From the Departments of Pharmacology, ** Internal
Medicine/Hypertension, and § Biological Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, ¶ Parke-Davis
Research Division of Warner Lambert Company, Ann Arbor, Michigan 48105, and
Department of Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Regulator of G protein-signaling (RGS) proteins
accelerate GTP hydrolysis by G subunits and are thought to be
responsible for rapid deactivation of enzymes and ion channels
controlled by G proteins. We wanted to identify and characterize
Gi-family
subunits that were insensitive to RGS
action. Based on a glycine to serine mutation in the yeast G
subunit
Gpa1sst that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey,
D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed
mutagenesis of
o and
i1 was done. G184S
o and G183S
i1 show kinetics of GDP
release and GTP hydrolysis similar to wild type. In contrast, GTP
hydrolysis by the G
S mutant proteins is not stimulated by RGS4 or
by a truncated RGS7. Quantitative flow cytometry binding studies show
IC50 values of 30 and 96 nM, respectively, for
aluminum fluoride-activated wild type
o and
i1 to compete with fluorescein
isothiocyanate-
o binding to glutathione
S-transferase-RGS4. The G
S mutant proteins showed a
greater than 30-100-fold lower affinity for RGS4. Thus, we have
defined the mechanism of a point mutation in
o and
i1 that prevents RGS binding and GTPase activating
activity. These mutant subunits should be useful in biochemical or
expression studies to evaluate the role of endogenous RGS proteins in
Gi function.
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INTRODUCTION |
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Receptor-mediated activation of heterotrimeric guanine
nucleotide-binding proteins initiates signals elicited by numerous hormone, neurotransmitter, and sensory stimuli (1). Receptors activate
G proteins by stimulating the release of GDP from the subunit,
allowing GTP to bind and to induce dissociation of the G protein
and
subunits, which interact with effector proteins to modulate
cellular responses (2-5).
The duration and strength of receptor-generated physiological responses
are regulated by the rate at which GTP is hydrolyzed by subunit (6,
7). It has been known for some time that the physiological turn-off of
some G protein-mediated signals is faster than would be predicted from
the in vitro GTPase activity of isolated G protein subunits
(8, 9). The solution to this paradox appears to reside in the newly
recognized family of regulator of G protein signaling
(RGS)1 proteins, first
identified genetically in the yeast Saccharomyces cerevisiae
and in the nematode, Caenorhabditis elegans (10-13). At
least 19 RGS protein cDNAs have been identified in mammalian tissues, all sharing a homologous carboxyl-terminal region of ~120
amino acid residues termed the RGS domain (13-15). Biochemical studies
with
i and
q family of G proteins
demonstrated that RGS4 and G
-interacting protein (GAIP) act as
GTPase accelerating proteins (GAPs) (16, 17), which could account for
inhibition of G protein-mediated responses (15). GAP activity of Sst2
for Gpa1 has also been recently demonstrated (18). The mechanism by
which GTPase activity is enhanced by RGS appears to be the stabilization of the transition state conformation of G
for
nucleotide hydrolysis (19, 20). RGS4 also directly inhibits the
interaction of the GTP
S-bound
q subunit with
phospholipase C
, presumably by binding to the effector region of
activated
q (16).
A mutant yeast G subunit, Gpa1sst, was recently identified
in a screen for novel strains showing the "supersensitive to
pheromone" (sst) phenotype. It has a single glycine to
serine mutation and escapes from negative regulation by the RGS
protein, Sst2 (21). Since many RGS proteins affect
Gi-family G proteins, and the crystal structure of the
RGS4·
i1 complex was recently reported, we wanted to
see if the corresponding G
S mutation in
i1 and
o would produce insensitivity to RGS. A major objective
was to obtain a detailed biochemical and mechanistic analysis of this
newly identified class of mutations.
We report that G i1 and
o subunit G
S
mutants are insensitive to GTPase activation by two different RGS
proteins. Quantitative flow cytometry studies demonstrated that a
>30-100-fold reduction in affinity of RGS for the
subunit
transition state is the mechanism of the insensitivity. In future
studies, these mutant
subunits should be useful for evaluating the
role of endogenous RGS proteins in the kinetics and function of
Gi family members. Given the existence of nearly 20 RGS
proteins of which at least 5 act on Gi-family proteins, it
would be difficult to inactivate all of them to determine the
physiological role of endogenous RGS proteins in Gi
signaling. Thus, a Gi
subunit insensitive to RGS
proteins can be used to assess the combined role of all RGS proteins in
Gi function in vivo.
