From the
In order to understand the specific interactions between
receptors and guanine nucleotide-binding regulatory protein (G
proteins), we attempted to delineate the
Molecular cloning has revealed the existence of genes encoding
at least 16 G
In general, four methods have been used to detect the
specific interactions between receptors and G proteins: 1) the use of
specific antibodies to block ligand-induced activation of PLC in
membrane preparations or to immunoprecipitate G proteins that are
photolabeled with a GTP analog. A number of laboratories have used
these approaches to detect specific coupling of receptors, including
those for vasopressin
(4) ,thromboxane A2
(5) ,
bradykinin, vasopressin, angiotensin
(6) , and
thyrotrophin-releasing hormone
(7) , to G
A lot of work has been done to
understand the molecular basis of the specificity in receptor-G protein
interactions
(20) . Amino acid sequences that are involved in
activation of G
The
half-maximal inhibition value (IC
We have previously demonstrated that the
One of the interesting questions
is whether the sequences required for activating G
In this report we used the site-directed mutagenesis and
cotransfection approaches to analyze the third inner loop of the
The BB XXB motifs
are commonly found in G protein-coupled receptors. There are two such
motifs in the third inner loop of the
Although
our data focus on the third inner loop of the
The
Unfortunately, in this study
we were unable to delineate the sequences involved in activating
G
In summary, our work has provided useful
information for understanding of specific interactions between
receptors and G proteins. The data suggest that we may be able to
create receptors with limited, but defined G protein-coupling
specificity. Such receptors would be very useful in determining the
specific in vivo function of signal transduction pathways
mediated by specific receptors and G proteins.
1B-adrenergic receptor
sequences involved in activation of the
subunits of the Gq class
of G proteins. A number of specific mutations were introduced into the
third inner loop of the receptor, and the mutants were tested for their
abilities to activate different G
subunits of the Gq class. Our
results indicate that the receptor sequences required for activating
G
q/11, G
14, or G
16 are different. The sequence extending
from residues Lys
to His
is
required for activation of G
q/11, but not for activation of
G
14 or G
16. Two segments in the third loop of the receptor
are required for activation of G
14: one is located at the N
terminus of the loop ending at residue Asn
, and the other
is located at the C terminus of the loop starting from residue
Ser
. The latter contains a BB XXB motif, which is
apparently critical for G
14 coupling, but not for G
16 or
G
q/11 coupling. Furthermore, the three amino acids stretch
(Tyr
to Val
) included in the N-terminal
segment is not only required for G
14 coupling, but also for
G
q/11 coupling. It may be involved to some extent in G
16
coupling as well.
subunits, 5 G
subunits, and 7 G
subunits
in mammals. These can form a variety of heterotrimers that serve to
connect specific cell surface receptors to a large number of different
effectors, including at least four PLC
(
)
isoforms and many adenylylcyclases as well as several specific ion
channels
(1, 2, 3) . One of the intriguing
questions posed by this apparent complexity is how signal transduction
circuits are organized so that different kinds of receptors can be
connected to effectors through various G proteins and coordinate a
variety of responses in a large number of different cells. The
specificity of some of the circuits is no doubt determined by
developmental regulation of the expression of genes that encode the
receptors, G proteins, and effectors. In addition, subcellular
localization may contribute to the specificity to a certain extent.
However, the primary determinant for formation of a specific signal
transduction circuit, we believe, lies in specific protein-protein
interactions.
q and/or G
11.
2) The use of antisense oligonucleotides to inhibit expression of
various subunits of G proteins. This approach has been successfully
applied to reveal the specific interactions among the receptors,
G
, G
, and G
in GH3 cells
(8, 9) . 3) The
use of purified proteins in reconstituted systems
(2, 10, 11, 12) . 4) The use of the
cotransfection system, in which cells are transfected with cDNAs
encoding receptors and G proteins with subsequent measurement of
ligand-induced responses. This approach has been used to study coupling
of the IL-8
(13) , C5a
(14) and
-adrenergic
(15, 16, 17, 18) receptors to the
subunits of the Gq and Gi classes. By using these methods, we and
others found that there is clear specificity in receptor-G protein
interactions. For instance, the
1-adrenergic receptors
preferentially couple to the
subunits of the Gq class of G
protein to activate inositide-specific PLC, while the
-adrenergic
receptors couple to the G
proteins
(19) .
