From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026
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ABSTRACT |
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G protein subunits consist of two domains, a
GTPase domain and a helical domain. Receptors activate G proteins by
catalyzing replacement of GDP, which is buried between these two
domains, with GTP. Substitution of the homologous
i2 residues for four
s residues in
switch III, a region that changes conformation upon GTP binding, or of
one nearby helical domain residue decreases the ability of
s to be activated by the
-adrenergic receptor and by
aluminum fluoride. Both sets of mutations increase the affinity of
s for the
-adrenergic receptor, based on an increased amount of high affinity binding of the
-adrenergic agonist,
isoproterenol. The mutations also decrease the rate of
receptor-mediated activation and disrupt the ability of the
-adrenergic receptor to increase the apparent affinity of
s for the GTP analog, guanosine
5'-O-(3-thiotriphosphate). Simultaneous replacement of the
helical domain residue and one of the four switch III residues with the
homologous
i2 residues restores normal receptor-mediated
activation, suggesting that the defects caused by mutations at the
domain interface are due to altered interdomain interactions. These
results suggest that interactions between residues across the domain
interface are involved in two key steps of receptor-mediated
activation, promotion of GTP binding and subsequent receptor-G protein
dissociation.
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INTRODUCTION |
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Heterotrimeric G proteins transmit signals from cell surface
receptors to effector proteins that modulate a wide variety of cellular
processes (1, 2). The and
subunits of G proteins are
associated in the inactive GDP-bound form. Receptors activate G
proteins by catalyzing replacement of GDP by GTP on the
subunit. Receptor-catalyzed nucleotide exchange is thought to involve an "opening" of the guanine nucleotide binding pocket that facilitates GDP release and increases the relative affinity for GTP compared with
GDP (3, 4). The transient empty state of the G protein has a high
affinity for the hormone-receptor complex. However, this state is
short-lived due to the high intracellular concentration of GTP. Binding
of GTP leads to dissociation of the receptor from
·GTP and
,
each of which can transmit signals to effectors. Hydrolysis of GTP by
the
subunit regulates the timing of deactivation and reassociation
of
with
.
subunit structures consist of two domains, a GTPase domain that
resembles the oncogene protein p21ras and a helical domain
consisting of
helices and connecting loops. The bound GDP is buried
between the two
subunit domains, suggesting that the helical domain
may present a barrier to GDP release. Three regions in the GTPase
domain (switches I-III) assume different conformations in the
structures of GTP
S1-bound
versus GDP-bound
subunits (5-8). Switches I and II
correspond to conformational switch regions in the structures of both
p21ras and EF-Tu. Like the helical domain, switch III,
which is located at the interface of the two domains, is unique to the
structures of heterotrimeric G protein
subunits. The conformational
switch regions are important for the interaction of
subunits with
effectors (9),
(10, 11), and RGS (regulators of
G protein signaling) regulators of G proteins
(12). Most likely, they play a role in receptor-mediated activation as
well.
We previously identified a cluster of four switch III
residues2 in s
at the interface between the GTPase and helical domains in which
substitutions with
i2 homologs in the mutant construct N254D/M255L/I257L/R258A
s decreased receptor-mediated
activation of adenylyl cyclase in transiently transfected cells (13).
The activation defect caused by substituting
i2 residues
for these
s residues was corrected by replacing the
helical domain of
s with that of
i2 in a
chimera,
sis, in which
i2 homologs were substituted for
s residues 62-235, extending from the
end of the
1 helix to the end of the
2 helix (13). Thus, matching
i2 residues on both sides of the domain interface of
s restored receptor-initiated activation.
We now report a detailed analysis of the activation defects caused by
these switch III substitutions and of a mutation that replaces a nearby
helical domain residue, Asn167, with the homologous
i2 residue, arginine. Measurements in stably transfected
cells of isoproterenol binding to the
-adrenergic receptor, and the
time course and dose-dependence of adenylyl cyclase stimulation by the
hydrolysis-resistant GTP analog, GTP
S, in the presence and absence
of isoproterenol indicate that the mutations increase the affinity of
s for the
-adrenergic receptor, decrease the rate of
receptor-mediated activation, and block receptor-stimulated increases
in GTP
S affinity. Additional mutational analysis refines the nature
of the interdomain interactions that play a role in receptor-mediated
activation by demonstrating that of the switch III substitutions, R258A
alone causes a defect in receptor-mediated activation, that this defect
is corrected when the helical domain of
s is replaced
with that of
i2, and that the defect caused by the N167R
substitution is corrected when combined with the N254D substitution but
not the R258A substitution. These results suggest that interdomain
interactions are involved in the transmission of signals between the
receptor and the guanine nucleotide binding pocket.