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EXPERIMENTAL PROCEDURES |
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DNA Construction and Mutagenesis--
The G184S mutation in the
o sequence was introduced by the megaprimer polymerase
chain reaction mutagenesis technique using mutagenic antisense primer
5'-GGTTTCTACGATCGAAGTTGTTTTGAC-3' as described (22, 23). The G183S
mutation in
i1 was constructed by overlap-extension
polymerase chain reaction using sense and antisense mutagenic primers
5'-AGTGAAAACGACGTCAATTGTGGAAACC-3' and
5'-GGTTTCCACAATTGACGTCGTTTTCACT-3', respectively. The coding region
of rat RGS4 was amplified by polymerase chain reaction from an
Expressed Sequence Tag obtained from The Institute of Genomic Research
and cloned into the pGEX-2T expression vector. A restriction fragment
comprising nucleotides 913-1358 of the complete human RGS7
cDNA2 was cloned into the
GST expression vector pgGSTag2. The expressed protein contained the RGS
domain (nucleotides 985-1341) but only a portion of the long amino
terminus of RGS7.
Purification of His6 Subunits and GST-RGS
Proteins--
All G
subunits in this paper were expressed in
Escherichia coli and purified as amino-terminal
His6 constructs by a modification of the method of Lee
et al. (24). The GST-RGS4 and -RGS7 fusion proteins were
purified as described (25). The bacterial supernatant was incubated
with glutathione-agarose beads (Amersham Pharmacia Biotech) at 4 °C
overnight. After washing with phosphate-buffered saline, the GST-RGS4
was eluted with 10 mM glutathione in phosphate-buffered saline and dialyzed against 50 mM Tris and 1 mM
EDTA, pH 7.4. The fusion proteins were cleaved by incubation overnight
at 4 °C with 10 units of thrombin/mg of fusion protein followed by
incubation with glutathione-agarose to remove GST and any
uncleaved GST-RGS4.
GAP Assays--
[-32P]GTP (1 µM)
was allowed to bind to 50 nM
o for 20 min at
room temperature (23-24 °C) or 50 nM
i1
for 15 min at 30 °C. After lowering the temperature to 4 °C, the
hydrolysis reaction was started by the addition of MgSO4
and GTP
S to final concentrations of 15 mM and 200 µM in the presence or absence of 100 nM RGS4 or RGS7. Aliquots (50 µl) were diluted in 1 ml of 15% (w/v) charcoal solution (50 mM NaH2PO4, pH 2.3, 0 °C) at the indicated time points. The amount of
[
-32P]Pi released at each time point was
fit to an exponential function, cpm(t) = cpmo +
cpm × (1
e
kt).
Binding of to GST-RGS4-agarose--
Wild type or mutant
GDP-bound
(1 µM) was mixed with GST-RGS4 fusion
protein (1 µM) bound to glutathione-agarose beads (5 × 105 beads/ml) in a final volume of 100 µl of HEDML
buffer (50 mM Hepes, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 10 mM MgSO4, 20 ppm deionized Lubrol) with 0.05% bovine serum albumin in the presence or absence of 20 µM AlCl3 and 10 mM NaF (HEDML/AF). After 2 h, the beads were washed
three times with 1 ml of ice-cold HEDML/AF buffer with bovine serum
albumin, and bound products were separated by SDS-polyacrylamide gel
electrophoresis and visualized by Coomassie Blue stain.
Competition Binding of the Subunit to GST-RGS4--
Labeling
of purified His6
o with fluorescein
isothiocyanate (FITC) was conducted as described (26).
Glutathione-agarose beads (Amersham) with sizes between 35 and 78 µm
were prepared for flow cytometry by filtering through stainless steel
Tyler sieves in HEDML buffer. Two nM GST or GST-RGS4 fusion
protein was bound to the beads (5 × 104/ml) for 20 min at room temperature in HEDML buffer. Beads were then washed and
incubated with 4 nM FITC-
o with the
indicated amounts of unlabeled
subunit in 100 µl of HEDML/AF
buffer at room temperature for 2.5 h. The amount of
FITC-
o bound to the GST-RGS4 was quantitated on a Becton
Dickinson FACScan as described (26). Data are presented as a fraction
of the control fluorescence after subtracting nonspecific binding
(~20% of the total). Results were fit to a one-site competition
binding function, and the IC50 for the competitors were
calculated using Prism version 2.01 for Windows 95 (Graphpad Software,
San Diego, CA).
