From the Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
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ABSTRACT |
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GAIP is a regulator of G protein signaling (RGS)
that accelerates the rate of GTP hydrolysis by some G protein Heterotrimeric G proteins associate with the cytoplasmic surfaces
of 7-transmembrane spanning receptors and function to transduce signals
from receptors activated by extracellular ligands to intracellular effectors (1). One of the most recent developments in the study of G
protein regulation is the identification of a novel family of proteins
known as regulators of G protein signaling or RGS proteins (2). RGS
proteins are characterized by the presence of an RGS domain that is
structurally conserved across evolution (3, 4). These molecules
function to desensitize G protein-coupled responses in organisms from
yeast to man by directly interacting with the Most of the initially described RGS proteins showed both binding and
functional selectivity for the G To evaluate the structural basis for the selectivity of the RGS
GAIP for individual members of the G Generation of Yeast Two-hybrid Fusion Constructs--
Rat G
protein
Human GAIP was PCR amplified from a human heart cDNA library using
oligonucleotides containing a 5' NarI restriction site and a
3' SalI restriction site. PCR products were subcloned and sequenced as above, then removed from pCRII with Nar I and
SalI, and subcloned into the pGAD Gal 4 activation domain
fusion vector (CLONTECH).
Generation of G Protein
G Site-directed Mutagenesis of G Protein Transformation of Competent Yeast--
Saccharomyces
cerevisiae of strain HF7c Immunoblotting--
Yeast transformants were grown overnight to
high density in 4-ml cultures, harvested, and resuspended in binding
buffer (0.2 M Tris, pH 8.0, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 20 µg/ml
pepstatin A). Cells were lysed by vortexing three times for 1 min in
the presence of glass beads at 4 °C and spun for 10 min at
12,000 × g to remove cell debris. 50 µg of lysate
was loaded per lane onto SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose, and probed with rabbit antibody common
to G protein Liquid Histidine Growth Assays--
5-ml cultures of yeast
transformants were grown to an A600 of 1.0 and
then 3 µl of 1:10 serial dilutions of confluent growths were spotted
on either SC-Leu-Trp or SC-Leu-Trp-His agar plates and allowed to grow
at 30 °C for 3 days.
Protein Expression and Purification--
Full-length G protein
GTP GTPase Assays--
100 nM purified GST-tagged G
protein Interactions of G Protein Fusions with GAIP--
To explore the
structural basis for the differences in GAIP binding by the different
members of the G
Given such a similar background of G protein fusion expression, a
measure of the strength of interaction between various G proteins and
GAIP can be estimated from the relative activation of Gal
4-dependent reporters. The yeast strain HF7C
According to both histidine and Chimeras--
Because G
To discriminate among these possibilities, chimeras composed of the
initial two-thirds of Site-directed Mutants--
As a next step, site-directed
mutagenesis of G
In addition to the C-terminal mutants shown in Fig. 3A,
three N-terminal G GTPase-deficient Mutants--
To determine whether different
nucleotide-dependent conformations of these G proteins
affected their relative GAIP affinities, GTPase-deficient mutants of
G Nucleotide Binding Affinity--
To explore the mechanism of the
selectivity of GAIP for G GAP Activity--
Finally, to determine whether any functional
differences might correlate with selective binding capacity, we tested
the ability of GAIP to catalyze the GTPase activities of
G RGS proteins are a family of G protein regulators that
down-regulate G protein-coupled responses by stimulating the GTPase activity of the G Elucidation of the sites on G proteins with which RGS proteins interact
and the selectivity of RGS proteins for different forms of G The sites on G protein To extend the characterization of RGS/G protein specificities and their
structure/function relationships, we sought to identify regions in the
G To further localize the region in the G protein C terminus responsible
for GAIP selectivity, site-directed mutants were generated in which
residues in G Closer inspection of the RGS4-G Finally, to determine whether there is also selectivity by GAIP for
G To determine whether the ability of GAIP to discriminate between
G The functional selectivity displayed by GAIP and other RGS proteins for
G protein partners in vivo remains to be explored. The
contributions of additional interacting partners, including C-terminal
tails of GPCRs (36) and additional effector proteins (18-20), and
post-translational modifications (37) will have to be considered to
determine how individual RGS proteins modulate specific G protein
signaling pathways.
subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the
G
i family. G
i1, G
i3,
and G
o interacted strongly with GAIP, whereas G
i2 interacted weakly and G
s did not
interact at all. A chimeric G protein composed of a G
i2
N terminus and a G
i1 C terminus interacted as strongly
with GAIP as native G
i1, whereas a chimeric N-terminal
G
i1 with a G
i2 C terminus did not
interact. These results suggest that the determinants responsible for
GAIP selectivity between these two G
is reside within the
C-terminal GTPase domain of the G protein. To further localize residues
contributing to G protein-GAIP selectivity, a panel of 15 site-directed
G
i1 and G
i2 mutants were assayed. Of the
G
i1 mutants tested, only that containing a mutation at
aspartate 229 located at the N terminus of Switch 3 did not interact
with GAIP. Furthermore, the only G
i2 variant that
interacted strongly with GAIP contained a replacement of the
corresponding G
i2 Switch 3 residue (Ala230)
with aspartate. To determine whether GAIP showed functional preferences
for G
subunits that correlate with the binding data, the ability of
GAIP to enhance the GTPase activity of purified
subunits was
tested. GAIP catalyzed a 3-5-fold increase in the rate of GTP
hydrolysis by G
i1 and G
i2(A230D) but no
increase in the rate of G
i2 and less than a 2-fold
increase in the rate of G
i1(D229A) under the same
conditions. Thus, GAIP was able to discriminate between
G
i1 and G
i2 in both binding and
functional assays, and in both cases residue 229/230 played a critical
role in selective recognition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of
heterotrimeric G proteins and increasing their rate of GTP hydrolysis
(5). Direct interaction between G protein
subunits and RGS
molecules was first demonstrated by DeVries et al. (6), who
isolated the cDNA for the RGS GAIP (G alpha
interacting protein) using a yeast two-hybrid
screen for G
i3-interacting proteins. A number of studies
quickly followed revealing GAP (GTPase-activating
protein)1 activity to be the
mechanism by which RGSs turned off G protein activation (7-10). Both
the structural interaction between RGS and G
subunits and the
mechanism of RGS GAP activity were further elucidated by the
co-crystallization of RGS4 with G
i1 (11). However, much
remains to be revealed about the function of individual members of the
RGS family, their specificities for interacting proteins, and the
structural determinants that define these interactions.
i family of G proteins (7-9, 12). More recently, a number of RGS molecules have demonstrated binding or functional interactions with G
q and/or
G
s signaling pathways (13-17), and p115RhoGEF was shown
to be a functional RGS for the G
12/G
13
family of G proteins (18-20). However, there has been little
information about the ability of any RGS to discriminate among the
closely related members of the G
i family. Evidence for
some specificity of RGS binding to distinct G
i family
members was demonstrated by DeVries et al. (6), who showed
strong interaction of GAIP with G
i1, G
i3,
and G
o but weak interaction with G
i2 and
no interaction with G
s. The differential binding
characteristics of G
i1 and G
i2 are
particularly intriguing because these two G proteins are highly
homologous, having an amino acid sequence identity of 88%. Differences
in RGS binding may reveal structural differences in these two G
proteins that have implications for their ability to differentially
activate divergent downstream signaling pathways.
i family, we have
expressed native, chimeric, and mutant G
proteins and compared their
abilities to bind GAIP and act as substrates for GAIP GAP activity. The results show a preference of GAIP for G
i1 over
G
i2 in both binding assays and GAP assays. This
preference was reversed by mutating residue Asp229 in
G
i2 to alanine and making the reciprocal mutation
(A230D) in G
i1. Interestingly, the selectivity of GAIP
for G
i1 over G
i2 was lost when
GTPase-deficient mutants of these two G
is were tested
for GAIP binding. Thus, the structural preference of GAIP for
G
i1 versus G
i2 in their ground
(presumably GDP-bound) states has functional consequences in their
respective GAP activities.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits were PCR amplified with oligonucleotides
containing 5' EcoRI restriction sites and 3' SalI
restriction sites. PCR products were then subcloned into the pCRII
vector (Invitrogen, Carlsbad, CA) and sequenced to ensure fidelity to the template. Inserts were excised with EcoRI and
SalI and subcloned into the pGBT9 Gal 4 DNA-binding domain
fusion vector (CLONTECH).
Subunit Chimeras--
The
G
s/i3 chimera was generated by removing the N-terminal
BamHI site in the G
s cDNA via
site-directed mutagenesis (see below) and then ligating the
BamHI-digested N-terminal 700-bp fragment of
G
s to the 430-bp C-terminal fragment of
BamHI-digested G
i3 cDNA. The
G
i3/s chimera was generated by ligating the N-terminal 630-bp G
i3 fragment to the C-terminal 516-bp
G
s fragment of the same digestions. Both chimeras were
subcloned into the pGBT9 vector and characterized with BamHI
and EcoRI as well as with BamHI and
SalI digestions to ensure correct constructions.
i1/i2 and G
i2/i1 chimeras were made by
engineering a BamHI site into the G
i1
cDNA at the same site as a naturally occurring BamHI in
G
i2. G
i2 and mutant G
i1
cDNAs were digested with BamHI, and the N-terminal
635-bp fragment of G
i1 was ligated to the C-terminal
433-bp fragment of G
i2 to generate
G
i1/i2. Similarly, G
i2/i1 consists of the
N-terminal BamHI fragment of G
i2 ligated to
the C-terminal BamHI fragment of G
i1.