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EXPERIMENTAL PROCEDURES |
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Construction of Subunit Mutants--
s mutant
constructs were generated from rat
s cDNA (14).
Chimeric
subunits were constructed from rat
s
cDNA and mouse
i2 cDNA (15). Subcloning and
mutagenesis procedures were verified by restriction enzyme analysis and
DNA sequencing. All
subunit constructs produced in this study
contain an epitope, referred to as the EE epitope (16), which was
generated by mutating
s residues
DYVPSD (189-194) to
EYMPTE and
i2
residues SDYIPTQ (166-172) to
EEYMPTE (single letter amino acid
code; mutated residues are underlined). This epitope does not affect
the ability of
s to activate adenylyl cyclase in
response to stimulation by the
-adrenergic receptor (17).
Preparation of Stable Cell Lines--
s
constructs were subcloned as HindIII fragments into the
retroviral vector pMV7 (20) and then stably expressed as described (21), in a subclone of cyc
S49 lymphoma cells,
cyc
kin
(22), in which
cAMP-dependent protein kinase is inactivated. Single
colonies containing the pMV7 vector were obtained using limiting
dilution in microtiter wells and selection in G418 (1 mg/ml). Clones
expressing
s constructs were identified by
immunoblotting with the anti-EE monoclonal antibody. Cell membranes
were prepared after nitrogen cavitation as described (23).
Immunoblots-- 25 µg of membrane proteins were resolved by SDS-polyacrylamide electrophoresis (10%), transferred to nitrocellulose, and probed with a monoclonal antibody to the EE epitope (16). The antigen-antibody complexes were detected using an anti-mouse horseradish peroxidase-linked antibody according to the ECL Western blotting protocol (Amersham Pharmacia Biotech).
Adenylyl Cyclase Assay--
Conversion of
[32P]ATP to [32P]cAMP in the presence of
various activators was measured as described (23). Membranes were
incubated at 30 °C. Reactions shown in Figs. 1 and 4 were
preincubated for 5 min in the absence of [
32P]ATP and
then incubated for 30 min. For the time courses shown in Fig. 3,
membranes were preincubated in the absence of [
32P]ATP
and activators for 6 min. At time = 0, [
32P]ATP
and either GTP
S or GTP
S and isoproterenol were added and aliquots
were removed at the indicated times for cAMP determination. To
determine EC50 values for stimulation of adenylyl cyclase
by GTP
S shown in Fig. 4, the observed adenylyl cyclase activity was
fitted to the equation,
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(Eq. 1) |
Receptor Binding Assay--
Membranes were incubated with 75 pM [125I]ICYP in competition with a range of
concentrations of isoproterenol (1011 to
10
3 M) in the presence or absence of 300 µM GTP for 1 h at 30 °C as described (24). At the
end of this time, the membranes were diluted and washed on Whatman GF/C
filters, and bound [125I]ICYP was measured. The
experimental data were analyzed for competition at two sites by
nonlinear least-squares curve fitting as described (24).
KL and KH, the low and
high affinity dissociation constants, respectively, were assumed to be
the same in the presence and absence of GTP. When
KL and KH were allowed to
vary in the two conditions, improved fits to the data were obtained.
Therefore, the two-state model may be an oversimplification of receptor
behavior, as has been suggested (25).
cAMP Accumulation Assay in Transiently Transfected
cyc S49 Lymphoma Cells--
subunit constructs were
introduced by electroporation into a subclone of
cyc
S49 lymphoma cells (26) that stably
expresses Simian virus 40 large T antigen, and cAMP accumulation was
measured after labeling with [3H]adenine as described
(13). Nucleotides were separated on ion-exchange columns (27), and cAMP
accumulation was expressed as
[3H]cAMP/([3H]ATP + [3H]cAMP) × 1000. Receptor-independent cAMP accumulation was determined by
measuring basal cAMP levels in cells transfected with the
sRC versions of the mutant constructs.
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RESULTS |
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Mutations at the Domain Interface of s Decrease
Activation by the
-Adrenergic Receptor and by Aluminum
Fluoride--
The ability of N254D/M255L/I257L/R258A
s
to be activated by the
-adrenergic receptor and by
AlF4
, which mimics the
-phosphate of GTP,
was measured after expression in cyc
S49
lymphoma cells (26), which lack endogenous
s (28). At equal expression levels (Fig.
