Mutagenesis of the Conserved Residue Glu259 of
Gs
Demonstrates the Importance of Interactions
between Switches 2 and 3 for Activation*
Dennis R.
Warner
§,
Rianna
Romanowski
,
Shuhua
Yu¶, and
Lee S.
Weinstein¶
From the
Membrane Biochemistry Section, Laboratory of
Molecular and Cellular Neurobiology, NINDS and the ¶ Metabolic
Diseases Branch, NIDDK, National Institutes of Health, Bethesda,
Maryland 20892
 |
ABSTRACT |
We previously reported that substitution of
Arg258 within the switch 3 region of
Gs
impaired activation and increased basal GDP release
due to loss of an interaction between the helical and GTPase domains
(Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein,
L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G
protein
-subunits and is predicted to be important in activation. To
determine the importance of Glu259, this residue was
mutated to Ala (Gs
-E259A), Gln
(Gs
-E259Q), Asp (Gs
-E259D), or Val
(Gs
-E259V), and the properties of in vitro
translation products were examined. The Gs
-E259V was
studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gs
-E259D stimulated adenylyl cyclase normally in
the presence of GTP
S but was less efficient with isoproterenol or
AlF4
. The other mutants had
more severely impaired effector activation, particularly in response to
AlF4
. In trypsin protection
assays, GTP
S was a more effective activator than
AlF4
for all mutants, with
Gs
-E259D being the least severely impaired. For
Gs
-E259D, the
AlF4
-induced activation defect
was more pronounced at low Mg2+ concentrations.
Gs
-E259D and Gs
-E259A purified from
Escherichia coli had normal rates of GDP release (as
assessed by the rate GTP
S binding). However, for both mutants, the
ability of AlF4
to decrease the
rate of GTP
S binding was impaired, suggesting that they bound
AlF4
more poorly. GTP
S bound
to purified Gs
-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at
micromolar concentrations. Sucrose density gradient analysis of
in vitro translates demonstrated that all mutants except
Gs
-E259V bind to 
at 0 °C and were stable at
higher temperatures. In the active conformation Glu259
interacts with conserved residues in the switch 2 region that are
important in maintaining both the active state and
AlF4
in the guanine nucleotide
binding pocket. Although both Gs
Arg258 and
Glu259 are critical for activation, the mechanisms by which
these residues affect Gs
protein activation are distinct.
 |
INTRODUCTION |
Heterotrimeric guanine nucleotide-binding proteins (G
proteins)1 couple
heptahelical receptors to intracellular effectors and are composed of
three subunits (
,
, and
) (reviewed in Refs. 1-3). The
-subunits, which are distinct for each G protein, bind guanine
nucleotide and modulate the activity of specific downstream effectors.
For Gs, these include the stimulation of adenylyl cyclase
and modulation of ion channels (4, 5). In the inactive state, GDP-bound
-subunit is associated with a 
-dimer. Upon receptor
activation, the
-subunit undergoes a conformational change resulting
in the exchange of GTP for GDP and dissociation from 
. While GTP
is bound, the
-subunit interacts with and regulates specific
effectors. An intrinsic GTPase activity within the
-subunit
hydrolyzes bound GTP to GDP, returning the G protein to the inactive
state. Analogs of GTP, such as GTP
S and
GDP-AlF4
, lock the G protein in
the active state.
X-ray crystal structures reveal that G protein
-subunits have two
domains, a ras-like GTPase domain, which includes the
regions for guanine nucleotide binding and effector interaction, and a helical domain, which may prevent release of GDP in the inactive state
(6-12). Comparison of the crystal structures of inactive (GDP-bound)
and activated (GTP
S- or
AlF4
-bound)
-subunits
demonstrates three regions (named switches 1, 2, and 3), the
conformation of which changes upon switching from the inactive to
active state. The movement of switches 1 and 2 is directly related to
the presence of the
-phosphate group, whereas switch 3 has no direct
contact with bound guanine nucleotide. Upon activation, switches 2 and
3 move toward each other, and the two regions form multiple
interactions that presumably stabilize the active state (7, 10). Switch
3 residues also make contacts with the helical domain that are
important for high affinity guanine nucleotide binding (10, 15). At
least for transducin, this region may also be important in effector
activation (13).
We have previously shown that substitutions of the switch 3 residue
Arg258 impairs activation by receptor or
AlF4
(15).2 The latter effect was
the direct result of decreased GDP binding due to loss of contacts
between the Arg258 side chain and residues within the
helical domain. The adjacent residue (Glu259) is invariant
in all known G protein
-subunits and is predicted to be important in
activation, because it makes interactions with switch 2 residues in the
active state (7, 12). Moreover, this residue is mutated to a valine in
a patient with Albright hereditary osteodystrophy (16). In the present
report, we provide evidence that substitution of Glu259
also leads to impaired activation, particularly by receptor or AlF4
. However, impaired
activation of these mutants by
AlF4
is not the result of
decreased GDP binding (as is the case for the Arg258
mutants) but rather is the result of a decreased ability to bind the
AlF4
moiety. The crystal
structure of GTP
S-bound Gs
reveals interactions between the acidic side chain of Glu259 and basic residues
within switch 2 that are important in maintaining the active state and
in binding of AlF4
(12).
