'' The data are
expressed as means ± S.E. and are representative of a single
experiment carried out in triplicate; three to five additional
experiments gave similar results.
To examine whether the presence of
the VTIL motif was required for agonist-independent signaling,
Val, Thr
, Ile
, and
Leu
were replaced with the corresponding m3 receptor
residues (AALS; corresponding to Ala
, Ala
,
Leu
, and Ser
; Fig. 1) in the m2(390
+ 1A) and m2(390 + 2A) mutant receptors (resulting in m2(390
+ 1A/VTIL->AALS) and m2(390 + 2A/VTIL->AALS),
respectively). As illustrated in Fig. 4, this modification
completely abolished the ability of m2(390 + 1A) and m2(390 +
2A) to functionally interact with the mutant G
subunit, qo5 (in the absence or the presence of agonist). Similar
results were obtained when Ala
was deleted from the wild
type m2 receptor (yielding m2(
A391); Fig. 4).
Figure 5: Stimulation of PI hydrolysis mediated by wild type and mutant m3 muscarinic receptors. The structures of the various m3 receptor insertion mutants are given in Fig. 1. COS-7 cells transiently expressing the indicated wild type and mutant receptor constructs were incubated for 1 h (at 37 °C) either in the absence (basal) or the presence of the agonist carbachol (1 mM). The resulting increases in intracellular inositol monophosphate levels were determined as described under ``Experimental Procedures.'' The data are given as means ± S.E. and are representative of four independent experiments, each carried out in triplicate.
In addition, two m3 mutant receptors were created
(m3(487 + 1A) and m3(487 + 2A)) that contained one or two
extra Ala residues N-terminal of Ala (Ala
corresponds to Val
in the m2 receptor; Fig. 1). Consistent with the results obtained with the
structurally homologous m2 receptor mutants ( Fig. 3and Fig. 4), m3(487 + 1A) and m3(487 + 2A) retained the
ability to mediate a robust agonist-dependent PI response
(5-6-fold increase in phospholipase C activity; Fig. 5).
Based on the results of a previous study (Liu et
al., 1995a), we speculated that the insertion of one or more extra
Ala residues into the endofacial segment of TM VI of the m2 muscarinic
receptor immediately C-terminal of Leu might be able to
mimic (at least partially) the agonist-induced conformational changes
in the receptor protein. Radioligand binding studies showed that the
resulting m2 mutant receptors displayed agonist and antagonist binding
affinities that closely resembled those found with the wild type m2
receptor. Because the binding of muscarinic ligands is known to involve
specific residues on TM VI and several other TM domains (Hulme et
al., 1990; Wess, 1993), this observation indicates that the
insertion of extra Ala residues C-terminal of Leu
did not
interfere with the proper assembly of the TM helical bundle. The
presence of extra residues at the N terminus of TM VI is therefore
predicted to ``push'' the adjacent VTIL motif further away
from the lipid bilayer into the cytoplasm.
Consistent with our
working hypothesis, we found that all mutant m2 receptors that
contained additional (one to four) Ala residues after Leu were constitutively active. Even in the absence of agonist, all
four mutant receptors were able to mediate inhibition of adenylyl
cyclase (via activation of G
) and to efficiently stimulate
phospholipase C activity when coexpressed with a mutant G
subunit containing
(or
)
sequence at its C terminus. The addition of agonist had no or little
effect on the magnitude of these responses (E
50% of wild type m2), which, however, could be completely
prevented by incubation with atropine (5 µM). According to
the recently proposed ``allosteric ternary complex model'' of
ligand/receptor/G protein interactions (see below; Lefkowitz et
al., 1993), atropine can therefore act as an inverse agonist (see
also Blüml et al., 1994;
Högger et al., 1995).
Whereas insertion
of extra Ala residues C-terminal of Leu rendered the
resulting m2 mutant receptors constitutively active, deletion of
Ala
from the wild type m2 receptor resulted in a mutant
receptor (m2(
A391)) that was unable to interact with G proteins,
either in the absence or the presence of agonist. Taken together, these
results suggest a model of agonist-induced m2 receptor activation in
which agonist binding induces a movement of TM VI toward the cytoplasm,
thus enabling the VTIL motif to interact with the C terminus of
G
subunits.