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RESULTS AND DISCUSSION |
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Nucleotide Binding to and RGS-stimulated Hydrolysis by Subunit--
The time course of nucleotide binding to
subunits was
determined by use of the fluorescent nucleotide derivative,
methylanthraniloyl GTP
S as described (27). Similar rates of binding
were seen for both the G
S mutants and the wild type proteins. Rate
constants were 0.20 ± 0.06 and 0.21 ± 0.08 min
1 for
o and 0.049 ± 0.002 and
0.12 ± 0.03 min
1 for
i1, wild type
and mutant, respectively. The kcat values for
wild type and G
S mutant proteins in the absence of RGS were very
similar (Fig. 1 and Table
I). In the presence of 100 nM
RGS4, the reaction was completed by the first time point for wild type
o and
i1 (Fig. 1). Even at 4 °C, the
reaction was too fast to measure with a rate constant greater than 5 min
1. There was no effect of RGS4 on the
kcat of either G
S mutant (Fig. 1,
B and D, and Table I). The RGS domain fragment of
RGS7 (100 nM) increased the kcat of
wild type
i1 by ~9-fold, whereas there was no effect
on the
i1 G
S (Fig. 1C and Table I).
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GAP Activity at Increasing Concentrations of RGS4--
To
determine if GAP activity could be restored at higher concentrations of
RGS4, GTP hydrolysis at 1 min was measured with 50 nM G
subunit and 0-3 µM RGS4. The EC50 values for
RGS4 were 5 ± 1 and 10 ± 4 nM for wild type
o and
i1, respectively (Fig. 2). There was only a slight increase in
GTP hydrolyzed by either G
S mutant
subunit, even at the
maximum concentration of RGS4 (Fig. 2).
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Reduced GAP Activity Is Due to Reduced Affinity--
To determine
whether the marked reduction in sensitivity of mutant to RGS4 was
due to decreased binding, we tested co-precipitation of
and RGS4.
In the absence of AlF4
, GDP-bound
subunit
did not bind to GST-RGS4 (Fig. 3). In the presence of AlF4
, wild type
o and
i1 showed substantial binding to GST-RGS4 immobilized on
glutathione-agarose beads.3
In contrast, there was no detectable binding of either of the G
S
mutant
subunits. To more quantitatively characterize the interaction of
subunits with RGS4, we used a recently developed flow cytometry approach (26). Binding of FITC-labeled
o
to GST-RGS4 on glutathione-agarose beads was detected by measuring the
amount of fluorescence associated with the beads in a FACScan flow
cytometer (see "Experimental Procedures"). Unlabeled wild type and
mutant
subunits were added to compete for the binding of
FITC-
o to RGS4. All binding was done in
AlF4
-containing buffer with 4 nM
FITC-His6
o and 2 nM GST-RGS4.
Nonspecific binding in the presence of a 50-fold excess of unlabeled
o represented less than 20% of the total binding.
Similarly, GTP
S-bound FITC-
o did not show any
specific binding (data not shown). Wild type
o and
i1 reduced the binding of FITC-
o, with
IC50s of 30 and 96 nM, respectively (and 95%
confidence intervals of 18-35 and 60-150 nM; see Fig.
4). As observed for the effects of RGS4
on GTPase activity, we were unable to demonstrate significant
interactions of the
i1 G
S mutant protein with RGS4
up to 3 µM
subunit. Because of the limited purity and
amounts of
o G
S, the highest concentration used was
300 nM. At this concentration, there was an ~20%
decrease in bound FITC-
o. Thus the affinity for both G
S mutant
subunits is at least 30-100-fold lower than that of
wild type.
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Generality of G S Mutation in G
-RGS Interactions--
In
this report, we identify a G
S point mutation in the
conformationally flexible "switch I" region of
o and
i1 that completely eliminates the interaction of these
subunit with RGS proteins. This mutation should be useful in
determining the role of endogenous RGS proteins in the physiological
functioning of G proteins. This is especially important for
Gi, since the kinetics of Gi-mediated regulation of effectors (e.g. adenylyl cyclase (28) and
potassium channels (9)) are faster than expected for the in
vitro GTP hydrolysis rates in the absence of RGS proteins.