Subunits--
Site-directed mutants of G
i1 and
G
i2 were made using Stratagene QuickChange site-directed
mutagenesis kit according to the manufacturer's protocols. Template
pGBT9-G
i1 or pGBT9-G
i2 was amplified for
14 cycles of 12-min extensions, each using overlapping forward and
reverse primers encoding the applicable mutation. All mutants were
sequenced throughout the entire coding region to ensure desired
mutagenesis as well as to screen against unwanted PCR-induced mutations.
were co-transformed with pGBT9
(containing Trp marker) and pGAD (containing Leu marker) vector
constructions by standard lithium acetate procedures
(CLONTECH Matchmaker two-hybrid system). Briefly,
single yeast colonies were grown overnight at 30 °C with continuous
shaking to an A600 of 0.6. Cells were harvested
by centrifugation for 10 min at 3000 rpm, washed once in sterile
H20, and resuspended in 2 ml of cold 100 mM
lithium acetate. After shaking at 30 °C for 1 h, 100 µl of
competent cells was added to 1-2 µg of transforming DNA in the
presence of 5 µg of carrier salmon sperm DNA and 0.7 ml of 40%
polyethylene glycol. Cells were heat shocked at 42 °C for 15 min,
then collected with a quick spin, and plated on -Leu-Trp selective
dropout agar medium to grow for 3 days at 30 °C. Four colonies of
each construct were streaked on -Leu-Trp agar to propagate for assay.
subunits (Calbiochem, La Jolla, CA) at 1:500 dilution
in Tris-buffered saline/5% milk. Immunoreactivity was detected with
horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody
(1:2000 dilution) and developed using ECL reagents according to the
manufacturer's protocols (Amersham Pharmacia Biotech).
-Galactosidase Assays--
Single colonies of
transformed cells were innoculated into 5 ml of SC-Leu-Trp agar and
grown overnight to an A600 of 0.8. Cells were
collected by centrifugation, washed once in Z buffer (60 mM
Na2HPO4, 40 mM
NaH2P04, 10 mM KCl, 1 mM MgSO4), resuspended in 300 µl of the same,
and lysed by four freeze/thaw cycles. To start the assay, 100 µl of
this cell lysate was suspended in 0.7 ml of Z buffer containing 0.27%
-mercaptoethanol and then added to 0.16 ml of Z buffer containing 4 mg/ml o-nitrophenyl
-D-galactopyranoside substrate. Suspensions were vortexed and incubated for 2 h at 30 °C. Color reactions were stopped with 0.4 ml of
Na2CO3 and read at A420
after spinning out cell debris.
-Galactosidase units (21) were
calculated according to the manufacturer's protocols (CLONTECH), as follows:
-galactosidase
units = 1000 × A420/(t × v × A600), where t is 120 min of
incubation, v is 0.1 ml of reaction volume·concentration
factor, and A600 was 0.8 for the culture.
subunits G
i1, G
i2,
G
i1(D229A), and G
i2(A230D) and
full-length GAIP were expressed as GST fusion proteins by subcloning
cDNAs downstream of the GST tag using
EcoRI/SalI sites of the vector pGEX-6P-1
(Amersham Pharmacia Biotech). Each plasmid construct was transformed
into bacterial strain BL21, grown overnight, and induced to express
protein with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside. Cells were
harvested by centrifugation, sonicated in TE containing 0.1 mM phenylmethylsulfonyl fluoride and 1 mM
-mercaptoethanol, and solubilized with 1% Triton X-100. Lysates
were cleared by centrifugation at 12,000 × g for 10 min, and supernatants were applied to pre-washed glutathione-Sepharose
columns (Amersham Pharmacia Biotech). Columns were washed with TE
containing phenylmethylsulfonyl fluoride and
-mercaptoethanol and
GST fusion proteins eluted with 10 mM glutathione. Purified
proteins were buffer exchanged into TED buffer (20 mM
Tris-HCl, pH 8, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol), concentrated to 1 mg/ml in Nanosep spin columns (Pall
Filtron Corp.), and stored at
80 °C. Size and homogeneity of
purified proteins were verified via Coomassie-stained
SDS-polyacrylamide gel electrophoresis, and in-frame translation of G
proteins was verified via immunoblot using a
G
i1/G
i2-selective antibody (kind gift of
Dr. David Manning, University of Pennsylvania, Philadelphia, PA).
