(Received for publication, June 2, 1995)
From the
Currently, detailed structural information about the arrangement
of the seven transmembrane helices (TM I-VII) present in all G
protein-coupled receptors is still lacking. We demonstrated previously
that hybrid m2/m5 muscarinic acetylcholine receptors which contain m5
sequence in TM I and m2 sequence in TM VII were unable to bind
significant amounts of muscarinic radioligands (Pittel, Z., and Wess,
J.(1994) Mol. Pharmacol. 45, 61-64). By using
immunocytochemical and enzyme-linked immunosorbent assay techniques, we
show in the present study that these pharmacologically inactive mutant
receptors are present (at high levels) on the surface of transfected
COS-7 cells. Strikingly, all misfolded m2/m5 hybrid receptors could be
pharmacologically rescued by introduction of a single point mutation
into either TM I (m5Thr
m2Ala
) or TM VII (m2Thr
m5His
). All our experimental data
are consistent with the notion that the two altered threonine residues
face each other at the TM I/TM VII interface in the pharmacologically
inactive m2/m5 hybrid receptors, thus interfering with proper
helix-helix packing. Our data provide the first experimental evidence
as to how TM I and TM VII are oriented relative to each other and also
strongly suggest that the TM helices in G protein-coupled receptors are
arranged in a counterclockwise fashion (as viewed from the
extracellular membrane surface).
The seven transmembrane helices (TM I-VII) ()predicted to be present in all G protein-coupled receptors
(GPCRs) are thought to be sequentially arranged in a ringlike fashion,
thus forming a very tightly packed TM receptor core (Baldwin, 1993,
1994; Schwartz, 1994). Residues located on the inner surfaces of
different TM helices are known to be involved in the binding of a great
number of ligands that act on GPCRs (Strader et al., 1994;
Baldwin, 1993, 1994; Schwartz, 1994). Moreover, ligand-induced
conformational changes in the TM receptor core are thought to be
intimately involved in receptor activation (Dohlman et al.,
1991; Wess, 1993; Strader et al., 1994). Detailed structural
information about the membrane-embedded portion of GPCRs is therefore
essential for understanding how GPCRs function at a molecular level.
By using the atomic coordinates of the heptahelical bacterial membrane protein bacteriorhodopsin (Henderson et al., 1990) as a template, several three-dimensional models of the TM core of GPCRs have been proposed (Trumpp-Kallmeyer et al., 1992; Nordvall and Hacksell, 1993; Hibert et al., 1993). However, the usefulness of these models has been questioned by recent mutagenesis studies (Zoffmann et al., 1993; Blüml et al., 1994) and the availability of a low resolution (9 Å) electron density map of rhodopsin, a G protein-coupled photoreceptor (Schertler et al., 1993).
In the absence of high resolution structural data on any GPCR, we have chosen a mutagenesis approach to identify interhelical interactions in GPCRs. In a previous study (Pittel and Wess, 1994), we identified a series of hybrid m2/m5 muscarinic receptors (C1-C4; Fig. 1) that were unable to bind significant amounts of muscarinic radioligands. A common structural feature of these pharmacologically inactive mutant receptors was the presence of m5 receptor sequence in TM I and of m2 receptor sequence in TM VII. Interestingly, hybrid m2/m5 muscarinic receptors in which both TM I and TM VII contained m2 receptor sequence (such as C5 or C6; Fig. 1) regained the ability to bind muscarinic ligands normally, suggesting that molecular interactions between TM I and TM VII are required for proper GPCR folding (Pittel and Wess, 1994).
Figure 1:
Structure of mutant
muscarinic receptors. A, hybrid m2/m5 muscarinic receptors
(C1-C6). Transfection of COS-7 cells with the underlined constructs (C1-C4) did not result in an appreciable number
of [H]NMS or [
H]QNB binding
sites (Pittel and Wess, 1994). B, sites in C1-C4 that
were targeted by site-directed mutagenesis. C, comparison of
the TM I and TM VII amino acid sequences of the human m2 and m5
muscarinic receptors. #, positions at which the sequences of the m2 and
m5 receptors differ. Residues marked with an arrow were
targeted by site-directed mutagenesis. Numbers refer to amino
acid positions within the human m2 and m5 muscarinic receptors (Bonner et al., 1987, 1988). The N termini of the TM helices were
chosen as defined by Baldwin(1993).