1A), adenylyl cyclase activity
stimulated by both isoproterenol and by AlF4
was reduced by 80% in membranes of cells expressing
N254D/M255L/I257L/R258A
s compared with membranes of
s-expressing cells (Fig. 1B). Stimulation by
the hydrolysis-resistant GTP analog, GTP
S, not only was intact, but
increased by almost 2-fold.
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Mutations at the Domain Interface of s Increase the
Apparent Affinity of
s for the
-Adrenergic
Receptor--
Because N254D/M255L/I257L/R258A
s and
N167R
s exhibited decreased receptor-mediated activation,
we used a competitive binding assay to determine whether these mutant
subunits exhibit alterations in binding to the
-adrenergic
receptor. This assay measures an
s-dependent
increase in the affinity of the
-adrenergic receptor for the
agonist, isoproterenol (24, 29), which occurs in the absence of bound
guanine nucleotide. The high affinity hormone binding state of the
receptor is thought to reflect its interaction with Gs in
the nucleotide-free state. In the presence of GTP, receptors in
membranes of
s-expressing cells were predominantly in
the low affinity state (Fig.
2A). In the absence of GTP,
s caused the appearance of high affinity binding sites
for isoproterenol on the receptor (Fig. 2A). Like
s, both N254D/M255L/I257L/R258A
s and
N167R
s increased the affinity of the
-adrenergic
receptor for isoproterenol in the absence of GTP compared with in its
presence (Fig. 2, B and C). However, in membranes
of cells expressing these constructs, the affinity of the receptor for
isoproterenol in both the presence and absence of GTP was greater than
in membranes from
s-expressing cells, due to decreases
in KL and KH, the low and
high affinity dissociation constants, respectively, as well as
increases in the percentage of receptors in the high affinity form, % RH. This increase in hormone-receptor binding
was particularly striking in cells expressing N167R
s, in
which 50% of the receptors were in the high affinity form in the
presence of GTP. The simplest explanation of the increased amount of
hormone-receptor binding observed in the presence of
N254D/M255L/I257L/R258A
s and N167R
s is
that these mutant
subunits increase the affinity of Gs
for the receptor in both the nucleotide-bound and -free states.
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Mutations at the Domain Interface of s Decrease the
Rate of Activation by the
-Adrenergic Receptor--
Because
dissociation of Gs from the activated receptor must precede
adenylyl cyclase activation, we investigated whether
N254D/M255L/I257L/R258A
s and N167R
s
exhibited altered rates of receptor-mediated activation. To estimate
relative rates of receptor-mediated activation, we determined the
effects of isoproterenol on the time courses of adenylyl cyclase
activation by GTP
S. In membranes of cells expressing
s, GTP
S activated adenylyl cyclase with a time lag
that was greatly reduced by isoproterenol (Fig.
3A). This decreased time lag
reflects receptor-stimulated increases in the rates of GDP dissociation
and GTP
S binding. In the absence of isoproterenol, GTP
S activated
adenylyl cyclase in membranes containing
N254D/M255L/I257L/R258A
s or N167R
s with
somewhat shorter time lags than in
s membranes (Fig. 3,
B and C). Isoproterenol increased the activation
rates of these mutants, but not to the same extent as for
s. Thus, in the presence of isoproterenol, the time lags
of the mutants were longer than that of
s (Fig. 3,
B and C). N167R
s, which caused the
appearance of the greatest amount of high affinity binding to the
receptor (Fig. 2C), exhibited the longest time lag in the
presence of isoproterenol. Thus,
N254D/M255L/I257L/R258A
s and N167R
s
exhibit decreased rates of receptor-mediated activation, which could
reflect decreased rates of GTP-dependent dissociation from
receptors. Alternatively, or in addition, decreased rates of
receptor-mediated activation could be due to defects in
receptor-stimulated GTP binding, which we investigated as described
below.