S Competition Curves--
100 nM purified
GST-tagged G protein
subunits were shaken for 4 h at 30 °C
in the presence of 100 nM [35S]GTP
S and
serial dilutions of 1-100 µM competing unlabeled GTP
S in 50 µl of binding buffer (50 mM HEPES, pH 8, 1 mM EDTA, 2 mM
-mercaptoethanol, 10 mM MgSO4, 2 mM ATP, 30% glycerol,
1 mg/ml bovine serum albumin) (22). Reactions were filtered over BA85 nitrocellulose filters and washed three times with 2 ml of cold GTP
S
STOP buffer (20 mM Tris-Cl, pH 8, 25 mM
MgCl2, 100 mM NaCl). Filters were immersed
overnight in scintillation fluid before counting to determine amount of
[35S]GTP
S bound.
subunits were loaded with 1 µM
[
-32P]GTP (8000 cpm/pmol) for 20 min at 30 °C in
600 µl of GTPase buffer (0.1% lubrol PX, 50 mM HEPES, pH
7.5, 1 mM dithiothreitol, 5 mM EDTA). Reactions
were chilled at 4 °C for 10 min, and assays were conducted at
6 °C. A 50-µl aliquot was removed immediately before initiating
the reaction and quenched with 750 µl of 5% Norit activated charcoal
in 50 mM NaPO4, pH 3. To initiate the reaction,
100 µM cold GTP and 15 mM MgSO4
(final concentrations) in the presence versus absence of 500 nM GST-tagged GAIP were added to reaction mixtures, and
50-µl aliquots were removed after 10 s, 20 s, 40 s, 1 min, 2 min, 3 min, 4 min, and 5 min and stopped as just described.
Charcoal was precipitated by centrifugation for 15 min at 12,000 × g, and 400-µl free phosphate-containing supernatants
were counted to determine the amount of Pi released per reaction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i family, we engineered a panel of G
protein chimeras and mutants. As a first step, native and engineered G
proteins were assayed for the ability to bind GAIP using the yeast
two-hybrid system. To make use of this system, G
protein cDNA
constructs were subcloned downstream of a Gal 4-binding domain cDNA
and coexpressed with a GAIP-Gal 4 activation domain fusion in the
S. cerevisiae strain HF7C
. All fusions were immunoblotted
to control for relative expression levels. An anti-G protein
subunit antibody raised against the internal GTP-binding sequence
common to all heterotrimeric G protein
subunits recognized a
protein of the appropriate molecular mass (about 65 kDa) for a G
protein
subunit fused to the Gal 4-binding domain in each of the
clones transformed with a G protein fusion (data not shown). All of the
clones expressed comparable levels of G protein fusion, and no protein
of the same size was seen in clones transformed with pGBT9-binding
domain alone.
was stably transformed with cDNAs encoding both
-galactosidase and
histidine reporters downstream of a Gal 4 promotor. In this system, the promotor is activated in proportion to the degree of interaction between the Gal 4-binding domain and activation domain fusions (23).
Thus, two different reporters were used to measure the relative
strength of the interaction between the G protein-binding domain fusion
and the GAIP activation domain fusion.
-galactosidase reporter
systems, robust interaction of GAIP was seen with G
i1,
G
i3, and G
o, whereas the interaction with
G
i2 was weak, and the interaction with G
s
was undetectable (Fig. 1). These results
are consistent with those obtained by DeVries et al. (6).
Due to the quantitative nature of the assays, liquid
-galactosidase
assays were used for interaction comparisons henceforth. Because
G
i1 gave a strong interaction with GAIP in its native
conformation, which was statistically indistinguishable from that of
G
i3 and G
o, and because this G protein
was tested in every assay conducted, this level of interaction was
designated as 100% for comparison with all other G protein constructs.
100% interaction in these assays corresponds to 1.4
-galactosidase
units (21). The
-galactosidase activity generated by GAIP
co-transfected with pGBT9 vector alone (0.12
-galactosidase units)
was considered background and was subtracted from all values for G
protein-GAIP interactions before normalization.
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Fig. 1.
A, liquid -galactosidase assays of
native G protein
subunit interactions with GAIP. Yeast clones
co-expressing indicated Gal 4-binding domain-G
fusions with
activation domain-GAIP fusions were assayed for the
interaction-dependent activation of a lacZ
reporter. The amount of
-galactosidase released was measured
colorimetrically using the substrate ONPG. Two clones of each
transformant were assayed and normalized to the interaction of GAIP
with G
i1 defined as 100%. The results shown are the
mean ± S.E. for n = 4-18 in triplicate.
