From the Departments of Biochemistry and
¶ Pharmacology and the ** Howard Hughes Medical
Institute, the University of Texas Southwestern Medical Center,
Dallas, Texas 75390
Received for publication, December 12, 2002
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ABSTRACT |
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Structural requirements for function of
the Rho GEF (guanine nucleotide exchange
factor) regulator of G protein
signaling (rgRGS) domains of p115RhoGEF and
homologous exchange factors differ from those of the classical RGS
domains. An extensive mutagenesis analysis of the p115RhoGEF rgRGS
domain was undertaken to determine its functional interface with the
G The intrinsic guanosine triphosphatase (GTPase) activity
of the Structural (5, 7) and mutagenesis (16-19) studies have defined three
distinct regions of the RGS domain which interact with G The crystal structures of the N-terminally truncated rgRGS domain of
p115RhoGEF (26) and that of its homolog, PDZRhoGEF (27), were recently
determined. These domains are comprised of 11 Here, we describe an extensive mutagenic analysis performed to define
the residues in the rgRGS domain which interact with G Expression Plasmids--
Complementary DNA oligomers encoding
single or double amino acid changes from the wild type p115RhoGEF gene
were used to generate point mutants by PCR. The mutagenesis template
consisted of amino acids 1-252 of human p115RhoGEF (rgRGS domain)
subcloned into the pGEX-KG vector. The sequences of primers used are
available upon request. The QuikChange KitTM from Stratagene was used
in a four-step procedure to generate mutant cDNA. Plasmids encoding the desired mutations were selected and verified by sequencing. Selected mutated rgRGS DNAs were also subcloned into the full-length p115RhoGEF gene in the pFastbac1 vector (Invitrogen), which has been
modified to provide an N-terminal EE tag (EYMPME) (8) for production of
baculovirus and subsequent expression in Spodoptera frugiperda (Sf9) cells.
Expression and Purification of Proteins--
All p115RhoGEF
proteins were expressed in transformed BL21 (DE3) or DH5
Full-length p115RhoGEFs containing mutated rgRGS domains were expressed
in Sf9 cells during 48 h of infection with recombinant baculoviruses. The expressed proteins were purifed from lysates by
chromatography with immobilized antibodies to the EE tag (BabCO) as
described elsewhere (8).
G GAP Assays for G G Protein Binding Assays--
The purified GST-tagged rgRGS
domains were bound to 20 µl of glutathione-Sepharose 4B (packed
beads) by gentle mixing in 200 µl of solution C (20 mM
NaHepes, pH 8.0, 1 mM EDTA, 1 mM
dithiothreitol, and 100 mM NaCl) for 30 min at 4 °C.
During that time, G Mutations of the rgRGS Helical Domain That Affect GAP Activity
toward G
Initial experiments were conducted with constructs encompassing only
the p115rgRGS domain (residues 1-252). 21 mutated p115rgRGS domains
were expressed in E. coli. All of the mutants were expressed at a high level (>1 mg/liter) and as soluble proteins (data not shown)
as would be expected for properly folded protein domains. The GAP
activities of the recombinant p115rgRGS mutants were measured by the
single turnover GTPase assay for G
A summary of the results with the 21 mutant domains tested under the
conditions described above is shown in Fig. 2C. Several mutations, including R111A, E155K, E212A, E213A, K214A, and K214E, had
no effect on GAP activity. In contrast, three mutations, Q69A, F70A,
and D156A, appear largely to abolish GAP activity, whereas other
mutations had more modest effects (apparent decreases in GAP activity
of 20-60% of the wild type domain). GAP activities of mutants
designed to target specific G
The L3 loop of the rgRGS domain is a likely site for interaction with
G
Structural and mutagenic studies of RGS proteins indicate that the
residue corresponding to Asn-128 in RGS4 is important for GAP activity.
A proline residue occupies the analogous position (residue 113 in
p115RhoGEF) in the rgRGS domains with strong GAP activity but is
replaced by lysine in GTRAP48 (Fig. 1B). Mutation of Pro-113
in L5 to either an alanine or a lysine reduces GAP activity (Fig.
2C), but the effect is not as severe as that caused by
mutations in the L3 or
Residues outside the
To elucidate the extent to which certain mutations affect GAP activity
of the rgRGS domain, we measured the quantity of GTP hydrolyzed by
G A Map of the Contact Surface of the rgRGS Core Domain for
Interaction with G Mutations of Residues N-terminal to the RGS Box in the rgRGS
Domain--
Residues 15-41 of p115rgRGS are required for GAP activity
toward G Mutations of Residues Required for rgRGS GAP Activity Also Reduce
Affinity for G Activities of p115RhoGEF Containing Point Mutations in the rgRGS
Domain--
Several of the single residue mutations characterized for
the rgRGS domain were inserted into the holo-p115RhoGEF protein to
determine their effect in this context. As shown in Fig.
