From the Department of Integrative Biology and
Pharmacology, University of Texas Health Science Center at Houston,
Houston, Texas 77030 and the § B Cell Molecular Biology
Section, Laboratory of Immunoregulation, NIAID, National Institutes
of Health, Bethesda, Maryland 20892
Received for publication, October 18, 2002, and in revised form, January 14, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The production of cAMP is controlled on many
levels, notably at the level of cAMP synthesis by the enzyme adenylyl
cyclase. We have recently identified a new regulator of adenylyl
cyclase activity, RGS2, which decreases cAMP accumulation when
overexpressed in HEK293 cells and inhibits the in vitro
activity of types III, V, and VI adenylyl cyclase. In addition, RGS2
blocking antibodies lead to elevated cAMP levels in olfactory neurons.
Here we examine the nature of the interaction between RGS2 and type V
adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase,
RGS2 inhibited G Heterotrimeric G protein-mediated receptor signaling pathways are
pivotal parts of the intricate and diverse biological processes dictating cellular function. G proteins transduce signals to a variety
of effectors including adenylyl cyclase
(AC)1 (1). The
hormone-sensitive AC system is a typical archetype of G
protein-mediated signal transduction. Appropriate agonist-bound, heptahelical receptors activate Gs by catalyzing the
exchange of GDP for GTP. The GTP-bound RGS2 is a 211-amino acid protein that like other members of this family
mediates its GAP activity via its core domain (4-6). Experiments using
recombinant RGS2 and membranes prepared from insect cells (Sf9)
expressing different AC isoforms indicate that RGS2 suppresses the
activity of AC III, the predominant isotype in the olfactory system,
and the cardiac isoforms, V and VI (3). RGS2 has also been shown to
decrease cAMP accumulation in The mechanisms for RGS2 regulation are as yet unclear (8).
Phosphorylation of RGS2 by protein kinase C decreases its capacity to
negatively regulate phospholipase C The list of non-G protein-binding partners for RGS proteins continues
to grow (8, 16). It remains to be determined whether interactions of
RGS2 with G Materials--
The anti-HA (hemagglutinin A) and
Ni-NTA-horseradish peroxidase conjugate antibodies were purchased from
Roche Molecular Biochemicals and Qiagen, respectively.
Anti-G Plasmids--
The expression vector for G Tissue Culture and in Vivo cAMP Detection--
HEK293 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% (v/v) fetal calf serum, 1 mM glutamine, 50 µg/ml
streptomycin, and 50 units/ml penicillin at 37 °C with 5%
CO2. For transient expression of proteins, subconfluent
HEK293 cells were plated on 6-well plates and transfected using FuGENE 6 transfection reagent following the manufacturer's protocol with constructs expressing either
To measure cAMP accumulation in cells transfected with
G Western Blotting--
The cell lysates were boiled for 5 min in
Laemmli sample buffer and sonicated. The proteins were resolved by
SDS-PAGE and transferred to nitrocellulose. The protein expression
levels were determined by immunoblotting using anti-HA antibody.
Immunoreactive protein bands were detected by a horseradish
peroxidase-conjugated secondary antibody and enhanced chemiluminescence.
Purification of Recombinant Proteins--
RGS2 polymerase chain
fragments digested with BspHI and BamHI were
inserted in-frame with an N-terminal hexahistidine tag in pQE60 using
NcoI and BamHI. The proteins were expressed in Escherichia coli and purified under nondenaturing conditions
using Ni-NTA-agarose beads (24). The purified protein fractions were dialyzed in buffer containing 10 mM Hepes, pH 7.4, 10 mM
All G protein Generation of Adenoviral Recombinants--
Adenoviruses for RGS2
and RGS4 were generated as described (28, 29). RGS2 and RGS4 were
cloned into the adenoviral shuttle vector pACsk2.cmv using the
restriction enzymes KpnI and XbaI. Both
constructs contained C-terminal HA tags. To obtain recombinant viruses,
HEK293 cells were co-transfected with the recombinant shuttle vector
and pJM17. Viral plaques were isolated and propagated, and viral DNA
was sequenced. HEK293 cells (~70% confluent) were infected with
HA-tagged RGS2 and RGS4 adenoviruses (multiplicity of infection = 2). The cells were washed once in ice-cold phosphate-buffered saline
and harvested 24 h post-infection in 1 ml of hypotonic lysis
buffer (50 mM Tris, pH 7.5, 4 mM EDTA plus
protease inhibitors). The cell lysates were subjected to 50 strokes of
Dounce homogenization on ice, followed by low speed centrifugation for
5 min to eliminate cellular debris, and subsequently centrifuged at
100,000 × g at 4 °C for 20 min. The supernatant was
checked for RGS protein expression using anti-HA antibody and used for
AC binding assays as described below. Note that the high overexpression
resulting from adenoviral expression often leads to a doublet for both
RGS2 and RGS4. This is most likely due to proteolytic cleavage of the
triple HA tag present at the C terminus. This does not occur with
lowered expression of RGS2 and RGS4 in transient transfections.
Adenylyl Cyclase Binding Assays--
Pull-down assays were
conducted with pure recombinant proteins as well as with HEK293 cell
lysates. The lysates were obtained by infecting cells with recombinant
adenoviruses encoding RGS2 or RGS4 or by transfecting subconfluent
HEK293 cells with constructs expressing wild type or mutant RGS2. Cells
at 36 h post-transfection were washed once in ice-cold
phosphate-buffered saline and harvested in 300 µl of hypotonic lysis
buffer (50 mM Tris, pH 7.5, 4 mM EDTA plus
protease inhibitors), followed by Dounce homogenization and
centrifugation to remove cellular debris. The lysates prepared from
HEK293 cells expressing RGS proteins (50 µg) were incubated with 15 µg of VC1(670)-H6 or H6-VC2 for 30 min and
then layered over Ni-NTA-agarose beads. When purified RGS proteins were
utilized, GST or GST-VC1 (15 µg) was first bound to
glutathione beads. The beads were then washed and mixed with H6-RGS2 or
H6-RGS4 (2 µM). The reactions were incubated for 2-4 h
at 4 °C on a rotating mixer, washed twice with wash buffer (20 mM Hepes, pH 8.0, 2 mM MgCl2, 200 mM NaCl, 1 mM Membrane Localization--
HEK293 cells (~70% confluent) were
transfected with wild type, mutant, or truncated RGS2 constructs. After
36 h, the cells were harvested, briefly sonicated, and subjected
to a low speed spin to remove nuclei and cellular debris. The lysate
was then subjected to centrifugation (100,000 × g) at
4 °C for 30 min to separate membranes and cytosol. The crude
membranes thus obtained were washed twice with cold phosphate-buffered
saline, subjected to SDS-PAGE, and probed with anti-HA antibody for
detection of RGS2 distribution in the cytosol and membrane.