Based on these results, this study was designed to identify specific amino acids on TM I and TM VII, which are responsible for the folding defect present in C1-C4 (Fig. 1). The identification of such residues should allow predictions as to how TM I and TM VII are oriented relative to each other. In addition, immunocytochemical and ELISA studies were carried out to examine the subcellular localization and the expression levels of various mutant receptors.
We found that all misfolded m2/m5 hybrid receptors
(C1-C4) could be pharmacologically rescued by introduction of a
single point mutation into either TM I (m5Thr
m2Ala
) or TM VII (m2Thr423
m5His
). Based on a recent model of the
possible arrangement of TM I-VII in GPCRs (Baldwin, 1993, 1994),
our data provide the first direct experimental evidence as to how TM I
and VII are oriented relative to each other.
Binding data were analyzed by nonlinear least squares curve-fitting procedures, using the computer programs LIGAND (saturation binding data; Munson and Rodbard(1980)) or KALEIDAGRAPH (competition binding data; Synergy Software), respectively.
All wild type and mutant muscarinic receptor constructs were
transiently expressed in COS-7 cells. As reported previously (Pittel
and Wess, 1994), the C1-C4 hybrid receptors (Fig. 1) were
unable to bind significant amounts of muscarinic radioligands such as
[H]NMS or [
H]QNB, whereas
C5 and C6 (Fig. 1) could bind muscarinic ligands normally (Table 1). It is therefore likely that a conformational
incompatibility exists between the m5 receptor sequence in TM I and the
m2 receptor sequence in TM VII in C1-C4, leading to improperly
folded receptor proteins.
Immunocytochemical studies (confocal fluorescence microscopy) demonstrated that C3-HA showed a subcellular distribution similar to that found with the wild type m5 receptor. As shown in Fig. 2, non-permeabilized COS-7 cells expressing the C3-HA construct displayed a distinct staining of the plasma membrane in a fashion similar to the wild type m5 receptor.
Figure 2:
Immunocytochemical localization of wild
type and mutant muscarinic receptors. COS-7 cells were transfected with
the following DNA constructs: vector (control, A), wild type
m5 (B), C3 (C), C3(m5Thr
m2Ala
) (D), and
C3(m2Thr
m5His
) (E). All receptors contained an HA-tag at their N terminus.
Immunofluorescence studies were carried out with non-permeabilized
COS-7 cells grown on glass coverslips, as described under
``Experimental Procedures.'' Cells were treated with a
monoclonal antibody directed against the HA-tag (12CA5) and then
incubated with a FITC-linked goat anti-mouse IgG secondary antibody.
Fluorescence images were obtained with a confocal laser scanning
microscope (MRC-600, Bio-Rad). Each picture is representative of three
independent experiments.
To quantitate the amount of C3-HA present on the cell surface, an indirect cellular ELISA was employed (for details, see ``Experimental Procedures''). For ELISA measurements, non-permeabilized COS-7 cells transiently expressing C3-HA or the epitope-tagged version of the wild type m5 muscarinic receptor were first incubated with a monoclonal antibody directed against the HA-tag (12CA5), followed by addition of a peroxidase-conjugated secondary antibody and the photometric determination of peroxidase activity. Control experiments with non-permeabilized COS-7 cells expressing an m3 muscarinic receptor containing an HA-tag at its C terminus resulted in optical density readings that were similarly low as those found with the nontagged versions of the m3 and m5 muscarinic receptors (data not shown). This finding, together with the observation that the C-terminally tagged m3 muscarinic receptor could be easily detected in permeabilized cells, demonstrates that the employed ELISA procedure does not interfere with the intactness of the plasma membrane barrier.