Based on computational approaches
(Strader et al., 1989) and recent mutagenesis data (Liu et
al., 1995a), we speculated that the residues forming the VTIL
motif are located on one side of an -helical receptor segment. If
this is correct, one would expect (due to changes in helix register)
that the stepwise insertion of extra Ala residues C-terminal of this
sequence element should lead to a progressive rotation (in 100 °
increments) of the VTIL surface. Because the degree of constitutive
receptor activity was found to be virtually independent of the number
of inserted Ala residues, one might conclude that the C terminus of
G
subunits can interact with the VTIL site
independent of its precise spatial orientation. However, such a
mechanism does not appear very likely, because many studies suggest
that proper receptor/G protein coupling involves coordinated
interactions between several intracellular receptor domains (Dohlman et al., 1991; Savarese and Fraser, 1992; Hedin et
al., 1993; Strader et al., 1994) and at least three sites
on the G
subunits (including the C terminus; Conklin and Bourne,
1993; Rens-Domiano and Hamm, 1995). One may therefore speculate that
the insertion of multiple Ala residues between Leu
and
Ala
(m2 receptor) does not lead to a progressive register
shift (involving the residues N-terminal of the insertion point) but
rather results in a local disruption of the TM VI helix. Structural
studies with various insertion mutants of T4 lysozyme have shown, for
example, that extra Ala residues can be accommodated within
-helical protein domains by ``looping out'' of the
inserted amino acids (Matthews, 1995). However, the possibility can
also not be excluded that the receptor segment in which the VTIL motif
is located is not
-helically arranged (in contrast to predictions
made based on previous results by Liu et al. (1995a)) but is
perhaps relatively disordered. To distinguish between these
possibilities, high resolution structural information (obtained, e.g. by NMR or x-ray crystallography) would be required.
Pioneering work by Lefkowitz and co-workers has shown that
mutational modification of the C-terminal portion of the i3 loop of
several adrenergic receptor subtypes also leads to constitutive
receptor activity (Kjelsberg et al., 1992; Ren et
al., 1993; Samama et al., 1993). It could be
demonstrated, for example, that replacement of Thr (initially erroneously referred to as Thr
) in the
-adrenergic receptor (corresponding to Thr
in the m2 receptor; Fig. 1) with five different amino
acids (Ren et al., 1993) or substitution of Ala
in the
-adrenergic receptor (corresponding to
Ala
in the m3 receptor; Fig. 1) with all 19
possible amino acids (Kjelsberg et al., 1992) resulted in
mutant receptors that could activate G proteins in an
agonist-independent fashion (note, however, that introduction of
structurally homologous mutations into the m2, m3, and m5 muscarinic
receptors does not result in constitutive receptor activity) (Burstein et al., 1995). (
)Based on the functional properties
of such constitutively active adrenergic receptors, it was proposed
that residues in the C terminus of the i3 loop play a role in
constraining the adrenergic receptors in an inactive conformation and
that replacement of these residues removes this constraining function
allowing the receptor to ``relax'' into an active
conformation (Lefkowitz et al., 1993).
In the light of
these findings, one may argue that the agonist-independent activity
displayed by the four m2 receptor insertion mutants described in this
study could also simply be due to the loss of a constraining
interaction involving residues at the i3 loop/TM VI junction. We could
show, however, that replacement of the VTIL motif in such
constitutively active m2 receptors with the corresponding m3 receptor
residues (AALS) completely abolished agonist-independent (as well as
agonist-dependent) signaling. This observation is in agreement with
previous results suggesting that the VTIL site is directly involved in
G protein recognition and activation (Liu et al., 1995a). This
notion is further supported by the finding that a 19-amino acid
synthetic peptide (including residues at the i3 loop/TM VI junction)
corresponding to the C-terminal portion of the i3 loop of the
G-coupled m4 muscarinic receptor can activate G
proteins at nanomolar concentrations in a reconstituted system
(Okamoto and Nishimoto, 1992). Moreover, a short synthetic peptide
derived from the i3 loop/TM VI junction of the G
-coupled
-adrenergic receptor could be chemically cross-linked
to G
and
subunits in vitro (Taylor et al., 1994). As an extension of the model of receptor
activation proposed by Lefkowitz et al. (1993), these data
strongly suggest that agonist binding to G
-coupled
(muscarinic) receptors leads to structural changes at the i3 loop/TM VI
junction, allowing distinct residues located in this region to interact
with specific sites on the G protein(s).