Adenylyl Cyclase Activity Assays--
Adenylyl cyclase activity
was measured as described (30). The assays were performed at 30 °C
in a final volume of 50 µl in the presence of 5 mM
MgCl2. A limiting concentration of the full-length
C1 domain from type V adenylyl cyclase was reconstituted with the C2 domain (final concentration, 0.5 µM) in buffer containing 0.5 mg/ml bovine serum albumin
(21). RGS proteins or control buffer were preincubated with adenylyl
cyclase domains for 20 min on ice, followed by the addition of
GTP RGS2 but Not RGS4 Directly Binds to the C1 Domain of
Type V Adenylyl Cyclase--
Mammalian AC consists of a short N
terminus, a set of six transmembrane spans, a cytoplasmic domain
(designated C1), followed by a second set of six
transmembrane spans, and a C-terminal cytoplasmic domain
(C2) (2). Inhibition of the activities of types III, V, and
VI AC reported earlier (3) could be attributed to the direct binding of
RGS2 with either cytoplasmic domain of the enzyme. To demonstrate that
RGS2 directly binds to AC, we performed binding assays using
hexahistidine-tagged C1 and C2 domains of type
V AC. These cytoplasmic domains are fully active when reconstituted with each other and retain most of the properties of the native enzyme
(21, 31-33). An excess of His-tagged C1 and C2
domains from type V AC were incubated with HEK293 extracts containing C-terminal HA-tagged RGS2 or RGS4 (Fig.
2A). Nontagged
GTP
Because the above experiment utilized HEK293 extracts, it was still
possible that a second protein was required to mediate this binding
event. Therefore, we tested the direct interaction of E. coli purified His-tagged RGS proteins with the C1
domain fused to GST versus GST alone. Glutathione resin was
used to pull down the interacting proteins that were detected by
immunoblotting with the Ni-NTA-horseradish peroxidase antibody or an
antibody directed against GST. Because of the limited expression of
GST-VC1, an excess of purified RGS proteins was used in
these reactions. RGS2 associated with GST-VC1 but not with
GST alone (Fig. 2B). No interaction was observed with RGS4.
These results further support our hypothesis of a direct interaction
between RGS2 with type V AC.
RGS2 Inhibits Type V AC in Vivo Independent of RGS2 GAP
Activity--
Previous studies have shown that RGS2 inhibits
G
To test the role of RGS GAP activity, we have utilized a mutation
within the highly conserved GAP domain. Replacement of
Asn128 with Ala in RGS4 reduces GAP activity and binding to
G proteins by over 3 orders of magnitude (39, 40). Structural data also indicates that Asn128 in RGS4 plays a crucial role in the
interaction of RGS and G proteins (41). We made the corresponding
mutation in RGS2, Asn149 to Ala, and examined its effect on
cAMP production. This mutant form of RGS2 also suppressed cAMP
accumulation to the same level as wild type RGS2 (Fig. 3A).
Hence, a mutation created at the G
Similar results were observed in HEK293 cells, transfected with type V
AC and the RGS2 and RGS2-N149A but Not The N-terminal 19 Amino Acids of RGS2 Are Sufficient for Inhibition
of AC--
The RGS2 box domain is located from amino acids 72-199, as
defined by structural homology to the RGS4 box (41). Zeng et al. (42) have shown that the N-terminal residues (1-33) of RGS4 confer high affinity and receptor selective signaling via
Gq. Deletion of the N-terminal domain decreased the potency
of RGS4 inhibition of Gq signaling by 104-fold.
RGS2 and RGS4 share a limited homology outside the RGS box from amino
acid 42-72, but the extreme N terminus of RGS2 is 20 amino acids
longer and displays no homology with the N terminus of RGS4. Although
the N-terminal domain of RGS2 appeared to be a likely candidate to
interact with AC, we could not rule out additional sites of interaction.
RGS2 contains 12 amino acids at the C terminus that are unique to
this RGS protein. In addition, residues within the RGS core domain
could also play a role. This domain shares many hydrophobic amino acids
among all RGS family members; however, significant differences between
RGS2 and RGS4 lie within Mutational Analysis of RGS2 Identifies a Series of Residues
Important for Inhibition of Type V AC--
The N-terminal 19 amino
acids of RGS2 clearly play a pivotal role in inhibiting type V
AC-generated cAMP production. We conducted a limited alanine scanning
analysis of the first 19 amino acids in the N-terminal region of RGS2
to identify residues important for RGS2 function. Sets of three
residues along the 19-amino acid stretch were mutated to alanine as
shown in Fig. 6A. HEK293 cells were transfected with expression vectors for type V AC and
G Membrane Localization of Wild Type, RGS2 Does Not Interact with the G
The E411A and L472A mutations were next tested for their potential
roles in RGS2-mediated inhibition. HEK293 cells were transfected with
G
Finally, the IC50 for inhibition of the cytoplasmic domains
(C1 and C2) of type V AC by purified RGS2 was
determined for wild type C1 and L472A and E411A mutant
C1 domains upon reconstitution with the C2
domain from type V AC (Fig. 7D). RGS2 inhibits
G Conclusion--
AC is regulated by many different signals. The
present study reinforces the important link between RGS proteins and
cellular signaling and identifies a novel binding site for the
interaction of RGS2 and a downstream effector protein, type V AC.