Furthermore, the specificity lies not only with different classes of G
proteins, but also within the same class. For example, we found that
the IL-8 receptor can couple to G
16 and G
14, but not to
G
q or G
11
(13) .
q have been mapped to the third cytoplasmic (inner)
loops of the
1B-adrenergic receptor, the m1 muscarinic receptor,
and the glutamate receptors by using various chimeras
(21, 22, 23) . Although these sequences share no
significant amino acid sequence homology, they appear to be different
from the sequences involved in activating G
s
(24, 25) . It is, however, not known whether these
G
q-activating sequences are also involved in activating the other
members of the Gq class, such as G
14, G
15, and G
16, and
how receptors like the
1B-adrenergic receptor can couple to all
G
subunits of the Gq class, while receptors like the IL-8 receptor
couple to G
14, G
15, and G
16, but not to G
q/11. In
this report, we investigated these questions by using site-directed
mutagenesis on the third loop of the
1B-adrenergic receptor. Our
results indicate that different
1B-adrenergic receptor sequences
are involved in coupling to G
q/11, G
14, or G
16.
Cell Culture and Transfection
COS-7 cells were
cultured in Delbecco's modified Eagle's medium containing
10% fetal calf serum under 5% COat 37 °C. For
transfection, COS-7 cells were seeded into 12-well plates at a density
of 1
10
cell/well the day before transfection. The
media were removed the next day, and 0.5 ml of Opti-MEM (Life
Technologies, Inc.) containing 5 µg of lipofectamine (Life
Technologies, Inc.) and 1 µg of plasmid DNA was added to each well.
Five h later the transfection media were replaced by the culture media.
Then the cells were labeled with 10 µCi/ml of
myo-[2-
H]inositol the following day, and
the levels of inositol phosphates were determined 1 day later as
described previously
(26) . All the cDNAs used in this studies
were constructed in the pCMV expression vector
(26) .
SDS-Polyacrylamide Gel Electrophoresis and Western
Blot
Equal numbers of transfected cells were solubilized in the
SDS sample buffer and loaded to 12% SDS-polyacrylamide gels. The
proteins were then electroblotted to nitrocellulose membranes and
detected with antibodies indicated in the figure legends.
Binding Assays
COS-7 cells in 12-well plates were
transfected with the cDNA encoding the 1-adrenergic receptor or
its mutants. After 48 h, the cells were washed with phosphate-buffered
saline and incubated with varying amounts of
[
H]prazosin (76 Ci/mmol, PuPont-NEN) in
phosphate-buffered saline for 40 min at 4 °C. After washing three
times with ice-cold phosphate-buffered saline, the cells were lysed in
0.5 ml of 0.2 N NaOH, and 0.1-ml aliquots were taken for
counting in a scintillation counter. The nonspecific binding was
determined by measuring binding of [
H]prazosin to
nontransfected cells. The number of specific binding sites
( Bmax) were determined by the Scatchard analysis.
) was determined by
measuring binding of [
H]prazosin (5 pM)
to transfected COS-7 cells in the presence of varying amounts of
norepinephrine.
Construction of
All the 1-Adrenergic Receptor
Mutants
1-adrenergic receptor mutants listed in
Fig. 1
were generated by polymerase chain reaction with the high
fidelity DNA polymerase, pfu (Stratagene) and each of the
mutations was confirmed by DNA sequencing.
Figure 1:
Schematic representation of
1B-adrenergic receptor sequences required for activation of
subunits of Gq class. The amino acid sequence of the third inner loop
of the
1B-adrenergic receptor is shown. The various deletion
mutations are denoted and so is the residue Lys
, which is
substituted by a Leu residue in mutant
1BK282L. The ligand binding
activities were determined as described under ``Experimental
Procedures.''