The constitutively active
mutant m2 receptors described in this study displayed agonist binding
properties similar to those of the wild type m2 receptor. Unchanged
agonist binding affinities have also been reported for several
constitutively active glycoprotein hormone receptors (Kosugi et
al., 1995; Kopp et al., 1995). In contrast, virtually all
known mutant adrenergic receptors capable of agonist-independent
signaling (as well as a recently described constitutively active m1
(Glu
Ala) mutant muscarinic receptor)
(Högger et al., 1995) show considerably
higher agonist affinities than the corresponding wild type receptors
(Kjelsberg et al., 1992; Ren et al., 1993; Samama et al., 1993). Based on this finding, together with the
observation that the extent of this affinity increase is related to
agonist activity (Samama et al., 1993), an allosteric ternary
complex model (as an extension of the ``classical ternary receptor
model'') (De Lean et al., 1980) of ligand/receptor/G
protein interactions was proposed (Samama et al., 1993;
Lefkowitz et al., 1993). This model predicts that the receptor
exists in an equilibrium (characterized by the equilibrium constant J) between an inactive (R) and an active conformation (R*) and
that agonists, by preferentially binding to the R* form, shift this
equilibrium to the active receptor conformation. According to this
model, a similar shift in the equilibrium toward R* can also result
from mutations resulting in constitutive receptor activity. The lack of
increased agonist binding affinity observed with the constitutively
active mutant receptors described here may therefore be explained by
assuming that the proportion of mutant receptors that are present in
the R* state is too small to be detected in radioligand binding assays.
Alternatively, because the allosteric ternary complex model predicts
that an increase in J causes an increase in agonist affinity,
it is also possible that the activating mutations described here
primarily affect the affinity of R* for the G protein. The notion that
mutations can activate receptors by different molecular mechanisms is
also supported by the finding that the activity of the constitutively
active m2 mutant receptors described here, in contrast to the
functional properties of previously published mutationally activated
receptors (Lefkowitz et al., 1993), was not significantly
increased by the addition of agonist. Such a potential heterogeneity of
receptor activation mechanisms would also be consistent with the
observation that constitutively active G protein-coupled receptors can
result from mutations in various different receptor regions including
the TM helices and various extracellular and intracellular regions (for
recent reviews, see Coughlin(1994) and Shenker(1995)).
Interestingly, when extra Ala residues were introduced after
Ser into the G
-coupled m3 muscarinic
receptor, the resulting mutant receptors, in contrast to the
structurally homologous m2 receptor mutants, did not display
constitutive activity (even in the presence of coexpressed wild type
G
; data not shown) but were functionally completely
inactive. Given the high degree of sequence homology found among
different muscarinic receptor subtypes (Bonner et al., 1987;
Hulme et al., 1990), this finding may indicate that the
G
- and G
-coupled muscarinic receptors
interact with their cognate G proteins in a somewhat different fashion.
Such a notion would also be consistent with the observation that the
four m3 receptor residues (AALS, Ala
, Ala
,
Leu
, and Ser
) corresponding to the VTIL
motif in the m2 receptor are not essential for G
coupling (in contrast to the functional role of the VTIL motif in
the m2 receptor) (Liu et al., 1995a), although they contribute
to the efficiency of receptor-mediated G
activation
(Blin et al., 1995). Moreover, substitution of the AALS motif
(by itself) into the wild type m2 muscarinic receptor failed to
establish coupling to
(Blin et al., 1995).
However, consistent with previous mutagenesis studies (Kunkel and
Peralta, 1993; Högger et al., 1995), the
complete lack of agonist-dependent signaling observed with the mutant
m3 receptors containing additional Ala residues after Ser
suggests that the structural integrity of the C terminus of the
i3 loop is critical for proper receptor-G protein interactions.
In a set of control experiments, one or two extra Ala residues were also inserted into the wild type m2 and m3 muscarinic receptors immediately N-terminal of the VTIL and AALS motif, respectively. Second messenger assays showed that these modifications had little effect on the magnitude of the m2 and m3 receptor-mediated functional responses. Interestingly, the VTIL (or AALS) motif is preceded by a four-amino acid sequence element (B-B-Glu-B, where B is a basic amino acid) that is conserved among virtually all G protein-coupled receptors that bind biogenic amine ligands (Watson and Arkinstall, 1994). Loss-of-function mutagenesis studies have shown that one or more of these charged residues are generally important for efficient G protein coupling (Kunkel and Peralta, 1993; Högger et al., 1995). Our data therefore suggest that the precise spatial orientation of this highly charged sequence motif (such as its direct proximity to residues at the i3 loop/TM VI junction) is not essential for receptor-mediated G protein activation.
In conclusion, we have demonstrated that insertion mutagenesis can serve as a useful tool to study the molecular mechanisms involved in receptor/G protein recognition and receptor-mediated G protein activation. It should be of considerable interest to examine which functional effects mutations homologous to those described here can cause in other classes of G protein-coupled receptors.