Further studies are required to completely understand the mechanism of this inhibition and the exact site of interaction on the C1
domain of type V AC. Our appreciation of RGS proteins as more than
simply inhibitors of G protein signaling is ever expanding. The direct link between RGS2 and AC is yet another example of the diverse roles
that RGS proteins play in cellular signaling.
s-Q227L- or
2-adrenergic
receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation
and to bind adenylyl cyclase. Further mutational analysis indicated
that neither the C terminus, RGS GAP activity, nor the RGS box domain
is required for inhibition of adenylyl cyclase. Alanine scanning of the
N-terminal amino acids of RGS2 identified three residues responsible
for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C1 but not the C2
domain of type V adenylyl cyclase and that the inhibition by RGS2 is
independent of inhibition by G
i. These results provide
clear evidence for functional effects of RGS2 on adenylyl cyclase
activity that adds a new dimension to an intricate signaling network.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunit of Gs in
turn activates AC, increasing the rate of synthesis of cyclic AMP from
ATP (1, 2). All isoforms of mammalian AC are stimulated by the
heterotrimeric G protein Gs. Many other regulatory
influences including RGS (regulators of G
protein signaling) proteins have crucial effects on various AC isoforms (3). These enzymes thus serve critical roles as integrators
of diverse inputs.
TC3 insulinoma cells (7). When added
to purified recombinant type V AC cytoplasmic domains, recombinant RGS2
decreases cAMP production stimulated by either G
s or
forskolin. The structural basis for the inhibitory effect of RGS2
remains unknown, although it is likely a direct effect. In
vivo, odorant-elicited cAMP stimulation resulted in the opening of
cyclic nucleotide gated channels. The microinjection of RGS2 antibody
into olfactory neurons dramatically augments the currents observed in
whole cell voltage clamp recordings of odorant-stimulated olfactory
neurons. These results indicate that the level of RGS2 in an olfactory
neuron may regulate the responsiveness of that neuron to olfactory
stimulants (3). This study provided evidence for a novel role of RGS
proteins as direct regulators of AC activity.
activation (9).
Signal-induced redistribution may also regulate RGS2 function (10) and
have important effects on cellular signaling. RGS2 shares with RGS4 and
RGS16 a conserved N-terminal domain that is necessary and sufficient
for plasma membrane targeting (10). Until recently, most reports of the
intracellular localization of members of the RGS family of proteins
have found them to be associated with the membrane fraction or
distributed between the membrane and cytosolic fractions (11, 12);
however, there have been some reports indicating the nuclear
localization of RGS proteins (13, 14). In human astrocytoma 1321N1
cells, increases in cAMP and phosphoinositide signaling gives rise to a
rapid and transient increase in RGS2 expression (15). In these cells
both endogenous and transfected RGS2 is found largely, although not
entirely, localized in the nucleus (15). Thus, the localization of RGS2
may in fact be controlled in ways not yet appreciated.
q/11, G
i, AC, and coat protein
(
-COP) can explain the putative roles for RGS2 in control of
behavior, T cell function, and synaptic development in hippocampal CA1
neurons as revealed by RGS2-deficient mice (17). We describe herein a
direct interaction between RGS2 and the C1 domain of type V AC. In addition, we report the nature of the AC-binding site on RGS2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
i, anti-G
s, and anti-GST antibodies were purchased from Santa Cruz Biotechnology. The anti-RGS2 antiserum was generated in rabbits against a C-terminal peptide (PQITTEPHAT) coupled to keyhole limpet hemocyanin followed by affinity purification using immunizing peptide. Goat anti-rabbit IgG horseradish peroxidase conjugate was obtained from Bio-Rad, and sheep anti-mouse IgG horseradish peroxidase was from Amersham Biosciences. FuGENE 6 transfection reagent was purchased from Roche Molecular Biochemicals, and all other cell culture reagents were obtained from Invitrogen. The
cyclic AMP enzyme immunoassay kit was purchased from Assay Designs,
Inc. (Ann Arbor, MI).
s-Q227L
was provided by S. Gutkind (National Institutes of Health),
and the expression vector for RGS2 has been described (18, 19).
Full-length type V AC and the E411A and L472A mutant clones were
subcloned from the pVL1392 vector (20, 21) into the mammalian
expression vector, pcDNA3, using the restriction sites
BamHI and XbaI. The plasmid encoding
GST-VC1 was created by subcloning
VC1(670)H6 (22) using the restriction enzymes
NcoI and HindIII into a modified form of pGEX-cs
(23) containing a HindIII site in the 3' portion of the
polylinker. RGS2 N-terminal truncations were created using PCR by
deletion of DNA encoding amino acid residues 1-19 (
NT1), 1-39
(
NT2), and 1-62 (
NT3). All of the truncations contain an N-terminal initiating methionine and were created using the vector pcDNA3 (Invitrogen). RGS2-
Box was created by deletion of DNA encoding amino acid residues 72-211 employing the following
forward and reverse primers, 5'-GCCACCATGCAAAGTGCTATG-3' and
5'-GTTGCGGCCGCTACTTGATGAAAGCTTGCTGTTG-3'. RGS2-
C was constructed by
deleting the last 12 residues at the C terminus of RGS2. Mutations
within RGS2 were generated using a QuikChange mutagenesis kit
(Stratagene). Asparagine at position 149 of RGS2 was mutated to alanine
using the oligonucleotides 5'-GCTCCAAAAGAGATAGCCATAGATTTTCAAACC-3' and
5'-GGTTTGAAAATCTATGGCTATCTCTTTTGGAGC-3'. Four mutants were generated in
the first 19 amino acids at the N terminus of RGS2, namely RGS2-MFL,
RGS2-VQH, RGS2-DCR, and RGS2-PMD (see Fig. 6A). Residues
MFL, VQH, DCR, and PMD were mutated to alanine using the following
oligonucleotides: for RGS2-MFL, 5'-ATGCAAAGTGCTGCGGCCGCGGCTGTTCAACAC-3' and 5'-CGTGTTGAACAGCCGCGGCCGCAGCACTTTGCAT-3'; for RGS2-VQH,
5'-GCTATGTTCTTGGCTGCTGCTGCTGACTGCAGACCCATG-3' and
5'-CATGGGTCTGCAGTCAGCAGCAGCAGCCAAGAACATAGC-3'; for RGS2-DCR, 5'-GGCTGTTCAACACGCCGCCGCACCCATGGACAAGAGCG-3' and
5'-CGCTCTTGTCCATGGGTGCGGCGGCGTGTTGAACAGCC-3'; and for RGS2-PMD,
5'-ACACGACTGCAGAGCCGCGGCCAAGAGCGCAGGC-3' and 5'-GCCTGCGCTCTTGGCCGCGGCTCTGCAGTCGTGT-3'. The veracity of all of
the DNA constructs was verified by nucleotide sequencing. A schematic
showing the expected RGS proteins expressed from the constructs used is
shown (Fig. 1). All of the RGS2 plasmids
and G
s-Q227L were tagged at their C termini, with the
exception of two mutants of RGS2,
box (
72-211) and
C
(
199-211), which were tagged at the N terminus with three copies of
the HA epitope. Wild type RGS2 was obtained from the Guthrie cDNA
Resource Center.