Consistent with the microscopic studies described above, the ELISA experiments showed that C3-HA was properly expressed on the cell surface. As illustrated in Fig. 3, the optical density readings found with C3-HA were only 25-30% lower than those found with the epitope-tagged version of the wild type m5 muscarinic receptor. Taken together, the immunological studies suggest that C3 (and, most likely, other pharmacologically inactive m2/m5 hybrid receptors such as C1, C2, and C4) are stably incorporated into the plasma membrane. The virtual lack of ligand binding activity observed with C1-C4 thus appears to be due to a specific folding defect.
Figure 3:
ELISA determination of the amount of wild
type and mutant muscarinic receptors expressed on the cell surface.
COS-7 cells were transfected with the following constructs all of which
(except A, which was control, wild type m5, non-tagged)
contained an HA-tag at their N terminus: wild type m5 (B), C3 (C), C3(m5Thr
m2Ala
) (D), and
C3(m2Thr
m5His
) (E). ELISA measurements were carried out with
non-permeabilized COS-7 cells in 96-well plates as described under
``Experimental Procedures.'' Optical density values can be
considered a direct measure of the amount of receptor protein present
on the cell surface. Data are presented as means ± S.D. of three
independent experiments, each performed in
triplicate.
Figure 4:
Helical wheel model of the possible
orientation of TM I and VII in muscarinic receptors. A,
anticlockwise (proposed) connectivity of TM I-VII, as viewed the
extracellular surface of the membrane. The TM I (m5) and TM VII (m2)
sequences of the pharmacologically inactive m2/m5 hybrid receptors,
C1-C4 (Fig. 1), are shown. Amino acids that differ between
the m2 and m5 muscarinic receptors are highlighted (blackbars). The model shown here is consistent with a low
resolution electron density map of rhodopsin (Schertler et
al., 1993; Baldwin, 1993). It differs from the Baldwin projection
(Baldwin, 1993, 1994) in that TM VII has been rotated by about 30°
(counterclockwise) to allow Tyr (underlined) to
project into the ligand binding cavity formed by TM III-VII.
Site-directed mutagenesis data obtained with different muscarinic
receptor subtypes suggest that this tyrosine residue is critically
involved in ligand recognition (Wess et al., 1991; Matsui et al., 1995). Residues marked with an arrow were
targeted by site-directed mutagenesis. The size of the circles next to the individual amino acids reflects their relative depth
in the plasma membrane. B, clockwise
(``incompatible'') connectivity of TM I-VII (as viewed
from the extracellular surface of the membrane), based on the
assumptions made in A. The predicted positions of m5Thr
(TM I) and m2Thr
(TM
VII) in C1-C4 are highlighted. Positions at which the TM I and TM
VII sequences of the m2 and m5 muscarinic receptors differ are marked
with an asterisk.
In Fig. 4A,
all TM I and TM VII residues that differ among the m2 and m5 muscarinic
receptors are highlighted (blackbars). Strikingly,
if this model is correct, there are only two residues at the TM I/TM
VII interface that are not identical between the two receptors (TM I, m5Thr/m2Ala
; and TM VII, m2Thr
/m5His
) (Fig. 1C and 4A). Virtually all other
nonconserved residues are predicted to face the lipid bilayer.
Characteristically, in all pharmacological inactive hybrid m2/m5
receptors (C1-C4), a TM I threonine residue (m5Thr
) faces a TM VII threonine residue (m2Thr
). We therefore speculated that a novel
interaction (which does not occur in the wild type receptors) formed
between these two polar residues (e.g. involving hydrogen bond
interactions) might interfere with proper helix-helix packing. If this
is correct, it should be possible to ``pharmacologically
rescue'' C1-C4 by single point mutations such that m5Thr
(TM I) faces its ``natural
partner'', m5His
(TM VII), or, vice
versa, that m2Thr
(TM VII) is located
adjacent to m2Ala
(TM I). To test this
hypothesis, the corresponding mutations (m5Thr
m2Ala
and m2Thr
m5His
) were introduced into TM I
and VII of C1-C4. The ligand binding properties of the resultant
mutant receptors are summarized in Table 1. Whereas C1-C4
were unable to bind significant amounts of [
H]NMS
(highest concentration tested: 5 nM), mutational modification
of m5Thr
or m2Thr
resulted
in the appearance a considerable number of specific
[
H]NMS binding sites. Similar results were
obtained with a structurally different muscarinic radioligand,
(-)-[
H]QNB (data not shown). All mutant
receptors in which either m5Thr
or m2Thr
was modified were able to bind muscarinic
antagonists (NMS, 4-DAMP) and agonists (acetylcholine, carbachol) with
affinities similar to those found with the two wild type receptors (Table 1).
