From the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The N termini of two G protein subunits,
q and
11, differ from those of
other
subunits in that they display a unique, highly conserved
six-amino acid extension (MTLESI(M)). We recently showed that an
q deletion mutant lacking these six amino acids (in
contrast to wild type
q) was able to couple to several
different Gs- and Gi/o-coupled receptors,
apparently due to promiscuous receptor/G protein coupling (Kostenis,
E., Degtyarev, M. Y., Conklin, B. R., and Wess, J. (1997)
J. Biol. Chem. 272, 19107-19110). To study which
specific amino acids within the N-terminal segment of
q/11 are critical for constraining the receptor coupling
selectivity of these subunits, this region of
q was
subjected to systematic deletion and alanine scanning mutagenesis. All
mutant
q constructs (or wild type
q as a
control) were coexpressed (in COS-7 cells) with the m2 muscarinic or
the D2 dopamine receptors, two prototypical Gi/o-coupled
receptors, and ligand-induced increases in inositol phosphate
production were determined as a measure of G protein activation.
Surprisingly, all 14 mutant G proteins studied (but not wild type
q) gained the ability to productively interact with the
two Gi/o-linked receptors. Similar results were obtained when we examined the ability of selected mutant
q
subunits to couple to the Gs-coupled
2-adrenergic
receptor. Additional experiments indicated that the functional
promiscuity displayed by all investigated mutant
q
constructs was not due to overexpression (as compared with wild type
q), lack of palmitoylation, or initiation of translation at a downstream ATG codon (codon seven). These data are consistent with
the notion that the six-amino acid extension characteristic for
q/11 subunits forms a tightly folded protein subdomain
that is critical for regulating the receptor coupling selectivity of these subunits.
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INTRODUCTION |
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G protein-coupled receptors
(GPCRs)1 regulate the
activity of a large variety of effector systems via interaction with
specific classes of heterotrimeric G proteins (consisting of ,
,
and
subunits) that are attached to the cytoplasmic side of the
plasma membrane (1-6). In most cases, an individual GPCR can only
activate a distinct subset of the many structurally similar G proteins present in each cell (7, 8). How this selectivity is achieved at a
molecular level is currently being explored by a great number of
laboratories.
A large body of evidence indicates that multiple regions on the G
protein subunits play key roles in receptor binding and dictating
the selectivity of receptor/G protein interactions (2, 5, 6, 8, 9).
Specifically, recent studies have shown that residues at the extreme C
terminus of the G protein
subunits are of fundamental importance
for regulating receptor/G protein coupling selectivity (10-13),
probably by directly contacting the receptor protein (14, 15). However,
several lines of evidence indicate that other regions of G
also
contribute to receptor binding and the selectivity of receptor
recognition (2, 5, 6, 9). Biochemical studies suggest, for example,
that the N-terminal portion of G
may also be in contact with the
receptor protein (14, 16, 17). An early study demonstrated that a synthetic peptide corresponding approximately to the N-terminal
N-helix of
-transducin (Fig. 1) was able to prevent
rhodopsin/transducin interactions, presumably by directly binding to
rhodopsin (14). Moreover, Higashijima and Ross (16) showed that a
cysteine-substituted mastoparan, a receptor-mimetic peptide toxin, can
be cross-linked to the extreme N terminus of G
o
subunits. Similar findings were obtained with a photoaffinity
derivative of a receptor-mimetic peptide corresponding to the
C-terminal portion of the third cytoplasmic loop of the
2A-adrenergic receptor (17).