View larger version (15K):
[in a new window]
Fig. 1.
Representation of truncated RGS2
constructs. Wt, wild type.
2-adrenergic receptor (0.5 µg/well) or G
s-Q227L (0.5 µg/well) along with RGS2
(1.0 µg/well) and type V AC (0.2 µg/well). The vector pcDNA3
(Invitrogen) was used to normalize all of the DNA concentrations to 1.7 µg/well. After 36 h of transfection, the cells were starved
overnight in medium containing 1% fetal bovine serum, followed by
starvation in medium devoid of serum for 2 h.
s-Q227L or corresponding control DNA constructs, the
cells were treated with 1 mM 3-isobutyl-1-methylxanthine
for 15 min and subsequently harvested in 250 µl of hypotonic lysis
buffer (50 mM Tris, pH 7.5, 4 mM EDTA, plus
protease inhibitors). A portion of the cell lysate (50 µl) was used
for Western blotting to monitor protein expression, and the remaining
lysate was boiled for 10 min at 100 °C and spun at 14000 × g for 10 min to remove cellular debris. The supernatant was
used to measure the total cAMP accumulation by cAMP enzyme immunoassay
detection. For
2-adrenergic receptor activation and
corresponding controls, the cells were treated with isoproterenol (0.1 µM) for 15 min before subsequent harvesting, lysis, and
cAMP detection. The data are represented as the means ± S.E., and
statistical significance is determined by Student's t test
(p < 0.05 is considered significant).
-mercaptoethanol, 10% glycerol, 200 mM
NaCl, pooled, concentrated, and stored at
70 °C. Type V AC domains
were expressed with an N-terminal (VC2) or C-terminal
(VC1) hexahistidine tag and purified as previously described (25). GST-VC1 was purified using glutathione
affinity resin. BL21(DE3) cells containing the GST-VC1
expression plasmid were grown at 30 °C in T7 medium until the
A600 reached 1.0. Synthesis of
GST-VC1 was induced with 0.5 mM
isopropylthiogalactoside, and incubation continued for 4 h at
30 °C. The cells were harvested and resuspended in lysis buffer
containing 50 mM Tris, pH 7.7, 1 mM EDTA, 2 mM dithiothreitol, 120 mM NaCl, and protease
inhibitors prior to incubation with 0.2 mg/ml lysozyme for 30 min at
4 °C and then subsequent incubation with 5 mM
MgCl2 and DNase (0.01 mg/ml) for 30 min. Cellular debris
was removed by centrifugation (100,000 × g for 30 min). The supernatant was applied to a glutathione column, which was
first washed with lysis buffer containing 400 mM NaCl (25 column volumes) followed by lysis buffer without NaCl (5 column
volumes). The protein was eluted with 20 mM Hepes, 1 mM EDTA, 2 mM dithiothreitol, 100 mM NaCl, 5% glycerol, and 12 mM glutathione.
The protein was then concentrated and dialyzed overnight in elution
buffer lacking glutathione.
subunits were synthesized in E. coli and
purified as described (26, 27). Purified
subunits were activated by
incubation with [35S]GTP
S at 30 °C for 30 min
(G
s) or 2 h (G
i) (21). Free GTP
S was removed by gel filtration.
-mercaptoethanol, and 0.1%
C12E10), boiled in Laemmli buffer for 5 min,
subjected to SDS-PAGE (13% gel), and analyzed by immunoblotting
employing the appropriate antibody.
S-G
s and 0.5 mM
[
-32P]ATP to start the reaction. The reactions were
terminated after 10 min, and the products were separated by sequential
chromatography on Dowex-50 and Al2O3.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
S-G
i or GTP
S-G
s were used as
positive controls for the C1 and C2 domains,
respectively. Ni-NTA resin was used to pull down the interacting
proteins that were eluted with SDS sample buffer, followed by
immunoblotting for the respective proteins. Approximately 30% of the
HA-tagged RGS2 present in the HEK293 extracts was bound to the
C1 domain of type V AC. No binding of RGS4 to the
C1 domain was observed, although the C1 domain
tightly bound G
i. Neither RGS2 nor RGS4 bound to Ni-NTA
beads alone or to the C2 domain, although this protein was
capable of binding G
s (22).
View larger version (16K):
[in a new window]
Fig. 2.