Characteristically, in the case of each
pharmacologically inactive hybrid receptor (C1-C4), the two point
mutations had a similar effect on the number of ``recovered''
[H]NMS binding sites (B
; Table 1). The C1- and C2-derived mutant receptors yielded B
values (approximately 150 fmol/mg) that were
clearly lower than the corresponding wild type receptor levels. In
contrast, C3(m5Thr
m2Ala
) and C3(m2Thr
m5His
) as well as the corresponding C4-based
mutant receptors yielded B
values (418-556
fmol/mg) that almost reached the corresponding wild type m2 receptor
levels. Either of the two point mutations therefore fully mimicked the
effect of replacing long N-terminal segments in C3 and C4 (including TM
I and adjacent extramembranous regions) with the corresponding m2
receptor sequences (C5, C6; Fig. 1, Table 1).
Immunocytochemical and ELISA studies with epitope-tagged versions of
C3(m5Thr
m2Ala
) and
C3(m2Thr
m5His
)
showed that the two mutant receptors were present on the cell surface
at levels similar to those found with C3 ( Fig. 2and Fig. 3).
For control purposes, two additional point mutations (m5Thr
m2Phe
and m5Val
m2Ser
) were
introduced into one of the pharmacologically inactive hybrid receptors,
C4, either alone or in combination (Fig. 1). These two sites
were targeted based on the observation that they are located in the
N-terminal segment of TM I and that the m2 and m5 receptor residues
present at these positions clearly differ in their physicochemical
properties (hydrophobic versus hydrophilic; Fig. 1C). Consistent with the predicted localization of m5Thr
and m5Val
on the
outer surface of TM I (facing the lipid bilayer; Fig. 4),
expression of the three resultant mutant receptors did not result in
the appearance of a significant number of [
H]NMS
binding sites (B
< 25 fmol/mg).
To further
explore the molecular mechanisms by which introduction of the m5Thr
m2Ala
and m2Thr
m5His
point
mutations into C1-C4 can restore proper receptor folding, four
additional mutant receptors were created and pharmacologically
characterized. The results of these studies are summarized in Table 2and Table 3. Introduction of an additional m5Thr
m2Ala
point
mutation into C4(m2Thr
m5His
) did not interfere with the ability of
this mutant receptor to properly bind the muscarinic antagonist,
[
H]NMS. Interestingly, C4 could be
pharmacologically rescued not only by replacement of m2Thr
with histidine (see above) but also by
replacement with glutamate and asparagine, which are present at the
corresponding position in the m1 and m3 muscarinic receptors,
respectively (Table 2). In contrast, exchange of m2Thr
with alanine did not restore ligand
binding activity to C4.
Figure 5:
Carbachol-induced stimulation of PI
hydrolysis mediated by wild type m5 and mutant m2/m5 muscarinic
receptors. Transfected COS-7 cells transiently expressing the indicated
receptors were incubated in six-well plates for 1 h at 37 °C with 1
mM carbachol, and the resulting increases in intracellular
IP levels were determined as described (Berridge et
al., 1983; Blin et al., 1995). Data are presented as
percent increase in IP
above basal levels in the absence of
carbachol. Basal IP
levels for the wild type m5 receptor
amounted to 4630 ± 290 cpm/well. The basal IP
levels
observed with the various mutant receptors were similar to this value.
Data are given as means ± S.E. of a single experiment performed
in triplicate; one additional experiment gave similar
results.