1B-adrenergic
receptor can couple to all the G
subunits of the Gq class of G
proteins, including G
q, G
11, G
14, and G
16, to
activate PLC
(15) . As shown in Fig. 2 A, COS-7
cells transfected with the
1B-adrenergic receptor cDNA showed
norepinephrine-dependent increases in accumulation of inositol
phosphates (IP). This suggests that the
1B-adrenergic receptor can
couple to endogenous G proteins, which are probably G
q and
G
11, because G
q and G
11, but not G
14, G
15, or
G
16, were detected in COS-7 cells
(15) . When the cells
were cotransfected with the cDNA encoding G
14 or G
16, there
were further increases in ligand-dependent responses over the ones seen
in the cells transfected with the
1B-adrenergic receptor alone
(Fig. 2 A), suggesting that the
1B-adrenergic
receptor can couple to G
14 and G
16 as well. In order to
determine whether the
1B-adrenergic receptor sequences involved in
activating G
14 and G
16 are the same as the ones required for
activating G
q/11
(22) , we made a specific mutation in the
1B-adrenergic receptor by deleting amino acids from residues
Lys
to His
(Fig. 1). This
13-amino-acid segment was previously identified as involved in
G
q/11 coupling
(22) . As expected, the cells transfected
with the cDNA encoding this mutated
1B-adrenergic receptor,
designated
1B
2, unlike those transfected with the wild-type
receptor cDNA, showed little ligand-induced accumulation of IPs
(Fig. 2, B and C, open circles). Even
cotransfection with the cDNA encoding G
q or G
11 led to little
ligand-induced response (data not shown), confirming that this segment
of the
1B-adrenergic receptor is required for coupling to G
q
or G
11. However, when the cells were cotransfected with the
1B
2 cDNA and the cDNA encoding either G
14
(Fig. 2 B) or G
16 (Fig. 2 C), there
were marked increases in norepinephrine-induced accumulation of IPs.
The expression levels of the recombinant G
14 and G
16 proteins
in transfected COS-7 cells were determined by the Western analysis with
a polyclonal antibody (Gqcom), which was raised against a synthetic
peptide derived from the sequence shared by all the G
subunits of
the Gq class (Fig. 3 A). The Western analysis indicated
that G
14 is expressed at a slightly higher level than some
endogenous proteins detected by the Gqcom antibody in nontransfected
COS-7 cells; these endogenous proteins are presumably G
q and
G
11. The identities of the endogenous proteins and expression of
G
14 were further confirmed by the Western analysis using the
G
q/11- and G
14-specific antibodies. G
14 was detected
only in cells transfected with the G
14 cDNA by the
G
14-specific antibody (Fig. 3 A) and a clear band,
which has the same electrophoretic mobility as the recombinant G
q,
was detected by the G
q/11-specific antibody in nontransfected
COS-7 cells (Fig. 3 B). In addition, we determined the
expression levels of the wild-type
1B-adrenergic receptor and its
mutant
1BAR and their affinities for norepinephrine. The
expression levels were determined by binding of
[
H]prazosin to the cells expressing the
receptors, while the binding affinities are indicated by the
IC
, which is the concentration of norepinephrine showing
50% inhibition of binding of [
H]prazosin to the
cells expressing the
1B-adrenergic receptor or its mutants. We
found that the cells expressing the
1-adrenergic receptor or
1B
2 showed similar numbers of binding sites for
[
H]prazosin (about 500 fmol/1
10
cells) and similar IC
values (7 µM)
(Fig. 1). Furthermore, we found that coexpression of different
G
proteins does not affect expression of either
1B-adrenergic
receptor or
1B
2 in cotransfected COS-7 cells (data not
shown). Therefore, we conclude that the sequence extending from
residues Lys
to His
is required for
activating G
q and G
11, but not for activating the other
G
subunits of the Gq class, including G
14 and G
16, and
that there might be different sequences involved in coupling to
G
14 or G
16.
Figure 2:
Activation of the Gq subunits by
wild-type
1BAR and its mutants,
1B
2 and
1B
11.