Interactions between RGS2 and the cytoplasmic
domains of type V AC. A, extracts containing HA-tagged
RGS4 (R4) and RGS2 (R2) were obtained from
adenovirus-infected HEK293 cells (Load). Equal amounts of
cell lysates were added in the reactions and incubated with His-tagged
C2 (H6-VC2) and C1 domains
(VC1-H6) of type V AC at 4 °C for 30 min. Ni-NTA alone was incubated with RGS2 cell extract as a negative
control (Ni-NTA Beads). Nontagged GTP S-G
s
and nontagged GTP
S-G
i were incubated with the
C2 and C1 domain, respectively, as positive
controls in the absence of cell extracts. Ni-NTA resin was then added
to all of the reactions, mixed for 2 h at 4 °C and washed as
described under "Experimental Procedures." The proteins were eluted
with SDS sample buffer, run on SDS-PAGE, and blotted with anti-HA
antibody (Ab). The blots were then stripped and reprobed for
G
i and G
s. The load represents 30% of
the total protein present in these reactions, whereas 100% of the
elution is run on SDS-PAGE. RGS2 and RGS4 were run as a doublet because
of proteolytic clipping of the HA tag in adenovirus-infected cell
extracts (see "Experimental Procedures"). B, equal
amounts of purified His-tagged RGS4 and RGS2 (Load) were
used in additional binding reactions. His-tagged RGS proteins were
incubated with glutathione beads alone (Glut. Beads) or with
glutathione beads bound with GST (GST-alone) or GST-fused to
the C1 domain of type V AC (GST-VC1).
The proteins were incubated for 1 h at 4 °C, washed, eluted
with SDS sample buffer, run on SDS-PAGE, and blotted with
Ni-NTA-horseradish peroxidase antibody to detect both RGS2 and RGS4.
The load represents 20% of the total protein used in the binding
reactions, whereas 100% of the eluted RGS protein is shown.
s-induced type III AC activation in vivo
(3). To verify that RGS2 inhibits G
s-induced type V
activation in vivo, we transfected HEK293 cells with the
expression vectors for type V AC and a GTPase-deficient form of
G
s, G
s-Q227L, in the presence or absence
of RGS2. Co-expression of G
s-Q227L and type V AC
increased cAMP accumulation 3-fold (Fig.
3A). Additional expression of
RGS2 reduced G
s-Q227L-stimulated cAMP accumulation to
near basal levels. A similar result was obtained with nontagged RGS2,
ruling out any possible unwarranted effects of the HA tag (data not
shown). The inhibition by RGS2 is unusual because many RGS proteins
including RGS1, RGS4, and GAIP have no effect or show increased cAMP
accumulation (34-36). However, RGS-PX1, RGS3, and RGS13 have also been
reported to have inhibitory effects on cAMP accumulation by either
G
s GAP activity or other unknown mechanisms
(36-38).
View larger version (22K):
[in a new window]
Fig. 3.
Inhibition of cAMP accumulation by RGS2 and
N-terminal truncations in the presence of
G s-Q227L or the
2-adrenergic receptor.
A, production of cAMP was measured in HEK293 cells
transfected as indicated with type V AC, G
s-Q227L, and
wild type, mutant (N149A), and truncated (N-terminal
deletions NT1 (
1-19), NT2 (
1-39), and NT3 (
1-62)) RGS2.
Protein expression levels were determined by immunoblotting using
anti-HA antibody. The data are expressed as the means ± S.E. from
three different experiments each performed in duplicate. B,
cAMP production was measured as in A, except isoproterenol
stimulation of the
2AR was used instead of
G
s-Q227L for activation of type V AC. HEK293 cells
transfected with control constructs or
2AR were
stimulated with 0.1 µM isoproterenol for 15 min prior to
lysis, cAMP detection, and Western blotting. The data are expressed as
the means ± S.E. from three different experiments each performed
in duplicate. Wt, wild type.
-RGS binding interface in RGS2
that disrupts GAP activity does not limit its ability to inhibit cAMP.
However, deletion of the N-terminal amino acids of RGS2 (
NT1,
NT2, and
NT3) abolished its ability to inhibit cAMP generated by
type V AC. Even the shortest deletion of 19 amino acids (
NT1) was
sufficient to eliminate RGS inhibitory activity (Fig. 3A).
Wild type, mutant, and truncated forms of RGS2 were all expressed to
similar levels as detected by immunoblotting using anti-HA antibodies.
2-adrenergic receptor (
2AR).
Wild type and mutant (N149A) RGS2 inhibited isoproterenol-stimulated
cAMP accumulation to a similar extent (Fig. 3B), whereas
N-terminal truncations of RGS2 displayed no significant inhibition.
NT1 Bind to the C1
Domain of AC--
Using binding assays as described in Fig.
2A, we assayed whether mutant and truncated RGS2 proteins
were deficient in binding the C1 domain from type V AC.
Cell extracts from HEK293 cells expressing HA-tagged proteins were
incubated with His-tagged C1 domain. Strong interactions
were observed with the C1 domain for both wild type and
mutant RGS2-N149A but not the
NT1 truncated protein (Fig.
4). Incubation with the C1
protein is sufficient to completely clear the cytosol of wild type and
N149A-RGS2 protein, whereas almost all of the
NT1 truncated protein
remains. Hence, deletion of the N-terminal 19 amino acids is sufficient
to eliminate detectable interactions between RGS2 and AC.
View larger version (47K):
[in a new window]
Fig. 4.
Binding of wild type, mutant (N149A), and
N-terminal truncated ( 1-19) RGS2 with the
C1 domain of type V AC. HA-tagged wild type
(Wt), mutant (N149A), and truncated RGS2
(NT1,
1-19) were obtained from transfected HEK293 cells.
Equal amounts of protein were loaded in the reactions
(LOAD). His-tagged C1 domain of type V AC was
incubated with extracts containing HA-tagged wild type, mutant and
truncated RGS2 as described in the legend to Fig. 2A.
Unbound (SUPERNATANT) and the eluted bound proteins
(VC1-H6 Elution) were run on SDS-PAGE and
blotted with HA-antibody. The samples represent 100% of the load,
supernatant, and the eluted bound proteins present in the
reactions.
helices 3 and 6 of the RGS fold (41). To
determine the precise regions of RGS2 critical for inhibition of type V
AC, we created two additional truncations of RGS2. RGS2-
box is
devoid of the RGS core domain and contains only the N-terminal 71 amino
acids. RGS2-
C lacks the 12-amino acid overhang at the C terminus.