Although adjacent switch 3 residues in Gs
(Arg258 and Glu259) are both critical for
activation, the mechanisms by which mutations of these residues result
in defective activation are distinct.
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EXPERIMENTAL PROCEDURES |
Construction of Gs
Plasmids and in Vitro
Transcription/Translation--
To generate Gs
Glu259 mutants, polymerase chain reaction was performed as
described previously (15) using linearized vector containing wild type
Gs
cDNA as template. The upstream primer was
5'-GACAAAGTCAACTTCCACATGTTTGACGTGGGTGGCCAGCGCGATGAACG-3', and the
downstream mutagenic primers were as follows:
5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCACCCGGATGACCATGTTG-3' for E259V,
5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCGCCCGGATGACCATGTTG-3' for E259A,
5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCTGCCGGATGACCATGTTG-3' for E259Q, and
5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCGTCCCGGATGACCATGTTG-3' for
E259D. Each polymerase chain reaction product was digested with
HincII andSse8387I and ligated into the
transcription vector pBluescript II SK (Stratagene, La Jolla, CA) that
contained wild type human Gs
cDNA (splice variant
Gs
-1, Ref. 17) in which the same
HincII-Sse8387I restriction fragment had been
removed. Mutations were verified by DNA sequencing, and synthesis of
full-length Gs
from each construct was confirmed by
immune precipitation of in vitro translated products with RM
antibody, directed against the carboxyl-terminal decapeptide of
Gs
(18). In vitro transcription/translation was performed on Gs
plasmids as described previously
(15, 19) using the TNT-coupled transcription/translation system from
Promega, with the exception that in most experiments, no RNase
inhibitor was added.
Adenylyl Cyclase Assays--
Wild type and mutant
Gs
in vitro transcription/translation
products (10 µl of translation medium) were reconstituted into 25 µg of purified S49 cyc plasma membranes and tested for stimulation of
adenylyl cyclase in the presence of various agents as indicated in
Table I (15, 19, 20). Reactions were incubated for 15 min at 30 °C,
and the amount of [32P]cAMP produced was measured as
described previously (21).
Trypsin Protection Assays--
Limited trypsin digestion of
in vitro translated Gs
was performed as
described previously (15, 19). Briefly, 1 µl of in vitro
translated [35S]methionine-labeled Gs
was
incubated in incubation buffer (20 mM HEPES, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol) with or without 100 µM
GTP
S or 10 mM NaF/10 µM AlCl3
at various temperatures for 1 h and then digested with 200 µg/ml
tosyl-L-phenylyalanine chloromethyl ketone-trypsin for 5 min at 20 °C. In some experiments, GDP was also included in the
preincubation, and in other experiments the MgCl2
concentration was varied. Reactions were terminated by boiling in
Laemmli buffer. Digestion products were separated on 10%
SDS-polyacrylamide gels, and the amount of 38-kDa protected fragment
was measured by phosphorimaging. The percentage of protection is the
signal in 38-kDa protected band divided by the signal in the undigested
full-length Gs
band × 100.
Sucrose Density Gradient
Centrifugation--
[35S]Methionine-labeled
Gs
was synthesized, and rate zonal centrifugation was
performed on linear 5-20% sucrose gradients (200 µl) as described
previously (19, 22). Gradients were prepared in 20 mM
HEPES, pH 8.0, 1 mM MgCl2, 1 mM
EDTA, 1 mM dithiothreitol, 100 mM NaCl, 0.1%
Lubrol-PX. Six-µl fractions were obtained and analyzed by
SDS-polyacrylamide gel electrophoresis, and the relative amount of
Gs
in each fraction was quantified as described
previously (19). To assess the ability of Gs
to bind to
G
, in vitro translation products were preincubated for
1 h at 0 °C in the presence or absence of G
(20 µg/ml)
prior to centrifugation. In order to optimize separation between free
-subunit and heterotrimer, 0.15% (w/v) CHAPS was substituted for
Lubrol-PX in the preincubations and gradients, and the samples were
centrifuged at 120,000 rpm (627,000 × g at the maximum
radial distance from the center of rotation
(Rmax) in a TLA-120.2 rotor (Beckman). G
was isolated from bovine brain (23).
Expression and Purification of Gs
from Escherichia
coli--
Plasmid pQE60, containing the long form of bovine
Gs
cDNA with a hexa-histidine extension at the
carboxyl terminus, was a generous gift of A. G. Gilman and R. K. Sunahara. The Glu259 residue was mutated by
site-directed mutagenesis using the Quickchange kit (Statagene). Each
mutated cDNA was sequenced to confirm the presence of the desired
mutation and to rule out polymerase chain reaction artifacts. After
transformation into E. coli strain JM109, cultures
were grown, Gs
expression was induced, and
Gs
proteins were purified as described previously (15,
24), except that [GDP] was only 10 µM in the storage buffer.