B, histidine-minus growth assays of native versus
mutant G protein
subunit interactions with GAIP. Yeast clones
co-expressing indicated binding domain-G
fusions with activation
domain-GAIP were assayed for their interaction-dependent
activation of a histidine reporter. Each clone was grown and plated as
detailed under "Experimental Procedures." Plates on the
left show limiting dilutions of clones grown on tryptophan-
and leucine-lacking agar medium to control for noninteraction dependent
growth. Plates on the right show identical dilutions of
clones grown on tryptophan-minus, leucine-minus, and histidine-lacking
medium to assay for interaction-dependent histidine
reporter expression. This assay has been performed twice with identical
results.
i3 interacted strongly with
GAIP, whereas G
s did not interact at all, chimeras of
G
i3 with G
s were generated in an attempt
to localize the regions of G
i required for GAIP binding.
A BamHI site that cuts both cDNAs roughly two-thirds into the length of the coding region was used to generate both chimeras
(Fig. 2). This BamHI site
conveniently separates all of the N-terminal
-helical domain from
most of the GTPase domain (a small part of which is encoded at the very
N terminus of the cDNA). The binding characteristics of these
chimeras could thus substantiate the relative importance of these two
domains in GAIP binding. However, neither chimera bound to GAIP (Fig.
2A). These results potentially indicate that both domains of
G
i3 contribute important determinants for RGS binding,
but the divergence in
s sequence from that of the
i family presents a number of other possible
interpretations.
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Fig. 2.
Relative interaction of G
chimeras with GAIP. Liquid
-galactosidase assays were
conducted as described in the legend to Fig. 1A. Two clones
of each transformant were assayed. The results shown are the means ± S.E. for n = 2-18 in triplicate. A,
interaction-dependent release of
-galactosidase from
clones expressing chimeras of G
i3 and G
s.
A schematic diagram of the chimeras is shown at the bottom.
B, interaction-dependent release of
-galactosidase from clones expressing chimeras of G
i1
and G
i2. A schematic diagram of these chimeras is shown
at the bottom, where the asterisk indicates the
position of G
i1 Asp229.
i1 fused to the distal one-third of
i2 and the reciprocal G
i2/i1 chimera
were prepared using an engineered BamHI site (Fig.
2B). G
i2 is highly homologous to
G
i1, yet its interaction with GAIP is negligible
compared with G
i1. The G
i2/i1 chimera
interacted with GAIP just as strongly as native G
i1,
whereas the reverse G
i1/i2 chimera, like wild type
G
i2, showed little binding to GAIP (Fig. 2B).
These results suggest that the G
i1 C terminus is
required for GAIP interaction. The results may also imply that the
determinants contributing to GAIP binding are entirely contained within
the GTPase domain of the G protein, but there may be additional
determinants that are conserved between the N termini of
G
i1 and G
i2 that remain to be identified.
i1 and G
i2 was used to
further localize determinants contributing to the selectivity of GAIP
interaction. Because G
i1 and G
i2 are 88%
identical at the amino acid level but show vastly different GAIP
binding capacities in the yeast two-hybrid system, the primary
sequences of the two proteins were compared with identify candidate
residues that might contribute to differential GAIP binding. Of the
amino acids that differed between G
i1 and
G
i2, reciprocal mutants were generated at eight
different positions in the primary sequence based on the likelihood
that a given position would affect RGS binding given its location in
the three-dimensional crystal structure of G
i1 bound to
RGS4 (11). The effects of C-terminal mutants were of particular
interest due to the results of the chimeras, but a number of N-terminal
mutants were also studied because they appeared to be close to
potential RGS contact sites in the crystal structure (11). Of the five
G
i1 mutants C-terminal to the BamHI site that
were tested, several impaired binding to GAIP, but only D229A abolished
it (Fig. 3A). Even more
significantly, the reciprocal mutation in the corresponding residue in
G
i2
(G
i2(A230D))2
produced a variant G
i2 that bound to GAIP as strongly as
G
i1 (Fig. 3A). Thus, G
i1(D229)
appears to be particularly important for GAIP interaction.
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Fig. 3.
A, relative interaction of C-terminal
point mutants with GAIP. B, relative interaction of
N-terminal point mutants with GAIP. Liquid -galactosidase assays
were conducted as described in the legend to Fig. 1A. Two
clones of each transformant were assayed. The results shown are the
means ± S.E. for n = 2-18 in triplicate.
i1 mutants and the corresponding
reciprocal G
i2 mutants were also assayed for
-galactosidase activity. Consistent with the results of the
G
i1/i2 chimeras, all of the N-terminal G
i1 mutants bound to GAIP, and none of the corresponding
G
i2 mutants bound GAIP as strongly as G
i1
(Fig. 3B). Thus, none of these residues appears to be a
necessary determinant for GAIP binding.
i1 and G
i2 were generated to "trap" the
subunits in their GTP-bound forms and assayed for binding to
GAIP. In contrast to the wild type proteins, the "activated" forms
of both G
i1 and G
i2 interacted at least
as strongly with GAIP as wild type G
i1 (Fig.