Type V AC and G
s-Q227L were co-expressed in the presence
or absence of constructs directing the expression of full-length RGS2
and the
box and
C truncations in HEK293 cells (Fig.
5A). Both truncations
inhibited cAMP production to the same extent as wild type RGS2,
indicating that the N terminus is sufficient for RGS2-mediated
inhibition with no significant contributions from the RGS box or C
terminus. Similar results were obtained by co-transfection with the
2AR and stimulation with isoproterenol (Fig.
5B).
View larger version (30K):
[in a new window]
Fig. 5.
Inhibition of cAMP accumulation by deletions
of the box domain and C terminus of RGS2 with activation by either
G s-Q227L or
2AR. Production of cAMP was
measured in HEK293 cells transfected as indicated with type V AC, wild
type (Wt), mutant (N149A), and deletions of box
(
72-211) and C-terminal (
199-211) domains of RGS2 constructs in
the presence of G
s-Q227L (A) or
2AR (B) as described in the legend to Fig. 3
and under "Experimental Procedures." The protein expression levels
were determined by immunoblotting using anti-HA antibody. Because of
the small size of RGS2-
box (
72-211), it was run on a 20% gel
and is shown separately. The data are expressed as the means ± S.E. from a single experiment (n = 2) and are
representative of three different experiments each performed in
duplicate.
s-Q227L, in the presence of full-length wild type RGS2
or mutant RGS2 (MFL, VQH, DCR, and PMD). With the exception of VQH, the
RGS2 mutants displayed similar inhibition of cAMP accumulation as wild
type RGS2 (Fig. 6B). The mutation VQH displayed no
inhibition of cAMP production by type V AC, although it expressed as
well as if not better than any of the other mutant proteins. In
addition to blocking inhibition of cAMP production by RGS2, mutation of
the residues VQH to alanine also prevented binding to the
C1 domain of type V AC (Fig. 6C). The N-terminal
19-amino acid stretch of RGS2 displays no homology with RGS3 or RGS13,
which have been reported to impair Gs signaling (36, 37),
nor with any other mammalian protein sequence in the
GenBankTM data base.
View larger version (34K):
[in a new window]
Fig. 6.
Alanine scanning mutations of the N-terminal
19 amino acids of RGS2: cAMP accumulation, localization, and binding
assays. A, a series of mutations were constructed
within the N-terminal 19 amino acids of RGS2. Groups of three amino
acids were mutated to alanine as shown. B, cAMP production
was measured in HEK293 cells transfected as indicated with type V AC,
G s-Q227L, and wild type (Wt) and mutant RGS2
constructs (RGS2-MFL, RGS2-VQH, RGS2-DCR, and RGS2-PMD). The protein
expression levels were determined by immunoblotting using anti-HA
antibody. The data are expressed as the means ± S.E. from two
different experiments each performed in duplicate. C,
HA-tagged wild type and two mutants of RGS2 (N149A and VQH) were
obtained from transfected HEK293 cells. Equal amounts of each protein
was loaded in the reactions (LOAD). His-tagged
C1 domain of type V AC was incubated with all three protein
extracts at 4 °C for 30 min. Unbound proteins
(SUPERNATANT) and the bound eluted proteins
(VC1-H6 Elution) were run on SDS-PAGE and
blotted with HA-antibody. The load and supernatant represent 60% of
the protein present in the reaction, whereas 100% of the
VC1-H6 eluted protein is shown. D, the
subcellular localization of wild type, mutant (RGS2-VQH), and truncated
(
NT1) RGS2 was determined as described under "Experimental
Procedures." Equal amounts of protein from whole cell lysate
(LOAD), cytosol, and membrane fractions were loaded on
SDS-PAGE and detected by immunoblotting.
NT1 (1-19) RGS2, and
RGS2-VQH--
Localization can be a key determinant of RGS2 function
(10). One trivial explanation for the loss of inhibition by RGS2-
NT1 and the mutation of residues VQH would be a possible mislocalization of
the truncated or mutated protein to regions away from the plasma membrane. Earlier reports identified amino acid residues 33-67 as an
amphipathic
-helical domain that was sufficient for plasma membrane
targeting (10). To rule out possible additional effects from the
N-terminal 19 amino acids, we fractionated HEK293 cells expressing wild
type,
NT1 truncated RGS2, and the mutant RGS2-VQH. All three forms
of RGS2 were present in the plasma membrane and in the cytosol of
HEK293 cells (Fig. 6D). Therefore, it is unlikely that gross
mislocalization of the truncated or mutant RGS2 proteins results in a
loss of activity.
i-binding Site on
Type V AC and Is Independent of Inhibition by
G
i--
Because RGS2 bound to the C1 domain
of type V AC, we tested whether RGS2 may utilize the G
i
inhibitory binding site. We made use of two mutations in type V AC,
ACV-E411A and ACV-L472A, that showed a 60-fold reduction in the
IC50 for G
i-mediated inhibition of type V AC
(21). The G
i-binding site was defined as a cleft formed
by the
2 and
3 helices of the C1 domain. The amino
acids Glu411 and Leu472 reside on the
2 and
3 helices, respectively. Mutation of these residues had no effect on
the activation of type V AC by G
s, the synergy with
forskolin, or the Km for ATP. Although these mutant
proteins were extensively characterized in vitro, they had
not been tested in vivo. Therefore, we transfected
constructs expressing wild type or the mutant type V AC with
G
s-Q227L and tested for their ability to be inhibited by
the muscarinic agonist, carbachol (Fig.
7A). As expected, the wild
type AC was inhibited upon the addition of carbachol, but neither
mutant of type V AC was affected. Hence, these proteins are deficient
in G
i inhibition both in vitro and in
vivo.
View larger version (30K):
[in a new window]
Fig. 7.