Guanine Nucleotide Binding Assays--
Assays measuring the rate
of binding of GTP
S were performed as described previously (15, 25).
Briefly, 1-2 pmol of purified Gs
was incubated at
37 °C in a final volume of 2 ml containing 1 µM
[35S]GTP
S (5,000-10,000 cpm/pmol) in 25 mM HEPES, pH 8.0, 1 mM EDTA, 100 mM
NaCl, 10 mM MgCl2, 1 mM
dithiothreitol, and 0.01% Lubrol-PX with or without 10 mM
NaF/10 µM AlCl3. At various times, 50-µl aliquots were removed and diluted with 2 ml of ice-cold stop solution (25 mM Tris-HCl, 100 mM NaCl, 25 mM
MgCl2, and 100 µM GTP) and maintained on ice
until all samples were collected. Samples were then filtered under
vacuum through nitrocellulose filters (Millipore) and washed twice with
10 ml of stop solution without GTP, and filters were dissolved in 10 ml
of scintillation mixture. To determine the effect of Mg2+
on the rate of GTP
S dissociation, ~2.5 pmol of purified
Gs
was loaded with [35S]GTP
S at
30 °C for 45 min in the presence of various free Mg2+
concentrations. After addition of 100 µM cold GTP
S,
bound [35S]GTP
S was determined at various time points
as described above. koff for GTP
S
dissociation was determined by fitting the data to the function
y = ae
kt + b using the software GraphPad Prism, version 2.01. Free
Mg2+ concentrations were calculated as described (26).
 |
RESULTS |
Substitution of Gs
Glu259 Leads to
Decreased Activation--
Gs
Glu259
substitution mutants were cloned into the transcription vector
pBluescript, and the in vitro transcription/translation products were compared with those of Gs
-WT in various
biochemical assays. We substituted Glu259 with valine
(Gs
-E259V) because a mutation encoding this substitution has been identified in a patient with Albright hereditary
osteodystrophy (16), a human disorder associated with heterozygous
loss-of-function mutations of Gs
(27, 28). Because the
presence of an amino acid with a bulky and branched side chain (valine)
may introduce nonspecific steric effects, we also generated and
analyzed additional mutants in which Glu259 was replaced by
alanine (Gs
-E259A), glutamine (Gs
-E259Q),
or aspartate (Gs
-E259D). In Gs
-E259A, the
acidic side chain was removed, whereas in Gs
-E259Q it is
converted to a residue in which the carboxyl group is replaced by a
neutral amide group. In Gs
-E259D, the charge of the
residue at position Glu259 is maintained, but the length of
the side chain is shortened by one methylene group.
After reconstitution of translation products into purified S49 cyc
membranes (which lack endogenous Gs
),
Gs
-E259V had markedly decreased ability to stimulate
adenylyl cyclase in the presence of GTP
S,
AlF4
, or activated receptor
(isoproterenol + GTP) (Table I). For Gs
-E259A and -E259Q, the ability to stimulate adenylyl
cyclase was moderately reduced in the presence of GTP
S (~40% of
Gs
-WT) and more markedly reduced in the presence of
AlF4
or activated receptor.
Stimulation of adenylyl cyclase by Gs
-E259D was normal
in the presence of GTP
S but moderately reduced in the presence of
AlF4
or activated receptor. Although
the severity of the defect varied depending on which specific residue
replaced Glu259, for each
Gs
-Glu259 mutant, GTP
S was the most
effective activator and AlF4
the least
effective activator.
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Table I
Adenylyl cyclase stimulation by Gs mutants
In vitro transcription/translation products were mixed with
purified cyc- membranes and assayed for adenylyl cyclase stimulation as
described under "Experimental Procedures." Results are expressed as
the mean ± S.D. ( n 1) of triplicate
determinations and are corrected for the relative level of synthesis of
each mutant to Gs -WT. Gs -E259V, -E259A, -E259Q,
and -E259D were synthesized to 73, 78, 74, and 91% of Gs -WT
levels, respectively, as determined by in vitro translation
with [35S]methionine, SDS-PAGE, and phosphorimaging.
Background values determined from mock transcription/translation
reactions (in pmol of cAMP/ml of translation medium/15 min: GTP,
29 ± 1; isoproterenol, 39 ± 5; GTP S, 39 ± 2; and
AlF4 , 64 ± 6) were subtracted from each
determination.
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We next examined the ability of
AlF4
or GTP
S to protect each
mutant from trypsin digestion, which measures the ability of each agent
to bind to Gs
and induce the active conformation (29). In the inactive, GDP-bound state, two arginine residues within switch 2 (most likely Arg228 and Arg231, based upon
sequence homology with transducin) are sensitive to trypsin digestion,
leading to the generation of low molecular weight fragments. When
Gs
attains the active conformation, these residues are
inaccessible to trypsin digestion (7) and therefore trypsinization of
activated Gs
generates a partially protected 38-kDa
product. Gs
-WT was well protected by
AlF4
or GTP
S at temperatures up to
37 °C (Fig. 1, Table
II). At 30 °C,
Gs
-E259V, -E259A, and -E259Q showed little protection by GTP
S and no protection by AlF4
(Fig. 1). In contrast, both GTP
S and
AlF4
were able to protect
Gs
-E259D, with GTP
S being a more efficient activator
than AlF4
(Fig. 1, Table II).