Deactivation of potassium currents was recently shown to be accelerated
by overexpressed RGS1, 3, or 4 in oocytes and Chinese hamster ovary cells (29, 30). These data show that exogenous RGS proteins can alter G
protein function, but the question of whether normal cellular
concentrations of RGS proteins alter the kinetics of ion channel
function has not been answered experimentally. With the
subunit
mutation described here, it is now possible to directly address that
question.
Structural Basis of Glycine to Serine Effects--
In the crystal
structure of the i1·RGS4 complex (19), the switch I
region of
interacts with three of the four different segments of
the RGS consensus domain, but there are also contacts with switch II
and switch III. Glycine 183 is located in the switch I region of
i1, forming a turn just before the
1 strand (31, 33).
Interestingly, Natochin and Artemyev (32) recently found that the
mutation of Ser-202 in switch II of transducin prevents interaction
with retinal specific RGS. Glycine 183 provides a substantial
contribution to the buried surface area between
i1 and
RGS4 (see Ref. 21 for details). There is direct contact of glycine 183 with the C
and C
of serine 85 and C
of tyrosine 84 of the RGS
protein. Introduction of the hydroxymethyl side chain of serine would
sterically hinder the formation of a tight complex of
and RGS.
Also, threonine 182, which is directly adjacent to glycine 183 mutated
in the
i1 G
S mutant, exhibits the greatest change
in accessibility of any
residue upon forming the
i1·RGS4 complex (19). Thus the G
S mutation may
also disrupt local protein conformation and prevent threonine 182 from
interacting with the highly conserved residues in RGS.
Binding of Subunits to GST-RGS4--
Both in co-precipitation
and fluorescence competition studies, the affinity of the
AlF4--bound mutant
subunits for RGS4 are
dramatically reduced. With the flow cytometry method, we obtained
quantitative measures of subunit affinities. The IC50
values of wild type
o and
i1 (30 and 96 nM, respectively) are similar to the KD
of 45 nM determined by surface plasmon resonance for
transducin binding to retinal-specific RGS (37). Our IC50
is significantly higher than the KD estimated from
on and off rates by plasmon resonance for RGS4 and
i1
(38). The differences between the latter data and ours may be due to
methodological differences (kinetic versus equilibrium
determination or direct binding versus competition methods).
In any case, the 30-100-fold lower affinity of the G
S mutant
G
proteins is very clear.
Function of RGS7--
Our data also include the first biochemical
demonstration of GAP activity by RGS7. At 100 nM, the
effect of the RGS7 RGS domain on GTP hydrolysis by i1 is
significantly less than the effect of RGS4. Interestingly, the
FITC-
o did not show detectable binding to the GST-RGS7
fusion protein (data not shown). These results are probably due to a
lower affinity of RGS7 for
o compared with RGS4. This
may be due in part to the lack of full-length RGS7 in the expression
construct.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Albrecht Moritz for
providing the GST-RGS7 expression construct and Masakatsu Nanamori for
expression and purification of the o protein. The
expression vectors pQE/
o and the
pQE6/His6
i1 were generously provided by Dr.
M. E. Linder (Washington University).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM 39561 (to R. R. N.), GM 53645 (to R. T.), and GM 55316 (to H. G. D.).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 and reprint requests should be
addressed: Dept. of Pharmacology, 1301 MSRB III, 1150 W. Med. Ctr. Dr., Ann Arbor, MI 48109-0632. Tel.: 313-763-3650; Fax: 313-763-4450; E-mail: RNeubig{at}umich.edu.
1
The abbreviations used are: RGS, regulator of G
protein signaling; FITC, fluorescein 5-isothiocyanate; GAP, GTPase
activating protein; GST, glutathione S-transferase; GTPS,
guanosine 5'-O-(3- thiotriphosphate).
2 A. Moritz and R. Taussig, unpublished information.
3
The apparent excess of bound G over GST-RGS4
protein was due to incomplete elution of the GST-RGS4 from the beads in
this experiment.
4 M. Nanamori, K-L.. Lan, and R. R. Neubig, unpublished information.
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REFERENCES |
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