In addition, functional analysis of an N-terminally truncated
q subunit suggests that the N terminus of G
subunits
is also involved in modulating the fidelity of receptor/G protein
recognition (18). G
q and G
11 contain a
unique six-amino acid extension (MTLESI(M)) that is not found in other
G
subunits (Fig. 1). This short sequence is highly conserved among
all vertebrate species from which these subunits have been cloned so
far (19-23). When this sequence was removed by deletion mutagenesis
(18), the resulting mutant
q subunit (corresponding to
q(
2-7) in Fig. 1) gained the ability to be activated by various
Gi/o- and Gs-coupled receptors which normally
do not couple efficiently to wild type (full-length)
q
(qWT). Based on these results, we speculated that the N-terminal
extension characteristic for
q/11 subunits may act as a
"filter" by selectively preventing Gi/o- and
Gs-coupled receptors from contacting functionally critical
q/11 residues. Alternatively, it is conceivable that the
N-terminal extension exerts indirect conformational effects on adjacent
q/11 domains, such as the functionally critical C
terminus, thereby constraining the selectivity of
receptor/
q/11 interactions.
To examine which specific amino acids within the N-terminal segment of
q/11 are critical for maintaining the selectivity of
receptor recognition, we generated a large number of mutant
q subunits in which the N-terminal residues were
progressively deleted or systematically replaced, either individually
or in combination, with alanine. The ability of these mutant subunits to be activated by different Gi/o- and
Gs-coupled receptors was examined in cotransfected COS-7
cells. Surprisingly, all 14 mutant
q subunits studied,
but not qWT, displayed pronounced receptor coupling promiscuity. These
results indicate that each single amino acid within the N-terminal
extension of
q/11 subunits is critical for constraining
the receptor coupling selectivity of these subunits. The most
straightforward explanation for this finding is that the N-terminal
portion of
q/11 forms a well defined protein subdomain
that regulates the fidelity of receptor/
q/11 interactions.
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EXPERIMENTAL PROCEDURES |
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Creation of Mutant G Protein Constructs--
A mouse
q cDNA (19) cloned into the pcDNAI expression
vector (24) was used as a template for polymerase chain reaction mutagenesis. All wild type and mutant G
q subunits
contained an internal hemagglutinin (HA) epitope tag (DVPDYA; Ref. 24).
The presence of the epitope tag, which replaced
q
residues 125-130, did not affect the receptor and effector coupling
properties of qWT (10, 11, 24). The construction of the q(
2-7)
deletion mutant (formerly referred to as
6q) has been described
previously (18). A silent BssHII site was introduced into
q(
2-7) at amino acid codons 18-19 to facilitate the generation of
additional mutant G proteins. All mutant
q subunits were
constructed by replacing a 57-base pair
BamHI-BssHII fragment of q(
2-7) with the
corresponding DNA fragments (generated via polymerase chain reaction)
containing the desired deletions or substitutions (Fig. 1). In the wild
type and all mutant
q plasmids, the
BamHI-site of the pcDNAI polylinker was immediately
followed by the initiating ATG codon. The correctness of all polymerase
chain reaction-derived DNA sequences was verified by dideoxy sequencing
of the mutant plasmids (25).
Coexpression of Receptor and G Protein Constructs--
COS-7
cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum at 37 °C in a humidified 5%
CO2 incubator. For transfections, 1 × 106
cells were seeded into 100-mm dishes. About 18-24 h later, cells were
cotransfected with the indicated G protein (1 µg of DNA/dish) and
receptor constructs (4 µg of DNA/dish) by using a DEAE-dextran procedure (26). The following receptor expression plasmids were used:
human m2 muscarinic receptor in pcD (27), human D2 dopamine receptor in
pcDNAI (28), and rat 2-adrenergic receptor in pSVL (29).
Phosphatidylinositol Hydrolysis Assays-- About 18-24 h after transfections, cells were split into six-well dishes (approximately 0.4 × 106 cells/well) and labeled with 3 µCi/ml [3H]myo-inositol (20 Ci/mmol; American Radiolabeled Chemicals Inc.). After a 24-h labeling period, cells were preincubated for 20 min at room temperature with 2 ml of Hanks' balanced salt solution containing 20 mM HEPES and 10 mM LiCl. Cells were then stimulated with the appropriate agonists for 1 h at 37 °C, and increases in intracellular inositol monophosphate (IP1) levels were determined by anion exchange chromatography as described (30).