Consistent with the results of the cyc reconstitution assays,
AlF4
was less effective than
GTP
S in protecting all Gs
-E259 mutants from trypsin
digestion.

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Fig. 1.
Trypsin protection of in vitro
translated Gs -E259 mutants
in the presence of GTP S or
AlF4 . In vitro
translates were digested with tosyl-L-phenylyalanine
chloromethyl ketone-trypsin (200 µg/ml) for 5 min at 20 °C after
1-h preincubations at 30 °C in the presence of GTP S or
AlF4 . For each
Gs , the full-length undigested Gs (52 kDa) is shown in the far left lane (no trypsin),
and complete digestion in the absence of activators is demonstrated in
the second lane (no adds.). The two right
lanes show the amount of the 38-kDa protected band generated after
trypsin digestion in the presense of either GTP S (100 µM) or AlF4 . The
smaller products in the left lane are due to initiation of
protein synthesis at downstream methionine codons. Quantitation of
trypsin protection assays for Gs -E259D is presented in
Table II.
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Table II
Effect of temperature and GDP on
AlF4 -induced trypsin protection
These data were obtained from experiments of the type presented in Fig.
1. The amount of the 38-kDa trypsin-stable Gs fragment was
determined by phosphorimaging, and for Gs -WT, it is
expressed as a percent of undigested Gs (mean ± S.E.).
No protection was observed when
AlF4 and GTP S were excluded.
Maximum trypsin protection has a theoretical limit of 71%, based on
the removal of 2 of 7 total methionine residues by trypsin. For
Gs -E259D, the data are expressed as percentage of wild type
at each condition (mean ± S.E.). The number of experiments
performed for each condition is shown in the right column.
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Because the Gs
-E259D encoded the most subtle structural
change and had the smallest activation defect, we studied the ability of this mutant to be protected by GTP
S and
AlF4
at various temperatures and in
the presence or absence of excess GDP (Table II). For
Gs
-R258 mutants, the activation defect in the presence
of AlF4
was more severe at higher
temperatures and was reversible in the presence of excess GDP (15).
Although raising the temperature had little effect on the ability of
GTP
S to protect Gs
-E259D from trypsin protection,
temperature had a profound effect on protection by
AlF4
, being 85, 49, and 7% of
Gs
-WT at 25, 30, and 37 °C, respectively. At
37 °C, addition of 2 mM GDP was able to somewhat reverse
the defect in activation by AlF4
,
although not to the extent that it was able to reverse the defect in
the Gs
-R258 mutants (15). Interestingly, addition of GDP lowered the ability of AlF4
to protect
Gs
-E259D at 25 and 30 °C (Table II). Although this effect was consistently observed, we have no good explanation for this observation.
Substitution of Gs
Glu259 Has Little
Effect on the Rate of GDP Release in the Basal State--
The impaired
activation of Gs
-Glu259 mutants by
AlF4
could result from decreased
affinity for AlF4
, decreased
ability for the GDP-AlF4
complex to activate the mutant Gs
s, or decreased ability
of the mutant Gs
s to maintain the GDP-bound state
because GDP binding is a prerequisite for
AlF4
binding and activation. For the
Gs
-Arg258 mutants, impaired activation by
AlF4
is primarily the result of
impaired GDP binding (15). The inability of GDP to significantly
reverse the AlF4
-induced
activation defect in Gs
-E259D suggests that this defect is not due to defective GDP binding.
To directly evaluate the rate of GDP release in the basal state, we
expressed and purified bovine Gs
-WT, -E259A, and -E259D, each with a carboxyl-terminal hexahistidine tag, from E. coli and examined the time course of GTP
S binding. The rate of
GTP
S binding has been shown to be limited by the rate of GDP
dissociation, and the experimentally determined values of these two
rates are essentially identical (30, 31). This assay has also been
previously used as a measure of the GDP dissociation rate in other
Gs
mutants(15, 32). Substitution of Glu259
had little effect on the rate of GDP release in the basal state, as the
time course of GTP
S binding at 37 °C (in the absence of AlF4
) is essentially identical for
Gs
-WT and -E259D, whereas the rate of GTP
S binding
for Gs
-E259A is only increased minimally (Fig.
2). Consistent with these results, the
rate of increase of trypsin protection of Gs
-WT and
-E259D in vitro translation products in the presence of
GTP
S was also identical (data not shown). These results demonstrate
that unlike substitutions of Arg258, the rate of GDP
release is not significantly altered by substitution of
Glu259, and therefore the impaired activation by
AlF4
is not primarily due to decreased
GDP binding.