4). Both G
i1(Q204L) and
G
i2(Q205L) generated about a 4-fold increase in GAIP
binding activity relative to that seen with wild type (nonactivated)
G
i1, so that the selectivity of GAIP for
G
i1 over G
i2 appears to be restricted to
the interaction with their ground state (presumably GDP-bound)
conformations. Two additional GTPase-deficient mutants,
G
i1(R178C) and G
i2(R179C), were also
tested and interacted very strongly with GAIP although less strongly
than the Q204L/Q205L mutants.
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Fig. 4.
Relative interaction of GTPase-deficient
mutant versus native G
subunits with GAIP. Liquid
-galactosidase assays were
conducted as described in the legend to Fig. 1A. Two clones
of each transformant were assayed. The results shown are the means ± S.E. for n = 6-18 in triplicate.
i1 over G
i2 in
their GDP-bound states, the position of G
i1 aspartate 229 in relation to the bound RGS4 molecule in the published crystal structure was examined (Fig. 5). In the
AlF4-activated state in which this G protein was
crystallized, Asp229 appears closer to the
nucleotide-binding site than to the RGS-binding site of this G protein.
Therefore, we examined the relative GTP
S affinities of both
G
i1 and G
i2 to determine whether there
were differences in nucleotide binding affinity that in turn might affect their affinities for GAIP. Recombinant full-length
G
i1, G
i2, G
i1(D229A), and
G
i2(A230D) were GST-tagged, expressed in bacteria, and
purified to homogeneity over glutathione affinity columns. The ability
of unlabeled GTP
S to displace [35S]GTP
S from each
of the proteins was measured over a range of GTP
S concentrations.
The IC50 for [35S]GTP
S displacement was
the same for all four proteins (Fig. 6),
so differences in GAIP binding are not reflective of differences in
nucleotide binding affinities.
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Fig. 5.
Position of
G i1 Asp229 in relation
to bound RGS4 and GDP-Mg2+-AlF4 molecules.
PDB 1AGR (2) showing the cocrystallization of G
i1 with
RGS4 was downloaded from the Brookhaven National Labs Protein Data Bank
and viewed using RasMol. The G
i1 subunit is shown in
dark blue bound to a cyan RGS4 molecule. GAIP
binding specificity determinant G
i1(Asp229)
is pictured in yellow at the top of the pink
Switch 3 region of G
i1. The bound GDP-AlF4
is the adjacent structure in green. G
i
residues Arg178 and Gln204 are highlighted in
red.
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Fig. 6.
Competition between
GTP S and
35S-GTP
S for binding to native and
mutant G protein
subunits. Purified
GST-tagged G protein
subunits (100 nM) were incubated
with 100 nM [35S]GTP
S (200,000 cpm/50 µl
assay volume) and indicated concentrations of unlabeled GTP
S and
filtered as described under "Experimental Procedures." Each value
is the mean ± S.E. of three experiments performed in
triplicate.
i1, G
i2, G
i1(D229A), and
G
i2(A230D). GAIP catalyzed a 5-fold increase in the rate of GTP hydrolysis by G
i1 (Fig.
7A) but caused no increase in the GTPase rate of G
i2 (Fig. 7B) under the
same conditions. In addition, GAIP only slightly increased the GTPase
activity of G
i1(D229A) (from Kobs
of 2.1 in the absence of GAIP to Kobs of 3.7 in
the presence of GAIP) (Fig. 7C). Of particular interest, the
rate of GTP hydrolysis seen for this mutant form of G
i1
in the presence of GAIP is similar to the GTPase rate of
G
i2 in the presence of GAIP (Kobs = 4.2). Similarly, G
i2(A230D) now behaves more like
G
i1 in that there is a significant increase in GAIP
activation, and the GTPase rate seen in the presence of GAIP is similar
to that seen for G
i1 in the presence of GAIP (Kobs = 5.2 for the former and 5.6 for the
latter) (Fig. 7D). Therefore, the ability of GAIP to act as
a GAP for these two G
i proteins and their reciprocal
mutants correlates with its affinities for these proteins in their
"ground states" as measured in the yeast two-hybrid assay.
View larger version (23K):
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Fig. 7.
Effect of GAIP on single turnover GTPase
activity of purified GST-tagged native and mutant G
subunits. Squares, G protein alone;
triangles, G protein in the presence of GAIP. GST-GAIP (500 nM) was added to 100 nM
[32P]GTP-loaded G
subunits in the presence of
Mg2+ and excess unlabeled GTP to initiate reactions.