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Mutations at the Domain Interface of s Disrupt the
Ability of the
-Adrenergic Receptor to Promote Binding of
GTP
S--
Receptors stimulate guanine nucleotide exchange on G
proteins by increasing the rate of GDP release and by causing a
preference for GTP compared with GDP (3, 4). For
s, this
results in an approximately 8-fold decrease in the half-maximal
effective concentration (EC50) for GTP
S stimulation of
adenylyl cyclase in the presence of isoproterenol compared with in its
absence (Fig. 4A). In the
absence of isoproterenol, N254D/M255L/I257L/R258A
s and
N167R
s exhibited EC50 values for GTP
S
stimulation of adenylyl cyclase that were slightly lower than that of
s (Fig. 4, B and C). However,
these EC50 values were unchanged by isoproterenol (Fig. 4, B and C) so that in the presence of
isoproterenol, their apparent affinities for GTP
S were less than
that of
s. Thus, although isoproterenol increases the
rate at which these mutant
subunits exchange nucleotide (Fig. 3),
it does not increase their apparent affinities for GTP.
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Localization of a Single Switch III Residue on the GTPase Side of
the Domain Interface That Is Important for Receptor-mediated Activation
of s--
We individually tested each of the four
switch III residues that were mutated in
N254D/M255L/I257L/R258A
s to determine their roles in
receptor-mediated activation. Receptor-dependent
stimulation of cAMP synthesis was measured in transiently transfected
cyc
S49 lymphoma cells. The only substitution
that decreased receptor-mediated activation was R258A (Fig.
5A). Receptor-independent cAMP
accumulation was also measured after introducing a second mutation (the
RC mutation) that substitutes cysteine for the arginine at position 201 (19).
sRC has decreased GTPase activity and is
constitutively activated. R258A
sRC produced
receptor-independent cAMP accumulation similar to that of
sRC, indicating that, as is the case for
N254D/M255L/I257L/R258A
s, R258A
s can
activate adenylyl cyclase when it is in the GTP-bound form.
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Complementation of the Activation Defect of
R258As--
Because the activation defect of
N254D/M255L/I257L/R258A
s was corrected by replacing the
helical domain of
s with that of
i2 in a
chimera,
sis, in which
i2 homologs were
substituted for
s residues 62-235, (Fig.
6) (13), we investigated whether introducing the single homolog substitution (R258A) responsible for the
defect of N254D/M255L/I257L/R258A
s into
sis would result in normal activation properties.
R258A
sis exhibited activation properties similar to
those of
s rather than those of R258A
s (Fig. 5A). Thus, the defect produced by the R258A
substitution appears to be due to an alteration in interactions with
s residue(s) in the helical domain.
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Combining the N167R and R258A Substitutions Results in an Additive
Defect in Receptor-mediated Activation--
We investigated the effect
of combining the substitutions on each side of the domain interface,
N167R and R258A, that caused significant decreases in receptor-mediated
activation. Although Asn167 and Arg258 are both
at the domain interface, they are not close enough to make contact in
the x-ray crystal structures of subunits (see Fig. 6).
N167R/R258A
s exhibits a more severe activation defect (Fig. 5B) than either N167R
s (Fig.
5C) or R258A
s (Fig. 5A) does, although receptor-independent activation by
N167R/R258A
sRC is normal. Because the defects of the
N167R and R258A substitutions are additive, the contributions of these
substitutions to defects in receptor-mediated activation are
independent. Furthermore, some other residue(s) in the helical domain
other than the
i2 homolog of Asn167 must be
responsible for the suppression of the R258A defect in R258A
sis.
Combining the N167R and N254D Substitutions Corrects the Defect of
the N167R Substitution--
According to the x-ray crystal structures
of subunits, Asn167 is close to Asn254 (see
Fig. 6). Therefore, we hypothesized that the N167R substitution might
cause a conditional defect, depending on the identity of the residue at
position 254. According to this hypothesis, replacing Asn254 with aspartate should correct the defect caused by
the N167R mutation. Indeed, we found that an
subunit with both
substitutions, N167R/N254D
s, exhibits activation
properties similar to those of
s (Fig. 5B).
Thus, the N254D substitution, which on its own does not disrupt
receptor-mediated activation, corrects the activation defect caused by
the N167R substitution.
Substitution of Asn167 by Alanine Does Not Cause a
Defect in Receptor-mediated Activation of s--
To
further investigate the mechanism by which the N167R substitution
causes a defect in receptor-mediated activation, we determined the
effect of mutating Asn167 to alanine. Alanine substitutions
eliminate the side chain beyond the
carbon but generally do not
alter the main chain conformation or impose significant electrostatic
or steric effects (30). Therefore, if the activation defect resulting
from the N167R substitution is due to a steric or electrostatic
incompatibility with Asn254, then alanine substitution
might not cause an activation defect. However, if the N167R
substitution removes a favorable interaction between Asn167
and Asn254, then alanine substitution should also cause a
defect. Because N167A
s exhibited normal
receptor-stimulated cAMP production (Fig. 5C), the
activation defect of N167R
s appears to be due to a
steric or electrostatic incompatibility that is reversed by the N254D substitution.