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Fig. 2.
Time course of GTP S
binding to purified Gs s in the
presence or absence of
AlF4 . Bovine
Gs -WT, -E259A, and -E259D, each with a carboxyl-terminal
hexahistidine extension, were expressed and purified from E. coli, and the time course of GTP S binding for each was
determined either in the presence (filled symbols) or
absence (open symbols) of
AlF4 . Gs -WT ( and ), Gs -E259A ( and ), and
Gs -E259D ( and ) were incubated
with 1 µM [35S]GTP S (~10,000
cpm/pmol) at 37 °C for varying times, and the amount of bound
GTP S was determined as described under "Experimental
Procedures." For each Gs , each data point
(with or without AlF4 ) was
normalized to maximal binding at 10 min in the absence of
AlF4 . Each data
point is the mean ± S.D. of triplicate determinations. This
experiment was representative of three experiments. The
Bmax values in the absence of
AlF4 were as follows:
Gs -WT, 3 pmol; Gs -E259A, 2 pmol; and
Gs -E259D, 1 pmol.
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Substitution of Gs
Glu259 Decreases
AlF4
Binding--
We next examined
the ability of AlF4
to interact with
mutant Gs
s in the GDP-bound state to determine whether
the decreased activation of Gs
-E259 mutants by
AlF4
is due to impaired
AlF4
binding. It has been shown
previously that the rate and extent of GTP
S binding to G
-subunits is markedly reduced in the presence of
AlF4
, presumably because the
GDP-AlF4
complex bound to G
is more stable than GDP alone (8). Because Gs
-WT,
-E259D, and -E259A have similar rates of GTP
S binding in the absence
of AlF4
, the time course of GTP
S
binding in the presence of AlF4
should
reflect the ability of each form of Gs
to interact with AlF4
. Similar to previously reported
observations (8), the rate and extent of GTP
S binding to
Gs
-WT was markedly reduced in the presence of
AlF4
(Fig. 2). In contrast,
AlF4
only partially reduced the rate
and extent of GTP
S binding to Gs
-E259D and had a
minimal effect on the GTP
S binding curve for Gs
-E259A
(Fig. 2). These results are consistent with the results of adenylyl
cyclase and trypsin protection assays, which demonstrate that
AlF4
-induced activation is severely
impaired in Gs
-E259A but only partially impaired in
Gs
-E259D and suggest that the decreased ability of
AlF4
to activate
Gs
-E259 mutants is primarily due to decreased ability of
the mutants to maintain AlF4
in the
guanine nucleotide binding pocket.
Effect of Mg2+ Concentration on Activation by
AlF4
and GTP
S
Binding--
Substitution of Gs
Arg231, a
residue in switch 2 that interacts with switch 3 residues in the active
state, leads to a defect in activation by
AlF4
that is more pronounced at low
Mg2+ concentrations (33). We therefore examined the effect
of varying Mg2+ concentration on the ability of
AlF4
to protect
Gs
-E259D from trypsin digestion. In the trypsin
protection experiments shown in Fig. 1 and Table II, the
MgCl2 concentration was 10 mM (~9
mM free Mg2+). Lowering the MgCl2
concentration to 2 mM (~1 mM free
Mg2+) had no effect on the ability of
AlF4
to protect Gs
-WT
at 30 °C (Fig. 3). In contrast,
lowering the MgCl2 concentration below 8 mM
(~7 mM free Mg2+) further impaired the
ability of AlF4
to protect
Gs
-E259D in a concentration-dependent
manner. Increasing the MgCl2 concentration up to 100 mM did not reverse the defect at 37 °C (data not shown).
These results are similar to those observed for the
Gs
-R231 mutant (33) and demonstrate that, like this
mutant, the GDP-AlF4
-bound form of
Gs
-E259D has a lower apparent affinity for
Mg2+ than Gs
-WT.

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Fig. 3.
Effect of MgCl2 concentration on
trypsin protection of Gs -WT and
-E259D in the presence of AlF4 .
Trypsin protection experiments were performed on Gs -WT
and -E259D after incubation for 1 h at 30 °C in the presence of
AlF4 and various concentrations of
MgCl2 ranging from 0 to 50 mM. The top
two panels show the gels from a representative
experiment. The bottom panel shows the percentage of
protection for Gs -WT and -E259D as a function of
MgCl2 concentration. Each data point represents
the mean ± S.D. of three experiments.
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We next examined the effect of lowering the Mg2+
concentration on the dissociation of GTP
S from
Gs
-E259D to determine whether or not the
Mg2+ dependence was specific for the
GDP-AlF4
-bound form. The apparent
Kd of GTP
S-Gs
-WT for
Mg2+ is very low (5-10 nM), and binding of
GTP
S is essentially irreversible in the presence of micromolar
concentrations of Mg2+ (34). Consistent with previously
published results (34), no dissociation of GTP
S from
Gs
-WT was observed at free Mg2+
concentrations of 30 µM or higher (Fig.