[3H]Palmitate Labeling and
Immunoprecipitation--
Approximately 48 h after transfections,
COS-7 cells were washed twice with 10 ml of serum-free DMEM and
incubated for 1 h in 5 ml of serum-free DMEM (37 °C, 5%
CO2). Following removal of the medium, cells were
metabolically labeled for 16 h with 200 µCi/ml
[9,10-3H]palmitate (50.0 Ci/mmol; American Radiolabeled
Chemicals Inc.) in 3 ml of DMEM containing 10% dialyzed fetal calf
serum (37 °C, 5% CO2). After the 16-h labeling period,
cells were washed twice with 10 ml of phosphate-buffered saline,
resuspended in 50 µl of buffer A (10 mM Tris-HCl, pH 8.0, and 150 mM NaCl) containing 1% SDS, and incubated at
70 °C for 10 min. Subsequently, 200 µl of extraction buffer
(composition: 10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 2 mM
EDTA, and 1 mM phenylmethylsulfonyl fluoride) was added,
and samples were tumbled for 30 min at 4 °C. Insoluble material and
nuclei were removed by centrifugation for 10 min at 14,000 rpm in a
refrigerated Eppendorf 5417R microcentrifuge. Supernatants were mixed
with an equal volume of buffer A and 20 µl of Sepharose 4B beads
(Sigma) coupled to the 12CA5 monoclonal antibody (Boehringer Mannheim) (the antibody was coupled to CNBr-activated Sepharose 4B according to
the manufacturer's (Sigma) instructions). Samples were tumbled for
2 h at 4 °C and then centrifuged for 1 min at 2,000 rpm in an
Eppendorf microcentrifuge to gently pellet the Sepharose beads. Pellets
were washed twice with 500 µl of a 1:5 dilution of extraction buffer
and resuspended in 20 µl of Laemmli sample buffer (Bio-Rad) without
reducing agents. After heating the samples for 10 min at 70 °C, the
beads were pelleted gently (same conditions as above), and supernatants
were used for SDS-PAGE (13%). Gels loaded with 10% of the total
sample volumes were subjected to Western blotting (see below). Gels run
with the remaining 90% of the samples were soaked in 20-30 ml of
Fluoro-Hance (Research Products International Corp.) for 30 min, dried,
and subjected to fluorography at 80 °C for 4-6 weeks.
Western Blotting--
All wild type and mutant Gq
subunits were detected with the 12CA5 monoclonal antibody directed
against the HA-epitope tag present in all G protein constructs. Samples
containing 20 µg of membrane protein prepared from transfected COS-7
cells were resolved by SDS-PAGE (13%), electroblotted onto
nitrocellulose, and probed with the 12CA5 antibody as described (11,
31). Immunoreactive proteins were detected by incubation with
horseradish peroxidase-conjugated sheep anti-mouse IgG antibody
(Amersham Pharmacia Biotech) and visualized using an enhanced
chemiluminescence system (Amersham Pharmacia Biotech).
Drugs-- All ligands used in this study were purchased from Research Biochemicals Inc. The remaining chemicals were obtained through Sigma.
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RESULTS |
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Construction and Expression of Mutant q
Subunits--
Initially we prepared two series of N-terminally
modified mutant
q subunits (Fig.
1). In one series, amino acids 2-7
(TLESIM) of mouse
q were progressively removed leading
to six
q deletion constructs (note that q(
2-7) was
previously referred to as -6q; Ref. 18). In the other series, these
residues were replaced, either individually (yielding six
q single point mutants) or in combination (resulting in
q(TLE
AAA) and q(SI
AA)) (Fig. 1). All wild type and mutant
q subunits contained an internal hemagglutinin (HA)
epitope tag replacing
q residues 125-130. Previous
studies demonstrated that the presence of the epitope tag does not
affect the receptor and effector coupling properties of qWT (10, 11,
24). Western analysis using an anti-HA monoclonal antibody (12CA5)
showed that all mutant
q subunits were properly expressed at levels similar to or somewhat lower than observed with qWT
(Fig. 2).