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DISCUSSION |
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Our analysis of s mutants with
substitutions at the interface of the GTPase and helical domains
suggests that interdomain interactions play a role in the
bi-directional transmission of signals between receptors and the
nucleotide binding site. Interaction between activated receptors and G
proteins promotes GTP binding by accelerating GDP release and
increasing the relative affinity for GTP compared with GDP (3, 4).
Conversely, nucleotide binding decreases the affinity of G proteins for
receptors (24, 29). Substitution of the homologous
i2
residues for four
s residues (Asn254,
Met255, Ile257, and Arg258) in
switch III of the GTPase domain or of one nearby helical domain residue
(Asn167) in the
D/
E loop causes defects in both
directions of this communication process. Signal transmission from the
receptor to the nucleotide binding site is defective in that the
affinities of these
s mutants for GTP
S are unchanged
by isoproterenol. Conversely, altered communication between the guanine
nucleotide binding pocket and the receptor binding site(s) is
demonstrated by high affinity hormone-receptor binding in the presence
of 300 µM GTP.
Contacts between the D/
E loop in the helical domain and three
regions of the GTPase domain have been implicated as playing a role in
receptor-mediated activation. In the heterotrimer-based
subunit
model shown in Fig. 6, the helical domain side of the interface
"above" the GDP consists of the
D/
E loop. Moving up from the
GDP toward the top of the
subunit, the corresponding GTPase side of
the interface consists of the
5/
G,
4/
3, and
G/
4
loops. Closest to the GDP, a salt bridge interaction between Asp173 in the carboxyl-terminal portion of the
D/
E
loop and Lys293 in the
5/
G loop (Fig. 6, dark
blue) is required for activation by the
-adrenergic receptor
and by AlF4
, but not by GTP
S (31). These
residues are highly conserved among
subunits, and
Lys293 is located in the NKXD motif, which is
important for GTP binding by monomeric GTPases. Mutation of
Asp173 increases GTP affinity, consistent with the idea
that the mutation "frees" Lys293 from
Asp173 to interact with GTP, whereas mutation of
Lys293 decreases GTP affinity. Because Asp173
and Lys293 are adjacent to the bound guanine nucleotide,
they are more directly involved in regulating guanine nucleotide
binding than the residues mutated in our study are. The effects of
mutating Asp173 and Lys293 on receptor affinity
and receptor-dependent changes in GTP affinity have not
been determined.
Arg258 (Fig. 6, red) is located further up, in
the 4/
3 loop, which includes switch III. Interestingly, a
mutation that substitutes tryptophan for Arg258 was found
in a patient with Albright hereditary osteodystrophy, and
R258W
s exhibited more severe defects than did
R258A
s.3 The
defect caused by the R258A substitution is suppressed (Fig. 5A) by substituting the entire helical domain of
i2 for that of
s in the
sis chimera (Fig. 6), but we have not identified the
helical domain residue(s) responsible. In addition to
Asn167, there are two other
s residues in
the
D/
E loop, Ile172 and Cys174, that
differ in the sequences of
s and
i2.
Cys174 is not close to Arg258 when modeled onto
the heterotrimeric G protein structures (10, 11) or in the structure of
s·GTP
S (32). However, in the latter structure, the
side chains of Ile172 and Arg258 are within
~4 Å of each other (see Fig. 7).
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Asn167 (Fig. 6, red) is located further away
from the GDP in the D/
E loop. The activation defect caused by
replacing Asn167 with its
i2 homolog
(arginine) is corrected (Fig. 5B) by simultaneously replacing Asn254 (Fig. 6, magenta) in the
4/
3 loop with its
i2 homolog (aspartate). In the
subunit structures, the corresponding residues are hydrogen bonded
to each other via the side chain of the residue corresponding to
Asn167 and either the side chain (in
t (5,
7) and an
t/
i1 chimera complexed with
t
t (10)) or the backbone carbonyl (in
s (9, 32) and
i1 (6)) of the residue
corresponding to Asn254 (see Fig. 7). The N254D
substitution in
s might correct the defect caused by the
N167R substitution via a charge neutralization mechanism. This idea is
supported by the fact that all
subunits with an arginine at the
position corresponding to Asn167 (
q,
11,
14,
15,
16, and
o) have an aspartate at the
position corresponding to Asn254 and by the observation
(Fig. 5C) that the N167A substitution in
s
leaves receptor-mediated activation intact. It is not surprising that
the activation defect of N167R/R258A
s is worse than
those of N167R
s and R258A
s (Fig. 5),
because these residues are not within contact distance. Because the two
mutations cause additive defects, the two residues also do not appear
to influence each other through electrostatic or steric effects
(33).