A, G
i1; B, G
i2;
C, G
i1(D229A); D,
G
i2(A230D). Aliquots were removed at the indicated
times, and free 32Pi released was measured. An
average of 470 fmol 32Pi was released per
assay, which was normalized to 100%. Values given are the means of
four experiments for A, B, and C and
the means of six experiments for D. The observed rate
constants (Kobs) for each reaction were
calculated based on an exponential association curve fit using GraphPad
Prism.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits to which they bind (3, 5). Both the G
protein binding and GAP activity of RGS molecules have been localized
to a 130-amino acid domain (RGS domain) that is conserved among all RGS
proteins (6, 10, 24). Within this domain, a number of residues have
been shown to serve as contact points for G
protein binding (11, 25,
26).
have
important implications for the mechanism by which RGSs stimulate
subunit GTPase activity. The observation that RGS4 binds more strongly
to the AlF4-GDP-Mg2+-bound state of
G
i than to the GDP or GTP-bound states suggests that
RGSs exhibit GAP activity by stabilizing the transition state for GTP
hydrolysis by G
(7-9, 27, 28). The crystal structure of
AlF4-GDP-Mg2+-G
i bound to RGS4
further reveals that the RGS interacts directly with the Switch regions
of G
i, reducing their flexibility in this transition
state mimic and thus further supporting this proposed GAP mechanism
(11). It has also been observed that the sites on G
to which RGS
proteins bind may interfere with the binding of the effector PLC
1,
suggesting another possible mechanism for G
i
down-regulation by RGSs (13).
subunits responsible for the selectivity
with which RGS proteins bind have been less well studied. DeVries
et al. (29) showed a significantly reduced GAIP interaction with a 10-amino acid truncation of G
i3, but a chimeric
G
q containing the last 10 residues of G
i3
did not bind to GAIP, indicating that other determinants remain to be
identified. More recently, Lan et al. (30) showed that a
G184S mutation in G
o and the equivalent mutation in
G
i1 prevents both binding to and activation by RGS4,
extending the observation by DiBello et al. (31) that a
mutant Gpa1 prevented a functional interaction with the yeast RGS sst2.
However, because this glycine is a highly conserved Switch 1 residue,
it appears to be required for all G
interactions with RGS molecules
rather than a determinant for specificity. Finally, Natochin and
Artemyev (32) showed that the interaction of G
t with
human retinal RGS could be abolished by mutating serine 202 to the
corresponding G
s aspartate, providing one candidate G
s site that might interfere with RGS binding. They
recently extended this finding by showing that mutation of this
G
s aspartate (G
s Asp229) to
the serine which occurs in G
i family members at the
corresponding Switch 1 position promotes binding to an RGS (33).
subunit that contributed to GAIP binding selectivity by testing
the relative interaction strengths of GAIP with a number of native G
protein
subunits, mutants, and chimeras using the yeast two-hybrid
system. In this system, GAIP interacts equally strongly with native
forms of G
i1, G
i3, and G
o
but very weakly with G
i2 and not at all with
G
s. Both G
s/i3 and G
i3/s
chimeras disrupted GAIP binding, indicating either that both the N and C termini of the G
i subunit contain determinants
required for binding or that divergent sequences in the
G
s protein relative to G
i may interfere
with GAIP contact points. G
i1/i2 and
G
i2/i1 mutants gave more interpretable results,
indicating that the C-terminal domain of G
i1 is required
for GAIP binding. This region constitutes most of the GTPase domain of
the G protein, which is consistent with reports showing that GAIP binds
in a groove within this domain (11). By comparison, the failure of
either G
s chimera to bind may indicate that N-terminal
inserts in the G
s sequence (such as amino acids 72-86)
relative to G
i interfere with the RGS-G
binding
surface or that other divergent residues in the G
s
N-terminal portion interfere with RGS contact. The interfering
aspartate (G
s residue 229) proposed by Natochin and
Artemyev (32, 33) is in fact in the N-terminal portion of our chimeras,
consistent with this possibility.
i1 and G
i2 were swapped.
Candidate residues were chosen on the basis of their conservation in
G
i1 and G
i3 and divergence in
G
i2. The mutation of aspartate 229 of G
i1 to the alanine present in G
i2 nearly abolished GAIP
binding. Conversely, when aspartate was substituted for the alanine
normally present at the same site in G
i2, the mutant
G
i2 bound GAIP to the same extent as native
G
i1. These results reveal the importance of aspartate
229 for the binding of G
i subunits in their native state
to GAIP and potentially suggest a site of physical contact with GAIP.