Mutations in the
G i-binding site of type V AC
prevent inhibition by carbachol but have no effect on inhibition by
RGS2. A, production of cAMP was measured in HEK293
cells transfected as indicated with G
s-Q227L and wild
type and mutant type V AC (E411A and L472A) constructs in the presence
or absence of carbachol (1 µM) for 15 min. B,
cAMP production was measured in HEK293 cells transfected with
constructs directing the expression of wild type and mutant type V AC
(E411A and L472A) and G
s-Q227L in the presence and
absence of wild type RGS2 (1 µg/well). The protein expression levels
were determined by immunoblotting using anti-HA antibody (data not
shown). The data are expressed as the means ± S.E. from three
different experiments each performed in duplicate. C, cAMP
production was measured in HEK293 cells transfected with constructs as
described for B, except that a variable amount of RGS2 DNA
was used (25 ng, 50 ng, 1 µg, and 2 µg). The data are shown as
percentages of the fold activation in the absence of RGS2 for each type
V AC construct, where the fold activation is simply the level of cAMP
of type V AC plus G
s-Q227L as compared with
G
s-Q227L alone. The data are expressed as the means ± S.E. from a single experiment (n = 2) and are
representative of two different experiments each performed in
duplicate. D, wild type (
, 60 nM), L472A
(
, 80 nM), and E411A (
, 225 nM) mutant
VC1(670) was reconstituted with 0.5 µM
VC2 and assayed with 400 nM activated
G
s in the presence of the indicated concentrations of
purified RGS2. The activities are expressed as percentages of control
values (1630, 1950, and 240 nmol/min mg for wild type and L472A and
E411A mutant VC1(670), respectively) and are representative
of two different experiments performed in duplicate. wt,
wild type.
s-Q227L and the constructs directing the expression of wild type or mutant type VAC, in the presence or absence of RGS2. Both
mutant proteins were inhibited by RGS2 expression to a similar extent
as wild type AC (Fig. 7B). However, these experiments were performed with maximal levels of RGS2. To determine whether the mutations at the G
i-binding site had any effects on the
IC50 for RGS2-mediated inhibition, increasing
concentrations of DNA-encoding RGS2 were used, resulting in a
~100-fold range of RGS2 expression as analyzed by Western blotting
(data not shown). In this case, the fold activation of type V AC by
G
s-Q227L is shown relative to G
s-Q227L
alone. Once again, the degree of inhibition by RGS2 is very similar for
wild type and L472A and E411A mutants of type V AC across a large range
of cellular RGS2 concentrations (Fig. 7C).
s-stimulated AC activity ~70% with very similar
IC50 values for the wild type and mutant C1
domains (0.4-1.0 µM). Therefore, the Glu411
and Leu472 residues, critical for G
i
inhibition, are not required for inhibition by RGS2. Thus, RGS2 most
likely utilizes a binding site on the C1 domain of type V
AC that is distinct from the G
i site of interaction. Although RGS2 is generally a poor GAP for G
i, it is
possible that AC may promote such interactions leading to either
reduced inhibition by G
i or possibly some type of
synergy between the two regulators. Under conditions where type V AC
was not fully inhibited by RGS2, no synergistic effects were observed
with both carbachol and RGS2 (data not shown). Hence, inhibition by
RGS2 appears to be independent of G
i regulation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kathy Graves for technical assistance and Dr. Joe Alcorn for his help with recombinant adenoviruses.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institute of Health Grant GM60419 (to C. W. D.) and funds from the National American Heart Association (to C. W. 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 should be addressed: Dept. of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, 6431 Fannin St., Houston, TX 77030. E-mail: Carmen.W.Dessauer@uth.tmc.edu.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M210663200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AC, adenylyl
cyclase;
2AR,
2-adrenergic receptor;
GTP
S, guanosine 5'-O-(2-thio)triphosphate;
HA, hemagglutinin;
Ni-NTA, nickel-nitrilotriacetic acid;
GST, glutathione S-transferase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sunahara, R. K., Dessauer, C. W., and Gilman, A. G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 461-480[CrossRef][Medline] [Order article via Infotrieve] |
2. | Smit, M. J., and Iyengar, R. (1998) Adv. Second Messenger Phosphoprotein Res. 32, 1-21[Medline] [Order article via Infotrieve] |
3. | Sinnarajah, S., Dessauer, C. W., Srikumar, D., Chen, J., Yuen, J., Yilma, S., Dennis, J. C., Morrison, E. E., Vodyanoy, V., and Kehrl, J. H. (2001) Nature 409, 1051-1055[CrossRef][Medline] [Order article via Infotrieve] |
4. | Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452[Medline] [Order article via Infotrieve] |
5. | Watson, N., Linder, M. E., Druey, K. M., Kehrl, J. H., and Blumer, K. J. (1996) Nature 383, 172-175[CrossRef][Medline] [Order article via Infotrieve] |
6. | Ingi, T., Krumins, A. M., Chidiac, P., Brothers, G. M., Chung, S., Snow, B. E., Barnes, C. A., Lanahan, A. A., Siderovski, D. P., Ross, E. M., Gilman, A. G., and Worley, P. F. (1998) J. Neurosci. 18, 7178-7188[Abstract] |
7. | Carr, S. A., Huddleston, M. J., and Annan, R. S. (1996) Anal. Biochem. 239, 180-192[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Hollinger, S.,
and Hepler, J. R.
(2002)
Pharmacol. Rev.
54,
527-559 |
9. |
Cunningham, M. L.,
Waldo, G. L.,
Hollinger, S.,
Hepler, J. R.,
and Harden, T. K.
(2001)
J. Biol. Chem.
276,
5438-5444 |
10. |
Heximer, S. P.,
Lim, H.,
Bernard, J. L.,
and Blumer, K. J.
(2001)
J. Biol. Chem.
276,
14195-14203 |
11. |
Druey, K. M.,
Sullivan, B. M.,
Brown, D.,
Fischer, E. R.,
Watson, N.,
Blumer, K. J.,
Gerfen, C. R.,
Scheschonka, A.,
and Kehrl, J. H.