4 and data not shown), although GTP
S
dissociated rapidly (koff = 2.5 min
1) in the absence of Mg2+ (5 mM EDTA). For Gs
-E259D, GTP
S binding was
essentially irreversible in the presence of 1 mM free
Mg2+, but in contrast to Gs
-WT, GTP
S
clearly dissociated from Gs
-E259D in the presence of 30 µM free Mg2+ (Fig. 4,
koff = 0.05 min
1). Dissociation of
GTP
S from Gs
-E259D (koff = 3.7 min
1) was similar to that of Gs
-WT in the
absence of Mg2+ (5 mM EDTA). Therefore, like
GDP-AlF4
-Gs
-E259D,
GTP
S-Gs
-E259D appears to have decreased affinity for
Mg2+, although the defects are apparent in the millimolar
range for the former and micromolar range for the latter.

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Fig. 4.
Effect of free Mg2+
concentration on dissociation of GTP S
from purified Gs s. Bovine
Gs -WT and -E259D, each with a carboxyl-terminal
hexahistidine extension, were expressed and purified from E. coli, and the time course of GTP S dissociation was determined
for each at various free Mg2+ concentrations.
Gs -WT (closed symbols) and
Gs -E259D (open symbols) were preloaded with
[35S]GTP S (5,000-10,000 cpm/pmol) at 30 °C for 45 min in the presence of 1 mM ( ), 30 µM ( and ), or no ( and ) free Mg2+ (5 mM
EDTA was added for the no Mg2+ condition). After addition
of 100 µM cold GTP S, the amount of bound
[35S]GTP S was determined at various time points. Each
data point is the mean ± range of duplicate
determinations. This experiment was representative of three
experiments. Maximum [35S]GTP S in the presence of 5 mM EDTA was 0.5 pmol for Gs -WT and 0.3 pmol
for Gs -E259D, and it was ~2.5 pmol for both in the
presence of Mg2+.
|
|
In contrast to Gs
-E259D, there is a slow rate of
dissociation of GTP
S from Gs
-R231H in the presence of
high Mg2+ concentrations (33). Another Gs
mutant (Gs
-G226A) also displays an abnormally high
apparent Kd for Mg2+ to prevent GTP
S
dissociation (34). Similar to Gs
-E259D, GTP
S dissociates from Gs
-G226A in the presence of micromolar
concentrations of Mg2+. There is also considerable
dissociation of GTP
S from Gs
-G226A even in the
presence of maximal (millimolar) concentrations of Mg2+,
because this mutant cannot attain the active conformation that stabilizes the Mg2+-GTP
S complex. The ability of
Gs
-E259D to irreversibly bind GTP
S in the presence of
1 mM Mg2+ suggests that this mutant can attain
the active conformation necessary to stabilize
Mg2+-GTP
S, consistent with the results obtained in the
adenylyl cyclase and trypsin protection assays (Table I and Fig.
1).
Gs
-E259Q, E259A, and E259D, but not
Gs
-E259V, Maintain Normal Overall Conformation and
G
Interaction--
We examined the ability of each
Gs
-E259 mutant to interact with 
by subjecting
in vitro translates to sucrose density gradient centrifugation in the presence or absence of purified bovine brain 
. We previously showed that Gs
has a sedimentation
coefficient of ~3.7 S (15, 19). When in vitro translates
of each Gs
-E259 mutant was held on ice, the gradient
profiles of all mutants were virtually the same as Gs
-WT
and consistent with the overall proper conformation (sedimentation
coefficient, ~3.7 S) (Fig.
5A). When preincubated on ice
with purified bovine brain 
, Gs
-WT, -E259Q, -E259A, and -E259D formed heterotrimers, as demonstrated by significant shifting of the peak toward the bottom of the gradient (Fig.
5B). In contrast, 
had no effect on the sedimentation
profile of Gs
-E259V, indicating that this mutant does
not interact with 
. After preincubation at 30 °C, gradient
profiles demonstrate that all mutants except Gs
-E259V
maintain an the normal 3.7 S conformation, whereas for
Gs
-E259V, the majority of the protein is a higher S
value material and is presumably denatured (19). Therefore, the valine
substitution probably alters the overall conformation and stability of
the protein due to nonspecific steric effects of its bulky hydrophobic
side chain. In contrast, the activation defect in
Gs
-E259A, E259Q, and E259D is not secondary to defects
in thermostability or 
binding.

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|
Fig. 5.
Sucrose density gradient centrifugation of
Gs in vitro translation
products. A, [35S]methionine-labeled
in vitro translates of both Gs -WT and -E259
mutants were preincubated for 1 h at 0 °C ( ) or 30 °C
( ) and subjected to sucrose density gradient centrifugation as
described under "Experimental Procedures." Fractions (6 µl each)
were collected, and odd-numbered fractions were analyzed by
SDS-polyacrylamide gel electrophoresis and phosphorimaging (15, 19).