A, COS-7 cells were cotransfected with the cDNA encoding
1B-adrenergic receptor and the cDNA encoding G
14 ( closed
triangle), G
16 ( closed circle), or the control
-galactosidase ( open square). B and C,
COS-7 cells were cotransfected with the cDNA encoding
1B
2
( circles) or
1B
11 ( triangles), and the cDNA
encoding G
14 ( B, closed symbols), G
16
( C, closed symbols), or the control
-galactosidase ( open symbols). The levels of inositol
phosphates were determined 20 min after addition of ligand. The level
of inositol phosphates in nontransfected COS-7 cells is taken as
100%.
Figure 3:
Western
analysis of expression of G subunits in COS-7 cells. Cells
transfected with the cDNA encoding G
14, G
16 ( A),
G
q ( B) or the control
-galactosidase (LacZ) were
solublized in the SDS sample buffer and separated by 12%
SDS-polyacrylamide electrophoresis gels. The proteins were then
electrotransferred to nitrocellulose membranes and analyzed by Western
blotting with the Gqcom antibody ( A, left panel),
G
14-specific antibody ( A, right panel), or
G
q/11-specific antibody ( B).
To delineate the 1B-adrenergic receptor
sequences involved in activating G
14 or G
16, we generated the
mutant
1B
11 by deleting the amino acids from residues
Arg
to Phe
in the
1B-adrenergic
receptor. Although this mutant showed altered ligand-binding
characteristic (lower IC
) and was expressed at a lower
level than the wild-type and
1B
2 (Fig. 1), it can still
activate G
14 and G
16 with capabilities comparable to the
1B
2 (Fig. 2, B and C). This indicates
that the
1B-adrenergic receptor amino acids extending from
residues Leu
to Phe
are not required for
coupling to G
14 or G
16.
16 can be
separated from those for activating G
14. To address this question,
we made another mutant,
1B
1, with a deletion extending from
residues Ala
to Ser
(Fig. 1). The
mutant
1B
1 was tested in the same way as for
1B
2,
and we found that
1B
1 could couple only to G
16, but not
to G
14 (Fig. 4 A). This tells us that the sequence
required for activating G
16 is different from the one required for
activating G
14. In addition, the fact that
1B
1 can not
couple to G
14, while
1B
11 can, suggests that the
sequence extending from residues Ala
to Asn
is required for activating G
14. In order to further
delineate the sequences required for activating G
14, we prepared
two additional mutants,
1B
3 and
4 (Fig. 1). The
inability of
1B
4 (Fig. 4 C) and the ability of
1B
3 to activate G
14 (Fig. 4 B) indicate
that the
1B-adrenergic receptor sequence extending from residues
Ser
to Ala
is also required for activating
G
14. Thus, there appear to be two segments involved in G
14
coupling: one is located near the C-terminal end and the other near the
N-terminal end of the third inner loop of the
1B-adrenergic
receptor. Although
1B
4 could not activate G
14, it as
well as
1B
3 could still activate G
16. Activation of
G
16 thus acts as an internal control for
1B
4, indicating
it retains overall structural integrity. All the
1B mutants were
tested for their expression levels and ligand binding affinities. The
numbers of binding sites and the IC
values shown by cells
expressing the mutants
1,
3, and
4 are between those
shown by
1B
2 and
11 (Fig. 1).
Figure 4:
Activation of the G subunits by
1-adrenergic receptor mutants. COS-7 cells were cotransfected with
the cDNA encoding one of the
1BAR mutants as indicated in the
figure and the cDNA encoding G
14, G
16, or the control
-galactosidase (LacZ, LZ). The levels of inositol
phosphates were determined 20 min after addition of norepinephrine (1
µM). The level of inositol phosphates in nontransfected
COS-7 cells is taken as 100%.