Yet, upon inspection of the G
i1-RGS4 crystal structure, this aspartate appears quite far from the sites of RGS4 interaction. Due to the location of G
i1 aspartate 229 at the far N
terminus of Switch 3, it is possible that the position of this amino
acid in the AlF4 transition state analogue in which it was
co-crystallized with RGS4 differs from its position in the nonactivated
state in which the G
is show selectivity for binding to
GAIP. That is, it may be that in its GDP-bound (ground state)
conformation, G
i1 Asp229 is in closer
proximity to GAIP than in its
AlF4-Mg2+-GDP-bound conformation.
i1 crystal structure
presents an alternative explanation. In this structure, aspartate 229 appears to be involved in a relay system that connects its carbonyl through a water molecule to lysine 270, which in turn maintains a
hydrophobic interaction with GDP in the RGS4-G
i1 crystal
structure. We hypothesized that removal of the carbonyl group at this
position by mutation to an alanine might disrupt this relay system,
destabilizing the binding of nucleotide and hence the binding of RGS,
because its binding is dependent on the nucleotide-bound state of the G
protein. To test this possibility, IC50 values for the
ability of GTP
S to compete [35S]GTP
S binding by
G
i1, G
i2, G
i1(D229A), and
G
i2(A230D) were compared. The displacement curves were
identical in all cases, implying that differences in nucleotide binding
capacities do not account for RGS binding differences.
i1 versus G
i2 in their
GTP-bound forms, GTPase-deficient mutants of both G
i1
and G
i2 were engineered and tested for GAIP binding in
the yeast two-hybrid system. Interestingly, both
G
i1(Q204L) and G
i2(Q205L) exhibited
similarly high binding affinities to GAIP (about four times the native
G
i1 interaction), consistent with an inability by GAIP
to discriminate between the two proteins in their GTP-bound states. The
G
i1(R178C) and G
i2(R179C)
GTPase-deficient mutants interacted less strongly than the Q204L/Q205L
mutants, although still more strongly than their native counterparts.
This may reflect the ability of RGS proteins to partially restore the GTPase activity of R178C mutants, but not Q204L mutants (7), such that
Q204L mutants remain in their GTP-bound states, but R178C mutants may
reflect a mixture of conformations. These data also bring up an
alternative explanation for the preferential binding of GAIP to
nonmutated G
i1 over G
i2, namely that
there is a greater population of GTP-bound G
i1 than
GTP-bound G
i2 in the yeast cell. This could result from
different rates of GTP/GDP exchange or GTP turnover by the two
subunits. Formally, that remains a possibility. However, because
mammalian G
proteins do not couple to yeast G protein-coupled
receptors (34) and because G proteins remain GDP-bound in the absence
of receptor stimulation (35), we find it more likely that there is a
structural difference between the two G
is that is
recognized by GAIP only in their nonactivated states.
i1 and G
i2 only in their GDP-bound
states has any functional significance, we measured the GAP activity of
GAIP with each of these proteins and their mutants. GAIP enhanced the
rate of GTP hydrolysis of G
i1 but not G
i2
under similar conditions. Furthermore, as predicted by the binding
studies, G
i1(D229A) was a poor substrate for GAIP GAP
activity compared with native G
i1, and
G
i2(A230D) was comparable with G
i1 as a
substrate for GAIP GAP activity. Although Berman et al. (7)
showed GAIP-catalyzed increases in GTPase activity of both
G
i1 and G
i2, Heximer et al.
(17) also showed a greater enhancement by GAIP of G
i1
over G
i2 GTPase activity. Our results indicate that GAIP
preferentially enhances G
i1 over G
i2
GTPase activity and that this activity correlates with the binding
selectivity shown for G
is in their ground state conformations. In addition, because GTPase-deficient mutants of both
i1 and
i2 subunits bind tightly to GAIP,
these results may imply that GAP binding is not sufficient for GAP
catalytic activity. Indeed, differential effects on G
binding
versus GAP activity were discerned by Chen et al.
(25) using various RGS mutants, consistent with this idea. It may be
that the difference in the binding affinities for GTP-bound
versus GDP-bound G
conformations drives GTP hydrolysis,
so that binding to the activated G protein conformation is not the only
indicator of RGS functional selectivity.
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ACKNOWLEDGEMENTS |
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We sincerely thank Skip Brass for much help with the manuscript, Dave Manning and Eliot Ohlstein for encouragement and advice, Katie Freeman for yeast expertise and many helpful discussions, and Cathy Peishoff for help with structural interpretations.
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FOOTNOTES |
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* 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: University of
Pennsylvania, 421 Curie Blvd., BRB-2, Rm. 913, Dept. Medicine,
Philadelphia, PA 19104; E-mail: woulfe{at}pharm.med.upenn.edu.
2
A one-residue insertion at amino acid 117 in the
primary sequence of Gi2 with respect to
G
i1 is responsible for the difference in numbering
between these two
subunits.
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ABBREVIATIONS |
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The abbreviations used are:
GAP, GTPase-activating protein;
PCR, polymerase chain reaction;
bp, base
pair;
GST, glutathione S-transferase;
GTPS, guanosine
5'-O-(thiotriphosphate).
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REFERENCES |
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