The data are expressed as the percentage of total Gs
present in each fraction. Fraction 1 represents the top of the
gradient. The position and S value of standard proteins are indicated
at the top of the Gs -WT gradients.
B, sucrose density gradient profiles of Gs -WT
( ), Gs -E259A ( ),Gs -E259Q
( ),Gs -E259D ( ), and Gs -E259V ( )
after preincubation for 1 h at 0 °C in the presence of purified
bovine brain G (20 µg/ml). The profile for Gs -WT
in the absence of G is also shown ( ). All
Gs -E259 (except Gs -E259V) mutants held at
0 °C in the absence of G had sucrose density gradient profiles
similar to that of Gs -WT (data not shown).
Gs -E259V had a somewhat broader peak at 0 °C that was
unaltered in the presence of G . Conditions were modified to
optimize separation between free -subunit and heterotrimer as
outlined under "Experimental Procedures." Similar results were
obtained with the detergent octyl- -glucoside (0.3% w/v).
|
|
 |
DISCUSSION |
We previously reported that substitution of the Gs
switch 3 residue Arg258 leads to impaired activation in the
presence of AlF4
or activated receptor
(isoproterenol + GTP) but normal activation in the presence of GTP
S
(15). The impaired activation by AlF4
was reversible in the presence of excess GDP, and further
characterization demonstrated a defect in GDP binding, presumably due
to loss of direct contact between Arg258 and a residue(s)
in the helical domain that would open the cleft through which guanine
nucleotide must exit. In this study, we examined the effect of
substituting the adjacent switch 3 residue (Glu259) on
Gs
function for the following reasons: 1) this residue
is strictly conserved among G protein
-subunits and therefore might have an important role in the biochemical function of these proteins; 2) upon activation, the Glu259 side chain interacts with
several residues in the switch 2 region (7, 12) and therefore
substitutions of this residue might be predicted to directly impair G
protein activation; 3) this Gs
residue is mutated to a
valine in a patient with Albright hereditary osteodystrophy (16), a
human disorder associated with heterozygous inactivating mutations
within the Gs
gene (27, 28).
Substitution of Gs
Glu259 to valine had a
marked effect on the conformation and stability of the protein. This
mutant was unable to interact with 
, even though
Glu259 is not within the 
interaction site (11). This
mutant was also more thermolabile. Presumably, the presence of a bulky
and branched side chain provided by valine introduces nonspecific steric effects that severely affect the conformation and stability of
the protein. We would predict that the primary biochemical defect in
the patient harboring this mutation is lack of expression of
Gs
-E259V in the membrane at physiological temperatures,
similar to what is observed in other patients with mutants encoding
unstable forms of Gs
protein (15, 19, 32).
In order to determine whether residue Glu259 is critical in
maintaining either the basal or activated state, we generated mutants with more subtle alterations of Glu259 side chain. The most
subtle mutation was Gs
-E259D, in which the charge of the
residue is maintained but the length of the side chain is shortened by
one methylene group. We also made two mutants in which the side chain
was either removed (Gs
-E259A) or converted from an
acidic to neutral amino acid (Gs
-E259Q). In all three of
these mutants, the overall conformation and stability, as well as the
ability to interact with 
, was maintained, as determined by
sucrose density gradient experiments. Based upon adenylyl cyclase and
trypsin protection assays, activation of Gs
-E259D by
GTP
S was normal, demonstrating that this mutant has not lost its
intrinsic ability to attain the active conformation and activate
adenylyl cyclase. However, this mutant had decreased ability of to be
activated by AlF4
or receptor.
Gs
-E259Q and -E259A showed a more severe phenotype, with
decreased activation in the presence of all agents. In all three
mutants, GTP
S was the most efficient activator whereas AlF4
was the least efficient. Mutation
of the analogous residue in transducin (Glu232) to leucine
had no effect on the ability of the G protein to interact with 
or its receptor (rhodopsin), but it did appear to decrease the ability
of GTP
S to mediate trypsin protection and effector activation
(13).
One possible mechanism for impaired activation by
AlF4
is decreased ability to maintain
the GDP-bound state, because binding of GDP is a prerequisite for
AlF4
binding and activation. This is
the primary mechanism by which substitutions of Gs
Arg258 lead to impaired activation by
AlF4
(15). However, the ability of
Gs
-E259 mutants to maintain the GDP-bound state was
similar to that of Gs
-WT, as demonstrated by both
Gs
-E259A and -E259D having a rate of GDP release that was similar to Gs
-WT, as well as an inability for excess
GDP to significantly reverse the
AlF4
-induced activation defect.
Consistent with normal guanine nucleotide binding, both
Gs
-E259A and -E259D were thermostable. Binding of
AlF4
to the GDP-bound
-subunit
results in formation of a stable and activated
GDP-AlF4
-protein complex that
mimics the transition state of the GTPase reaction and will slow the
rate of GTP
S binding, probably by inhibiting GDP release (8). The
ability of AlF4
to inhibit the rate
and extent of GTP
S binding to both Gs
-E259A and
-E259D was significantly reduced, suggesting that in these mutants the
activation defect in response to AlF4
is due at least in part to impaired
AlF4
binding. The fact that the
activation defect is greater for AlF4
than GTP
S suggests that mutation of Glu259 has a more
dramatic effect on stabilizing the transition
(AlF4
-bound) state than the activated
(GTP
S-bound) state.