(1998)
J. Biol. Chem.
273,
18405-18410 |
12. |
Dulin, N. O.,
Sorokin, A.,
Reed, E.,
Elliott, S.,
Kehrl, J. H.,
and Dunn, M. J.
(1999)
Mol. Cell. Biol.
19,
714-723 |
13. |
Bowman, E. P.,
Campbell, J. J.,
Druey, K. M.,
Scheschonka, A.,
Kehrl, J. H.,
and Butcher, E. C.
(1998)
J. Biol. Chem.
273,
28040-28048 |
14. |
Dulin, N. O.,
Pratt, P.,
Tiruppathi, C.,
Niu, J.,
Voyno-Yasenetskaya, T.,
and Dunn, M. J.
(2000)
J. Biol. Chem.
275,
21317-21323 |
15. | Zmijewski, J. W., Song, L., Harkins, L., Cobbs, C. S., and Jope, R. S. (2001) Biochim. Biophys. Acta 1541, 201-211[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Sullivan, B. M.,
Harrison-Lavoie, K. J.,
Marshansky, V.,
Lin, H. Y.,
Kehrl, J. H.,
Ausiello, D. A.,
Brown, D.,
and Druey, K. M.
(2000)
Mol. Biol. Cell
11,
3155-3168 |
17. |
Oliveira-Dos-Santos, A. J.,
Matsumoto, G.,
Snow, B. E.,
Bai, D.,
Houston, F. P.,
Whishaw, I. Q.,
Mariathasan, S.,
Sasaki, T.,
Wakeham, A.,
Ohashi, P. S.,
Roder, J. C.,
Barnes, C. A.,
Siderovski, D. P.,
and Penninger, J. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12272-12277 |
18. | Druey, K. M., Blumer, K. J., Kang, V. H., and Kehrl, J. H. (1996) Nature 379, 742-746[CrossRef][Medline] [Order article via Infotrieve] |
19. | Kehrl, J. H. (1998) Immunity 8, 1-10[Medline] [Order article via Infotrieve] |
20. |
Taussig, R.,
Quarmby, L. M.,
and Gilman, A. G.
(1993)
J. Biol. Chem.
268,
9-12 |
21. |
Dessauer, C. W.,
Tesmer, J. J. G.,
Sprang, S. R.,
and Gilman, A. G.
(1998)
J. Biol. Chem.
273,
25831-25839 |
22. |
Sunahara, R. K.,
Dessauer, C. W.,
Whisnant, R. E.,
Kleuss, C.,
and Gilman, A. G.
(1997)
J. Biol. Chem.
272,
22265-22271 |
23. |
Wang, C. R.,
Esser, L.,
Smagula, C. S.,
Sudhof, T. C.,
and Deisenhofer, J.
(1997)
Protein Sci.
6,
2264-2267 |
24. |
Hepler, J. R.,
Berman, D. M.,
Gilman, A. G.,
and Kozasa, T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
428-432 |
25. |
Dessauer, C. W.,
and Gilman, A. G.
(1996)
J. Biol. Chem.
271,
16967-16974 |
26. | Lee, E., Linder, M. E., and Gilman, A. G. (1994) Methods Enzymol. 237, 146-164[Medline] [Order article via Infotrieve] |
27. |
Linder, M. E.,
Ewald, D. A.,
Miller, R. J.,
and Gilman, A. G.
(1990)
J. Biol. Chem.
265,
8243-8251 |
28. | McGrory, W. J., Bautista, D. S., and Graham, F. L. (1988) Virology 163, 614-617[Medline] [Order article via Infotrieve] |
29. | Graham, F. L., and Prevec, L. (1991) in Methods in Molecular Biology (Murray, E. J., ed), Vol. 7 , pp. 109-128, Humana Press Inc., Clifton, NJ |
30. | Dessauer, C. W. (2002) Methods Enzymol. 345, 112-126[Medline] [Order article via Infotrieve] |
31. |
Yan, S. Z.,
Hahn, D.,
Huang, Z. H.,
and Tang, W.-J.
(1996)
J. Biol. Chem.
271,
10941-10945 |
32. |
Whisnant, R. E.,
Gilman, A. G.,
and Dessauer, C. W.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6621-6625 |
33. |
Dessauer, C. W.,
and Gilman, A. G.
(1997)
J. Biol. Chem.
272,
27787-27795 |
34. |
Scheschonka, A.,
Dessauer, C. W.,
Sinnarajah, S.,
Chidiac, P.,
Shi, C. S.,
and Kehrl, J. H.
(2000)
Mol. Pharmacol.
58,
719-728 |
35. |
Huang, C.,
Hepler, J. R.,
Gilman, A. G.,
and Mumby, S. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6159-6163 |
36. |
Tseng, C. C.,
and Zhang, X. Y.
(1998)
Endocrinology
139,
4470-4475 |
37. |
Zheng, B.,
Ma, Y. C.,
Ostrom, R. S.,
Lavoie, C.,
Gill, G. N.,
Insel, P. A.,
Huang, X. Y.,
and Farquhar, M. G.
(2001)
Science
294,
1939-1942 |
38. |
Chatterjee, T. K.,
Eapen, A. K.,
and Fisher, R. A.
(1997)
J. Biol. Chem.
272,
15481-15487 |
39. |
Srinivasa, S. P.,
Watson, N.,
Overton, M. C.,
and Blumer, K. J.
(1998)
J. Biol. Chem.
273,
1529-1533 |
40. | Posner, B. A., Mukhopadhyay, S., Tesmer, J. J., Gilman, A. G., and Ross, E. M. (1999) Biochemistry 38, 7773-7779[CrossRef][Medline] [Order article via Infotrieve] |
41. | Tesmer, J. J. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261[Medline] [Order article via Infotrieve] |
42. |
Zeng, W.,
Xin, X.,
Popov, S.,
Mukhopadhyay, S.,
Chidiac, P.,
Swistok, J.,
Danho, W.,
Yagaloff, K. A.,
Fisher, S. L.,
Ross, E. M.,
Muallem, S.,
and Wilkie, T. M.
(1999)
J. Biol. Chem.
273,
34687-34690 |