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Interaction of Mutant q Subunits with
Gi/o-coupled Receptors--
To study the effects of the
different mutations on the fidelity of receptor/G protein interactions,
all mutant
q subunits were initially coexpressed (in
COS-7 cells) with the m2 muscarinic receptor, a prototypical
Gi/o-coupled receptor (32, 33). Transfected cells were then
incubated with the muscarinic agonist, carbachol (0.5 mM),
and the ability of the m2 muscarinic receptor to couple to the
different G proteins was determined by measuring increases in inositol
phosphate production (due to
q-mediated activation of
PLC
; Refs. 34 and 35). Coexpression of the m2 receptor with either
vector DNA (pcDNAI) or qWT, followed by ligand stimulation, resulted only in a relatively small increase in PLC
activity (approximately 3-fold; Fig. 3), a
response known to be largely due to activation of PLC
isozymes by G
protein
subunits released upon m2 receptor-mediated activation
of endogenous Gi/o proteins (36, 37). Remarkably, the m2
muscarinic receptor gained the ability to productively couple to all 14 mutant
q subunits, resulting in a 6-11-fold stimulation
of PLC activity (Fig. 3).
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Interaction of Mutant q Subunits with a
Gs-coupled Receptor--
We next examined the ability of
the Gs-coupled
2-adrenergic receptor to interact
functionally with selected mutant
q subunits (q(
2-7),
q(T2A), q(L3A), q(E4A), q(S5A), q(I6A), and q(M7A)). Consistent with
its known coupling preference (1), this receptor subtype, when
stimulated with (
)-isoproterenol (200 µM), was unable
to activate efficiently qWT (Fig. 5),
mediating only an approximately 1.5-fold increase in IP production.
However, the
2-adrenergic receptor gained the ability to interact
productively with all examined mutant
q subunits,
leading to a 3-5-fold stimulation of PLC activity (Fig. 5).
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Translation Start Site in Mutant q Subunits--
As
described above, all investigated mutant
q subunits
showed similar functional properties, displaying coupling promiscuity upon coexpression with different Gi/o- and
Gs-coupled receptors. As shown in Figs. 1 and
6A, the N-terminal six-amino
acid extension (MTLESI) characteristic for
q subunits is
immediately followed by a second in-frame methionine codon (codon
seven). If this second rather than the first methionine codon is used
as a translation start site, all
q constructs would
direct the synthesis of the same N-terminally truncated protein
(corresponding to q(
2-7)). To exclude such a mechanism as a
potential cause of the observed functional promiscuity displayed by the
different mutant
q subunits, an additional experiment
was designed. Frameshift mutations were introduced between the two
N-terminal ATG codons (codons one and seven), using two mutant
q constructs, q(L3A) and q(E4A), as model systems (Fig.
6A). In q(L3A)-FS, the deletion of a single nucleotide (C)
within codon three in q(L3A) led to a shift of the amino acid reading
frame resulting in a premature stop codon after 10 amino acids of
nonsense sequence. In q(E4A)-FS, the insertion of a single nucleotide
(C) after codon four in q(E4A) led to a changed amino acid reading
frame and a premature stop codon after 59 amino acids of nonsense
sequence (Fig. 6A).
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Palmitoylation Pattern of Wild Type and Mutant q
Subunits--
Previous studies (24, 38-42) have shown that
q and
11, like most other G
subunits,
are palmitoylated at cysteine residues located near the N termini
(corresponding to Cys-9 and Cys-10 in Fig. 1). We therefore wanted to
examine whether the promiscuous mutant
q subunits
differed from qWT in their palmitoylation patterns. Toward this goal,
COS-7 cells transfected with qWT or selected mutant
q
subunits (q(
2-4), q(
2-7), q(TLE->AAA), q(SI->AA), q(T2A), and
q(M7A)) were metabolically labeled with [3H]palmitic
acid, followed by immunoprecipitation of
q subunits by
the 12CA5 monoclonal antibody, SDS-PAGE, and fluorography. Western
analysis showed that the amounts of immunoprecipitated wild type and
mutant
q proteins were similar (Fig.