Interestingly,
the two segments required for activation of G14 contain a
signature motif, BB XXB (B represents a basic amino acid and
X represents any amino acid) (Fig. 1). This motif occurs
frequently in the inner loops of many G protein-coupled receptors and
has been implicated as a part of consensus sequences involved in
coupling to G
i
(27) . To test whether this motif is
important for G
14 coupling, we introduced a point mutation into
each of the motifs. The mutant
1BK282L with substitution of
residue Lys
for a Leu residue at the C-terminal end of
the third inner loop can couple to the endogenous G
q/11 to the
same extent as the wild-type
1B-adrenergic receptor
(Fig. 5 A). To test whether this mutation has any effect
on coupling to G
14 or G
16, we introduced another mutation
into the mutant
1BK282L by deleting the amino acid sequence
extending from residues Lys
to His
, which
is also deleted in
1B
2 (Fig. 1), so that we can test
the mutated receptor without activating endogenous G
q/11. This new
mutant, designated
1B
2KL, couples to G
16 as well as
1B
2 does (Fig. 5 B), but it cannot couple to
G
14 (Fig. 5 C). This result, in addition to the fact
that both
1BK282L and
1B
2KL showed similar
ligand-binding characteristics and both are expressed at similar levels
to the wild-type receptor and to
1B
2 (Fig. 1), allows
us to conclude that the sequence motif (BB XXB) located at the
C-terminal end of the third inner loop is required for coupling to
G
14. A similar sequence motif that is located at the N-terminal
end of the third loop was also mutated by substituting residue
Lys
for an Ala residue in both the wild-type and
1B
2 (Fig. 1). However, this mutation had no effect on
coupling to G
q/11, G
14, or G
16 (data not shown),
suggesting that neither residue Lys
nor the entire motif
is required for G protein coupling.
Figure 5:
Activation of G subunits by mutants
1BK282L and
2KL. A, cells were transfected with the
cDNA encoding
1BK282L ( squares) or the
1B-adrenergic
receptor ( triangles). B, cells were cotransfected
with the cDNA encoding
2KL ( squares) or
1B
2
( triangles) and the cDNA encoding G
16. C, cells
were cotransfected with the cDNA encoding
2KL ( right two
bars) or
1B
2 ( left two bars) and the cDNA
encoding G
14 or
-galactosidase (LacZ, LZ). The
levels of inositol phosphates were determined 20 min after addition of
norepinephrine. 1 µM of norepinephrine was added in
panel C. The level of inositol phosphates in nontransfected
COS-7 cells is taken as 100%.
In an attempt to determine the
sequences that are directly involved in activation of G16, we made
the mutant
1B
5 by deleting the
1B-adrenergic receptor
sequence from residues Tyr
to His
. We found
that this mutant, when cotransfected with any of the G
subunits of
the Gq class, showed no ligand-induced activation of PLC
(Fig. 4 D). This suggests that it cannot couple to any of
the G
subunits, although it can still bind ligand albeit with a
decreased IC
and lower expression level (Fig. 1).
This result raised a possibility that the three amino acids extending
from Tyr
to Val
may be involved in coupling
to G
16 because the mutant
1B
5, which lacks these amino
acids, loses its ability to activate G
16, while the mutant
1
that contains these three amino acids can still activate G
16
(Fig. 4 A). To test this possibility, we generated two
1B-adrenergic receptor mutants: one, designated
1B
12,
was generated by deleting the three amino acids
(Tyr
-Val
) in the wild-type
1B-adrenergic receptor and the other, designated
1B
12
2, was generated by deleting these three amino acids
in
1B
2. The latter was made because it lacks the
G
q/11-coupling sequence so that it can be tested for coupling to
G
14 and G
16 in a background-free system. Both mutants were
tested for their abilities to activate the G
subunits of the Gq
class. We found that COS-7 cells transfected with either of the mutants
gave no ligand-dependent accumulation of IPs (Fig. 6), suggesting
neither of the mutants could couple to the endogenous G
q/11.
Moreover, these two mutants did not show ligand-dependent accumulation
of IPs when they were coexpressed with G
14 (Fig. 6).
However, ligand-dependent accumulation of IPs was found when these two
mutants were coexpressed with G
16, but the responses were much
lower than those found with the
1B
2 mutant (Fig. 6).
The binding assay indicates that
1B
12 (Fig. 1) and
1B
12
2 (data not shown) were expressed at lower levels
(160 fmol compared to 500 fmol expressed by the wild-type) with no
changes in their IC
values, suggesting that the deletion
somehow reduces the expression levels, without significantly changing
the conformation of the ligand-binding domains. We do not think that
reduction in the number of binding sites is sufficient to account for
the lower responses found in cells coexpressing G
16 because the
mutant
1B
11 that expresses at the similar level strongly
activates G
16. Thus, we conclude that though these three amino
acids extending from residues Tyr
to Val
may not constitute the major G
16-interacting domain, they
are involved in G
16 activation and that these three amino acids
are certainly required for activating G
q/11 and G
14.