However, cAMP assays with COS-7 cells coexpressing the
V2 vasopressin receptor and C1(m5Thr
m2Ala
) or C1(m2Thr
m5His
) showed that these two mutant receptors
gained the ability to inhibit AVP-stimulated cAMP production
(7-9-fold stimulation above basal levels in the absence of
carbachol) by 20 ± 4% (n = 2; stimulation with
0.1 mM carbachol). Under the same experimental conditions, the
wild type m2 muscarinic receptor mediated a reduction of AVP-stimulated
cAMP levels by 30 ± 4%, whereas C1 was completely inactive (n = 2).
In the absence of detailed structural information on any GPCR, we (Pittel and Wess, 1994) and others (Suryanarayana et al., 1992; Zhou et al., 1994; Rao et al., 1994) have recently employed mutagenesis approaches to identify molecular interactions between the seven TM helices predicted to be present in all GPCRs. In a previous study (Pittel and Wess, 1994), we found that hybrid m2/m5 muscarinic receptors (C1-C4; Fig. 1) containing m5 receptor sequence in TM I and m2 receptor sequence in TM VII were unable to bind significant amounts of muscarinic radioligands. However, immunocytochemical and ELISA experiments demonstrated (this study) that the folding defect present in C1-C4 does not affect proper targeting of these mutant receptors to the cell surface (shown for C3).
As reported previously (Pittel and Wess, 1994),
substitution of N-terminal m2 receptor sequences (including TM I) into
misfolded hybrid m2/m5 muscarinic receptors such as C3 or C4 (Fig. 1) could rescue ligand binding activity (Table 1),
suggesting that TM I and VII are located in close proximity to each
other and that specific molecular interactions between these two
helices are required for proper receptor folding. A similar conclusion
was reached in a study examining the pharmacological properties of a
series of hybrid /
-adrenergic
receptors (Suryanarayana et al., 1992). Although both studies
(Suryanarayana et al., 1992; Pittel and Wess, 1994) suggest
that TM I and TM VII are located next to each other in the TM receptor
core, they do not allow predictions as to how these two TM helices are
oriented relative to each other.
To address this question, we
assumed that the residues responsible for the misfolding of the
pharmacologically inactive m2/m5 hybrid receptors (C1-C4) are
likely to be located at the TM I/TM VII interface. To identify these
amino acids, we initially displayed the TM I and VII sequences of
C1-C4 in a helical wheel model (Fig. 4), based on the
recently published Baldwin projection (Baldwin, 1993, 1994). The
Baldwin model proposes a possible arrangement of TM I-VII in
GPCRs, which is consistent with a low resolution electron density map
of rhodopsin (Schertler et al., 1993). However, our model (Fig. 4) differs from the Baldwin projection in that TM VII has
been rotated by approximately 30° (counterclockwise as viewed from
the extracellular side of the membrane) to take into account data from
several recent mutagenesis studies (Wess, 1993; Baldwin, 1994;
Schwartz, 1994). This arrangement would allow a TM VII tyrosine residue
(corresponding to m2Tyr in Fig. 4) known
to be critically involved in the binding of muscarinic ligands (Wess et al., 1991; Matsui et al., 1995) to project into
the ligand binding cavity formed by TM III-VII. Moreover,
residues located at the homologous positions in many other GPCRs have
also been shown to be essential for high affinity ligand binding
(Baldwin, 1994; Schwartz, 1994). The model depicted in Fig. 4is
consistent with the general assumption that the (mostly lipophilic)
residues, which differ among functionally closely related GPCRs (in
this case, subtypes of muscarinic receptors), are located on the
lipid-facing side of the TM helices (Baldwin, 1993).