It is of interest that the biochemical phenotype of our
Gs
-Glu259 mutants is quite similar to that
previously described for another Gs
mutant present in a
patient with Albright hereditary osteodystrophy, in which the switch 2 residue Arg231 is mutated to histidine
(Gs
-R231H) (33, 35). Similar to the
Gs
-Glu259 mutants, this mutation leads to
normal GTP
S-mediated but decreased AlF4
- and receptor-mediated
activation. Moreover, similar to the Gs
-R231H mutant,
the AlF4
-induced activation defect in
Gs
-E259D was more pronounced at low Mg2+
concentrations (33). This is not surprising, based upon mutual interactions between Glu259 and Arg231 present
in the active (GTP
S-bound) conformation of Gs
(Fig. 6). Upon activation, interactions between
switches 2 and 3 stabilize the GTP-bound form of the G protein.
Specifically, Arg231 in switch 2 interacts with
Glu259 in switch 3 through a water molecule and directly
with Glu268 in the
3 helix (Fig. 6). Conversely,
Glu259 interacts with two basic switch 2 residues,
Arg228 and Arg231. Both Arg231 and
Glu259 interact with Gly226, a residue that is
critical for both AlF4
binding (36)
and conformational switching of switch 2 upon binding of GTP or
AlF4
(29). Therefore, the impaired
activation and AlF4
binding
observed in Gs
-Glu259 mutants might be the
direct result of loss of contacts with Gly226. Loss of
these contacts may also result in the apparent decreased affinity of
Gs
-Glu259 and R231H mutants for
Mg2+ because mutation of Gly226 to alanine also
lowers the apparent affinity of Gs
for Mg2+
(34).

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|
Fig. 6.
Crystal structure of
Gs -GTP S.
Detailed view of interactions between Glu259 in switch 3, Glu268 in 3, and residues in switch 2 and between
Gly226 and the phosphate of GTP S. Hydrogen bonds are
shown as dotted lines. The atom coloring scheme is as
follows: black, carbon; red, oxygen;
blue, nitrogen; and yellow, sulfur.
Mg2+ and water molecules are shown as magenta
and cyan spheres, respectively. The figure was generated
with MOLSCRIPT (39) and rendered with RASTER3D (37) using coordinates
for the short form of bovine Gs -GTP S (Protein Data
Bank accession code 1AZT (12)), although the numbering on the figure
corresponds to the long form of Gs (17). This view is
similar to that previously shown for transducin (6).
|
|
Mutation of Glu259 leads to a subtle defect in
receptor-mediated activation (at least when compared with activation by
GTP
S). Gs
-E259 mutants are able to bind 
, and
mutation of the analogous residue in transducin (Glu232)
has no effect on interactions with 
or receptor (13). It has been
proposed that decreased receptor activation of Gs
-R231H is due to a conditional defect in GTP binding, which is more pronounced in states in which guanine nucleotide binding is destabilized (such as
interaction with activated receptor (33)). Our results are consistent
with those observed with Gs
-R231H and support this hypothesis.
In conclusion, this study provides further evidence for the role of
switch 3 in the activation mechanism and demonstrates the importance of
interactions between Glu259 and switch 2 residues. Taken
together with the prior studies on Arg258 mutants (15, 38),
the present results demonstrate the importance of switch 3 in
maintaining both the basal and active states.
 |
ACKNOWLEDGEMENTS |
We thank J. Nagle for performing DNA
sequencing analysis, A. G. Gilman and R. K. Sunahara for
providing plasmid pQE60-
s-H6 and helpful technical advice on the
expression and purification of Gs
from E. coli, S. Sprang for providing the coordinates for the crystal
structure of Gs
, V. Rao and J. Hurley for assistance with Fig. 6, and P. Fishman for helpful advice.
 |
FOOTNOTES |
*
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: Bldg. 49, Rm. 2A28,
National Institutes of Health, Bethesda, MD 20892-4440. Tel.: 301-496-2007; Fax: 301-496-8244; E-mail: dwarner{at}helix.nih.gov.
 |
ABBREVIATIONS |
The abbreviations used are:
G protein, guanine
nucleotide-binding protein;
Gs, stimulatory G protein;
Gs
, Gs
-subunit;
Gs
-E259D, -E259A, -E259Q, and -E259V, Gs
mutant with
Glu259 substituted to aspartate, alanine, glutamine, and
valine, respectively;
AlF4
, mixture of 10 µM AlCl3 and 10 mM
NaF;
GTP
S, guanosine-5'-O-(3-thiotriphosphate);
WT, wild
type.
2
All numbering is based on the
Gs
-1 sequence reported by Kozasa et al.
(17).
 |
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