8A). The
q subunits were the only immunoprecipitated proteins, since no labeled proteins were detected when cells were transfected with "empty" vector DNA. As shown in Fig. 8B, qWT and all mutant
q subunits incorporated significant amounts of
[3H]palmitate. However, the strength of the
palmitoylation signal was generally weaker in the case of the mutant
q subunits (40-75% compared with qWT, as determined by
scanning densitometry).
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DISCUSSION |
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The fidelity of receptor/G protein interactions critically depends
on the ability of different GPCRs to discriminate between unique
structural features of the G subunits (2, 5-9). Characteristically, most G
subunits (when bound to
in a trimeric complex) can be
efficiently activated by only certain functional classes of GPCRs (7,
8). In this study, we have identified a series of 14 mutant
q subunits that display promiscuous receptor coupling. In contrast to qWT, all 14 mutant subunits investigated here could be
activated by both Gi/o- and Gs-coupled
receptors. In these mutant G proteins, residues within the six-amino
acid N-terminal extension that is characteristic for
q/11 subunits were systematically substituted with
alanine (either individually or in combination) or progressively
deleted.
The most straightforward explanation for the observed functional
promiscuity displayed by all mutant q subunits is that
the unique N terminus of
q/11 subunits adopts a well
defined three-dimensional structure that is critical for maintaining
the coupling selectivity of these G proteins. Based on this concept,
mutations within this structural motif are thought to interfere with
its proper folding and its ability to regulate receptor/G protein
coupling selectivity.
Although all mutant G proteins showed a qualitatively similar
functional profile, quantitative differences in their ability to be
activated by different Gi/o- and Gs-coupled
receptors (m2 muscarinic, D2 dopamine, and 2-adrenergic) were noted.
Moreover, the observed patterns of G protein activities also varied
among the three receptors used in this study. It is likely that the specific receptor residues involved in G protein binding differ between
the three receptors, thus providing an explanation for the observed
ability of these receptors to discriminate between different mutant
G
q subunits which, in turn, are also likely to display
subtle structural differences.
Recently, the atomic structures of two different G protein
heterotrimers, Gi1
1
2 (Ref.
43) and G
t/i1
1
1 (Ref. 44), have been resolved by x-ray crystallography. In these structures (but
not in the free
subunits), the N-terminal segment of the
subunit (corresponding to residues 6-26 in
t) is
-helically arranged and protrudes away from the "bulk" of G
.
This so-called
N-helix (Fig. 1) is engaged in interactions with the
complex via docking along the first blade of the
"propeller," thus determining the location of the acylated N
terminus of G
(note, however, that the G protein structures were
resolved using proteins devoid of lipid modifications). Unfortunately,
the extreme N termini of the
subunits (corresponding to amino acids
1-10 in
q; Fig. 1) were not observed in the x-ray
structures, probably due to their conformational flexibility. The
available structural information therefore provides little insight into
the potential arrangement of the N-terminal 10 amino acids of
q/11 subunits.
Accumulating evidence indicates that several C-terminal regions of G
(including the C-terminal tail, portions of the
5-helix, and the
4/
6 loop) are directly involved in receptor binding and play a
key role in dictating the selectivity of receptor/G protein
interactions (10-15, 45-49). X-ray crystallographic studies suggest
that the N- and C-terminal segments of G
subunits are conformationally linked (43, 50). One possibility therefore is that the
N-terminal extension characteristic for
q/11 subunits constrains their receptor coupling selectivity by preventing access of
Gi/o- and Gs-coupled receptors (either due to
steric hindrance or to allosteric effects) to functionally critical
C-terminal G
regions.