We previously found that substitution of i2 residues for
s residues 304, 305, and 307-311 in the
G/
4 loop
(furthest from the nucleotide on the GTPase side of the interface in
Fig. 6) disrupts receptor-mediated activation in the context of
s but not
sis (13). Of the mutated
s residues, only Lys305 and
Tyr311 are close to the interface in the structure of
s·GTP
S (32).
Although interactions between residues in switch III and the D/
E
loop are important for receptor-mediated activation, the known receptor
binding sites of
s, the carboxyl terminus of
5 (13,
21, 34) and possibly the
4/
6 loop (34), are not near this
interface.
subunits bind to
, which is required for
receptor-mediated activation, via switches I and II and the amino
terminus (10, 11), which are also not near this interface. Thus,
receptors initiate activating signals at a significant distance from
the domain interface, possibly via an interaction between switches II
and III.
N254D/M255L/I257L/R258As and N167R
s
exhibit two characteristics in the absence of receptor stimulation that
are not normal and that resemble those of wild-type
s
upon activation by hormone-bound receptors. They exhibit slightly
elevated basal rates of activation (Fig. 3) and somewhat increased
basal affinities for GTP
S (Fig. 4). The basal activation rates of
these
s mutants, which reflect basal nucleotide exchange
rates, are not nearly as elevated as in an
s mutant,
A366S
s, which is both thermolabile and constitutive activated (35). However, increased rates of basal GDP dissociation in
N254D/M255L/I257L/R258A
s and N167R
s would
account for their observed defects in activation by aluminum fluoride,
which requires the presence of bound GDP.
The defects in guanine nucleotide and receptor binding of
N254D/M255L/I257L/R258As and N167R
s may
be interrelated. For instance, decreased receptor-stimulated GTP
binding would stabilize the high affinity hormone-receptor-G protein
complex, which forms when the G protein is in the nucleotide-free
state. Conversely, higher affinity receptor-G protein interactions
could decrease nucleotide binding, because activated receptors can
cause dissociation of both GDP and GTP analogs (36, 37). However,
although there are other reported
s mutants with defects
in GTP binding and receptor-mediated activation, there is no precedent
for an associated increase in receptor affinity. R231H
s,
containing a mutation in the
2 helix (38), and S54N
s,
containing a substitution in the
1 helix (39), exhibit impaired
activation by receptors and AlF4
but can be
activated by GTP
S. The affinities of these
s mutants for GTP are decreased upon receptor stimulation. The affinity of
R231H
s for the receptor is normal, and the affinities of
S54N
s and of A366S
s, which exhibits
accelerated GDP release (35), were not determined. The defect of
S54N
s appears to be due to altered interactions with the
bound Mg2+, with which Ser54 interacts. Thus,
the activation defects of these
s mutants are distinct
from those of N254D/M255L/I257L/R258A
s and
N167R
s.
Further studies will be required to elucidate how the subunit
domain interface mediates communication between the residues responsible for receptor binding and the guanine nucleotide binding pocket. This type of regulation appears to be unique to heterotrimeric G proteins, which interact with seven-transmembrane-spanning receptors, compared with monomeric GTPases, which lack switch III and the helical
domain and which utilize different exchange factors.
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ACKNOWLEDGEMENTS |
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We thank David Lambright and Paul Sigler for
the coordinates of the t/
i1 chimera
complexed with
t
t, Stephen Sprang for the
coordinates of
s·GTP
S complexed with the catalytic
domains of adenylyl cyclase, Dennis Warner and Lee Weinstein for
sharing data prior to publication, and Thomas Hynes for helpful
discussions and critical reading of the text.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM50369 (to C. H. B.).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.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed. Tel.: 203-785-3202; Fax:
203-785-4951; E-mail: cathy_berlot{at}qm.yale.edu.
1
The abbreviation used is: GTPS, guanosine
5'-O-(3-thiotriphosphate).
2
Residue numbering throughout is according to the
long splice variant of s.
3 D. R. Warner and L. S. Weinstein, personal communication.
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