The proposed
arrangement of TM I and VII (Fig. 4) predicted that there are
only two amino acids located at the TM I/TM VII interface (TM I, m5Thr/m2Ala
; and TM VII, m2Thr423/m5His
, respectively) that
differ between the m2 and m5 muscarinic receptors. Characteristically,
all pharmacologically inactive m2/m5 hybrid receptors (C1-C4)
contain a threonine residue at these two positions. We therefore
speculated that a novel interaction (which may involve hydrogen bonds)
between these two residues interferes with proper helix-helix packing,
ultimately resulting in misfolded receptor proteins unable to bind
muscarinic ligands. Since both residues are predicted to be located
about 1-2 helical turns away from the membrane surface (Fig. 1C), such contact appears theoretically possible.
To test this hypothesis, single m5Thr
m2Ala
(TM I) and m2Thr
m5His
(TM VII) point mutations were
introduced into C1-C4, thus creating mutant receptors in which
the amino acid configuration at the TM I/TM VII interface mimicked that
found in the wild type m2 and m5 muscarinic receptors, respectively. We
found, consistent with our working hypothesis, that mutant receptors in
which either of the two threonine residues (m5Thr
or m2Thr
) was structurally modified gained
the ability to bind significant amounts of muscarinic radioligands. All
resulting mutant receptors were able to bind muscarinic agonists and
antagonists with wild type affinities and, with the exception of the
two C1-based mutant receptors, gained the ability to mediate a
pronounced stimulation of PI hydrolysis. The observed lack of
functional PI activity seen with the C1-based mutant receptors is
consistent with the previous observation (Wess et al., 1989)
that the presence of m2 receptor sequence in the N-terminal segment of
the third intracellular loop abolishes muscarinic receptor-mediated PI
hydrolysis.
However, in contrast to C1, both
C1(m5Thr
m2Ala
) and
C1(m2Thr
m5His
)
gained the ability to inhibit the increase in cAMP levels mediated by
stimulation of the cotransfected V2 vasopressin receptor. The degree of
inhibition observed with these two mutant receptors (20 ± 4%)
was somewhat smaller than that found with the wild type m2 receptor (30
± 4%), probably due to the fact that the presence of m2 receptor
sequence in the third intracellular loop and in the C-terminal
``tail'' is not sufficient to allow optimum coupling to
G
(Wess et al., 1990; Liggett et al.,
1991).
Immunocytochemical and ELISA studies showed that the m5Thr
m2Ala
(TM I)
and m2Thr
m5His
(TM VII) point mutations did not lead to an increase in the
amount of receptor protein expressed on the cell surface, suggesting
that either substitution can specifically ``cure'' a
conformational defect present in C1-C4. In contrast, exchange of
residues predicted to be located on the lipid-facing side of TM I (m5Thr
m2Phe
and m5Val
m2Ser
) did not
restore ligand binding activity.
Most strikingly, the C3- and
C4-derived m5Thr
m2Ala
and m2Thr
m5His
mutant receptors yielded B
values that
closely approached those found with the wild type m2 muscarinic
receptor. In contrast, the analogous C1- and C2-based mutant receptors
displayed about 4-6-fold lower B
values
than the two wild type receptors, indicating that additional
conformational incompatibilities, perhaps involving intracellular
receptor domains, exist in C1 and C2 and interfere with optimum
receptor folding and/or stability. This notion is consistent with the
previous observation that an m3 muscarinic receptor in which the third
intracellular loop was replaced with the corresponding m2 receptor
sequence was expressed at 5-11-fold lower levels than the two
wild type receptors (Wess et al., 1989).
Characteristically, in each case (C1-C4), replacement of
either of the two critical threonine residues ((m5Thr or m2Thr
) had a quantitatively similar
effect on the number of ``recovered''
[
H]NMS binding sites (Table 1). This result
would be expected if the virtual lack of ligand binding activity found
with C1-C4 is due to a direct (conformationally unfavorable)
interaction between these two residues. Most of the residues thought to
be located at the TM I/TM VII interface have a polar character (Fig. 4), suggesting that a complex network of hydrogen bond
interactions may be involved in the proper positioning of these two TM
helices. All our experimental data are therefore consistent with the
notion that, in C1-C4, the formation of a novel hydrogen bridge
between m5Thr
(TM I) and m2Thr
(TM VII) interferes with the proper formation of this hydrophilic
network.