As outlined in the Introduction, biochemical studies suggest that
residues within the N-terminal 30-amino acid segment of G may be
involved in receptor binding (14). Since this G
region is also known
to be critical for binding of G
to
complexes (43, 44),
binding of the receptor to an N-terminal G
site may contribute to
triggering dissociation of the G protein heterotrimer into free
and
subunits. An alternative possibility therefore is that the first
six amino acids characteristic for
q/11 subunits have a
"gate" function by regulating access of different classes of GPCRs
to an adjacent N-terminal G
region.
It has been noted that a negatively charged cavity that is predicted to
face the plasma membrane (and/or the receptor) is located at the
/
interface (43, 44). On the other hand, structure-function
analysis of a great number of different GPCRs has shown that several
positively charged residues located within the second and third
intracellular loops of these receptors play key roles in triggering G
protein activation (3, 8). Thus, another possibility is that the N
terminus of
q/11 subunits controls the access of
different functional classes of GPCRs to this anionic surface on
q/11. The resolution of the atomic structure of a Gq heterotrimer (that also includes the N-terminal
q sequences) is likely to provide clues that would allow
us to distinguish between these different possibilities.
Interestingly, the functional properties of the mutant q
constructs examined in this study resemble those of two other G protein
subunits,
15 and
16 (
15
and
16 appear to be the murine and human versions of the
same gene; Ref. 51). Recent studies have shown that
15
and
16, like the mutant
q subunits
described here, show little receptor selectivity and can be activated
by most GPCRs studied to date (52, 53). A sequence comparison indicates
that the N-terminal eight amino acids of
15/16
(MARSLTWG(R)) differ considerably from the corresponding
q/11 sequence (MTLESI(M)MA) (19, 51, 54). It therefore
remains to be elucidated whether the coupling promiscuity displayed by
15/16 and all mutant
q constructs
examined here is dependent on a similar molecular mechanism.
Like most other G subunits,
q and
11
are known to be reversibly palmitoylated at cysteine residues located
near their N termini (corresponding to Cys-9 and Cys-10 in Fig. 1;
Refs. 24 and 38-42). Studies with mutant
q constructs
in which both N-terminal cysteine residues were replaced with serine or
alanine suggest that palmitoylation facilitates membrane attachment of
this
subunit (24, 38, 39). However, palmitoylation does not appear to be an absolute requirement for membrane targeting of
q (39). In addition, palmitoylation-defective mutant
q subunits were found to be severely impaired in their
ability to mediate receptor-dependent increases in PLC
activity (24, 38). However, in a reconstituted system, enzymatic
removal of palmitate from purified
q had little effect
on the ability of this subunit to interact with receptors and
downstream effector enzymes (39), indicating that the N-terminal cysteine residues themselves rather than the palmitate moieties attached to them are of primary functional importance.
In the present study, all examined mutant q subunits,
similar to qWT, were found to incorporate significant amounts of
[3H]palmitate (Fig. 8B). We noted, however,
that the intensity of the palmitoylation signal was reduced in the case
of the mutant
q subunits (about 40-75% as compared
with qWT). On the other hand, Western analysis of immunoprecipitated,
[3H]palmitate-labeled G proteins indicated that most
mutant
q subunits were expressed at levels similar to
qWT (Fig. 8A). It is therefore conceivable that
palmitoylation plays an inhibitory role in constraining the receptor
coupling selectivity of
q, perhaps by specifying the
spatial orientation and fold of the N terminus of
q.
Consistent with this notion, biochemical studies have shown that the
myristoyl moiety attached to the N terminus of
t is
essential for the proper folding of the N-terminal
t
segment (55). In addition, a previous study analyzing the coupling
properties of a palmitoylation-defective mutant
z
subunit suggests that palmitoylation inhibits
z
signaling by an as yet unknown mechanism (56). Interestingly, Tu
et al. (57) recently showed that palmitoylation of
z and
i1 strongly inhibits the activity
of several RGS proteins (regulators of G protein signaling) which are
known to stimulate the GTPase activity of these
subunits. This
finding indicates that palmitoylation of
subunits may represent a
general mechanism for prolonging the lifetime of activated
subunits. However, such a mechanism cannot explain the functional
properties of the mutant
q subunits investigated in this
study, which appear to be palmitoylated less efficiently (as compared
with qWT) and would therefore be expected to show reduced rather than
increased functional activity.