Moreover, the same abilities of
1B
12 and of
1B
12
2 to activate G
16 indicates that the 13 amino
acids (Lys
-His
) deleted in the
2
mutants have no effect on specific G
16 activation, thus suggesting
that the 12-amino-acid sequence is essentially not involved in
G
16-coupling.
Figure 6:
Activation of G subunits by mutants
1B
12 and
1B
2
12. COS-7 cells were cotransfected
with the cDNA encoding
1B
2,
1B
12, or
1B
2
12 and the cDNA encoding G
14, G
16, or
-galactosidase (LacZ, LZ). The levels of inositol
phosphates were determined 20 min after addition of norepinephrine (1
µM). The level of inositol phosphates in nontransfected
COS-7 cells is taken as 100%.
1B-adrenergic receptor for its involvement in specific coupling to
the
subunits of the Gq class of G proteins and found that
different
1B receptor sequences are required for coupling to
G
q/11, G
14, and G
16. The amino acids extending from
residues Lys
to His
and from residues
Tyr
to Val
are required for activating
G
q/11, while the amino acids upstream of residue Asn
and downstream of residue Ser
in the third inner
loop of the receptor are required for activating G
14. It appears
that the third inner loop of
1-adrenergic receptor does not play a
crucial role in activation of G
16.
1B-adrenergic receptor. The
second basic amino acid, Lys
in the motif located at the
C-terminal end of the third inner loop is required for activating
G
14. However, we do not know whether the whole motif at the
C-terminal end of the third inner loop is part of the consensus
sequences involved in G
14 coupling. It is interesting to note that
the C5a receptor (data not shown), which lacks this sequence motif at
the C-terminal end of the third inner loop, cannot couple to G
14,
while the
1B-adrenergic receptor and IL-8 receptors, which have
this motif at the corresponding positions, can couple to G
14
(13) , suggesting that this motif may be part of the consensus
sequence involved in G
14 coupling. We will be able to test this
hypothesis by studying more G
14-coupling receptors.
1-adrenergic
receptor, we do not exclude the possibility that other segments of the
receptor may be involved in G protein binding and activation. In
addition, we do not know if loss of coupling activities caused by
specific mutations is a result of conformational changes or loss of
specific interacting or activating peptide sequences. The results that
all the mutated receptors retain ligand binding activities and that
most of them showed ligand-dependent activation of G
16 suggest
that the modified receptors retain their overall structural integrity.
In addition, we did not include G
in our study. G
has been shown to be involved in receptor-G protein coupling
(12) . In this study, endogenous G
subunits may be
involved. However, since there appears to be little specificity in the
interactions between G
and G
(28) or in
G
-mediated regulation of effectors
(29, 30) ,
we believe that our results in this report would be largely unaffected
by molecular differences in G
.
1B-adrenergic
receptor and some of its mutants apparently couple to G
16 better
than to G
14. The fact that G
14 is expressed at a lower level
than G
16 may partially explain this observation
(Fig. 3 A). It is also possible that the
1B-adrenergic receptor has a lower affinity for G
14 or/and
that G
14 is a less effective activator of PLC. Since it is
difficult to use the cotransfection assay system for kinetic or
quantitative studies due to the difficulties in manipulating the
expression levels of recombinant proteins, we were unable to determine
the precise mechanism. Moreover, it is worth noting that no differences
between G
q and G
11 have been observed in coupling to any of
the mutated receptors (data not shown).
16. However, our results presented in this report indicate that
the
1-adrenergic receptor sequences that determine the specificity
in G
16 coupling are unlikely to be located in the third inner loop
of the receptor. A number of reports have previously indicated that the
other inner loops and C-terminal end of G protein-coupled receptors
also play roles in G protein coupling
(31, 32) .
Currently, we are investigating the other inner loops of the
1-adrenergic receptor for their roles in coupling to G
16 by
using similar approaches.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.