Such an interaction cannot occur in the wild type m2
muscarinic receptor where m5Thr is replaced with an
alanine residue (m2Ala
). Likewise, introduction of an
additional m5Thr
m2Ala
point mutation into C4(m2Thr
m5His
) did not interfere with proper receptor
folding ( Table 2and Table 3), indicating that a hydrogen
bond between m5Thr
(TM I) and m5His
(TM VII) is not required for the formation
of a functional receptor protein.
To further explore by which
mechanism the m2Thr
m5His
point mutation can rescue the function of
C1-C4, m2Thr
(in C4) was replaced with
glutamate and asparagine (which are present at the corresponding
positions in the m1 an m3 muscarinic receptors, respectively) as well
as with alanine. Whereas the glutamate and asparagine substitutions,
similar to the histidine substitution, yielded fully functional
receptors, exchange of m2Thr
with alanine was
unable to correct the folding deficit present in C4. These data suggest
that a hydrophilic residue at position m5His
(TM
VII) is required for proper receptor folding, perhaps by stabilizing TM
VII via formation of an intrahelical hydrogen bond interaction with one
of the conserved polar residues located on the same side of TM VII (Fig. 4A). This stabilizing interaction cannot occur in
the inactive hybrid receptors (C1-C4) where m2Thr
is predicted to be engaged in a hydrogen
bridge with m5Thr
on TM I. In contrast to
threonine, other polar amino acids such as histidine, glutamate, and
asparagine, due to the presence of two heteroatoms in their side
chains, can form multiple hydrogen bridges (including the proposed
stabilizing interaction within TM VII). This may explain why
replacement of m2Thr
(in C4) with the latter
three residues can restore proper receptor folding.
In addition to
providing experimental evidence for the relative orientation of TM I
and TM VII, our findings also strongly suggest that the TM helices in
GPCRs are arranged in a counterclockwise fashion (as viewed from the
extracellular membrane surface), an arrangement that has been commonly
assumed but one for which no strong experimental evidence has been
available (Baldwin, 1993, 1994; Schwartz, 1994). In the case of a
clockwise connectivity of TM I-VII in C1-C4, m2Thr on TM VII would be predicted to be located
adjacent to TM VI (Fig. 4B) rather than TM I. However,
such an orientation is absolutely inconsistent with our experimental
data.
Interestingly, a possible interhelical contact site has
recently also been identified in the gonadotropin-releasing hormone
receptor (Zhou et al., 1994). In this receptor, two usually
highly conserved aspartate (TM II) and asparagine (TM VII) residues are
replaced with asparagine (Asn) and aspartate
(Asp
), respectively. Whereas an Asn
Asp mutant receptor completely lacked pharmacological activity, an
Asn
Asp/Asp
Asn double-mutant
receptor regained the ability to bind ligands with high affinity,
suggesting that these two residues are located next to each other
within the TM helical bundle (Zhou et al., 1994). Moreover,
valuable structural information about interhelical interactions in
GPCRs has also been obtained in biophysical and mutational studies of
constitutively active forms of rhodopsin (Robinson et al.,
1992; Rao et al., 1994). It could be demonstrated that the TM
VII Lys residue (Lys
), which acts as the retinal
attachment site is located in close proximity to both a TM III
glutamate residue (Glu
, the natural counterion for
Lys
) and a TM II glycine residue (Gly
).
Very recently, mutational analysis of the tachykinin NK-1 receptor
showed that replacement of two residues located at the top of TM V and
VI (Glu and Tyr
, respectively) with
histidine enabled Zn
ions to act as an antagonist on
this receptor (Elling et al., 1995). It has therefore been
proposed that the artificial creation of metal ion binding sites in
GPCRs may represent a novel approach to probe helix-helix interactions
(Elling et al., 1995).
In conclusion, by using a ``gain-of-function'' mutagenesis approach we could provide direct experimental evidence as to how TM I and VII are oriented relative to each other. Moreover, our data strongly support the concept that the TM helices in GPCRs are arranged in a counterclockwise fashion (as viewed from the extracellular membrane surface).