In eucaryotic organisms, translation initiation is usually restricted
to the first in-frame ATG (AUG in the case of mRNA) codon. However,
several examples are known where translation is initiated from a
downstream ATG codon (a process frequently referred to as "leaky
scanning"), particularly when the first in-frame ATG codon is located
in a "weak" context that considerably deviates from the consensus
sequence (5'-CC(A/G)CCATGG-3') for strong initiation codons
(58, 59). The residues that exert the strongest effects on the
efficiency of translation initiation are a 3 purine (A or G) and a +4
G (the A of the ATG codon is numbered +1, and nucleotides to the left
have negative numbers). Inspection of the nucleotide sequences of the
q constructs used in this study indicates that the
second N-terminal ATG codon (codon seven) is present in a clearly
better context than the first one (codon one) (Fig. 6A). It
was therefore important to demonstrate that the mutant
q
constructs analyzed in this study do not allow translation initiation
from codon seven, thus yielding the same, N-terminally truncated
protein (q(
2-7)) that is known to be functionally promiscuous (18). Several observations clearly argue against such a mechanism. First, since the first ATG in qWT is located in the same weak context as in
all mutant
q plasmids (all constructs contain identical 5'-flanking sequences), leaky scanning should also lead to the synthesis of the promiscuous q(
2-7) subunit in the case of qWT. However, no such promiscuous coupling was observed when qWT was coexpressed with different Gi/o- and Gs-coupled
receptors. Second, a mutant
q construct in which the
second ATG codon (codon seven) was replaced with an alanine codon (thus
allowing only translation from codon one) showed functional properties
similar to all other mutant
q constructs examined.
Third, we constructed two mutant
q subunits, q(L3A)-FS
and q(E4A)-FS, that differed from q(L3A) and q(E4A) only by the
deletion and insertion of a single nucleotide, respectively (Fig.
6A). To avoid changing the nucleotide sequences immediately
adjacent to the two ATG codons, these mutations were introduced 7-11
base pairs upstream of the second ATG codon. Functional studies showed
that the two frameshifted mutant
q constructs did not
allow the synthesis of (mutant)
q subunits capable of interacting with the m2 muscarinic receptor (Fig. 6B).
Consistent with this finding, no q(
2-7) protein could be detected
via Western blotting (Fig. 7). Taken together, these observations
clearly exclude the possibility that the functional promiscuity of the N-terminal
q mutants studied here is due to leaky
scanning and the use of the second methionine codon (codon seven) as a
translation start site.
In conclusion, our data suggest a novel mechanism by which receptor/G protein coupling selectivity can be regulated. Elucidating the precise molecular mechanisms involved in this process should eventually lead to new insight into the complex processes governing the selectivity of receptor/G protein interactions.
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ACKNOWLEDGEMENTS |
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We thank Drs. B. R. Conklin and C. M. Fraser for generously providing us with different receptor and G protein expression plasmids.
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FOOTNOTES |
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* This work was supported by a grant from the Deutscher Akademischer Austauschdienst (NATO) (to E. K.).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: NIDDK, Laboratory of
Bioorganic Chemistry, Bldg. 8A, Rm. B1A-05, Bethesda, MD 20892. Tel.:
301-402-3589; Fax: 301-402-4182; E-mail: jwess{at}helix.nih.gov.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium;
IP1, inositol monophosphate; PLC, phospholipase C; qWT,
wild type Gq; PAGE, polyacrylamide gel electrophoresis;
HA, hemagglutinin.
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REFERENCES |
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