6A, these mutations are as
debilitating to the GAP activity of the holoprotein as they are to that
of the rgRGS domain alone. Thus, mutations F31A and E27K caused almost
total loss of GAP activity, whereas three other mutations (D156A, D28A,
and E29K) showed impaired potency but substantial efficacy at higher
concentrations. As with the rgRGS domain, these mutations also reduced
the affinity of the holoprotein for activated G
In contrast to the impairment of binding and GAP activities, the point
mutations described above did not affect the ability of activated
G The rgRGS domains form a unique and distinct subgroup of the RGS
family. Although the rgRGS domains are, in three-dimensional structure,
apparent homologs of the RGS domains, their sequences bear little
similarity to the RGS consensus sequence (3). The mutagenesis
experiments that we have conducted are consistent with the hypothesis
that the G The segment extending from L3 to L5 is the most highly conserved
structural feature common to the rgRGS and RGS folds. Less well
conserved is the segment corresponding to Mutation of Asn-128 in the L5 loop of RGS4 (16) can result in a modest
reduction in GAP activity if replaced by serine (the cognate residue in
GAIP) but severe losses (>99.9% reductions) if replaced by bulkier
residues (18). Yet, mutation of the structurally equivalent residue in
p115RhoGEF, Pro-113, to either alanine or lysine, decreases GAP
activity only 50% at a 10:1 molar ratio of rgRGS to
G The N-terminal 41 residues of p115RhoGEF include a hydrophobic and
proline-rich sequence (residues 11-26) followed by a 15-residue segment (amino acids 27-41) containing 9 acidic residues (Fig. 8A). Although the first 12 residues of p115RhoGEF were shown to be dispensable for GAP activity,
deletion of the first 41 residues abolished this function (15). Here,
we show that the electronegative cluster encompassed by residues 27-30
is crucial to GAP function. Indeed, substitution of any of the first 3 acidic residues in the cluster caused a greater than 99% loss of GAP
potency and efficacy. Mutation of the aromatic residue, Phe-31, which
is adjacent to the acidic sequence, is equally deleterious. The only
mutation within the RGS domain of rgRGS which causes an equivalent
degree of impairment is that of Asp-156 in 13 subunit. Results indicate that there is global
resemblance between the interaction surface of the rgRGS domain with
G
13 and the interactions of RGS4 and RGS9 with their
G
substrates. However, there are distinct differences in the
distribution of functionally critical residues between these
structurally similar surfaces and an additional essential requirement
for a cluster of negatively charged residues at the N terminus of
rgRGS. Lack of sequence conservation within the N terminus may also
explain the lack of GTPase-activating protein (GAP) activity in a
subset of the rgRGS domains. For all mutations, loss of functional GAP
activity is paralleled by decreases in binding to G
13.
The same mutations, when placed in the context of the p115RhoGEF
molecule, produce deficiencies in GAP activity as observed with the
rgRGS domain alone but show no attenuation of the regulation of Rho
exchange activity by G
13. This suggests that the rgRGS
domain may serve a structural or allosteric role in the regulation of
the nucleotide exchange activity of p115RhoGEF on Rho by
G
13.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunits of certain heterotrimeric guanine
nucleotide-binding proteins (G proteins) can be stimulated by members
of the regulators of G protein signaling
(RGS)1 family (1-3). The
catalytic activity of these GTPase-activating proteins (GAPs) resides
in an ~120-residue
-helical domain that corresponds to a consensus
"RGS box" defined by amino acid sequence similarity. RGS box
sequences show considerable diversity and can be subdivided into
distinct homologous clusters (4), but all share conserved features
recognizable in the three-dimensional structures of representative
family members: RGS4 (5), GAIP (6) and RGS9 (7). p115RhoGEF, a guanine
nucleotide exchange factor (GEF) for the monomeric G protein RhoA (8,
9), is the prototype of a family that includes LARG (10), Lsc (11), PDZRhoGEF (12), and GTRAP48 (13). These proteins contain regions in
their N-terminal halves with low sequence identity to RGS domains (14).
In p115RhoGEF, this region extends from residue 42 to 163. Recombinant
p115RhoGEF and fusion proteins containing the first 246 (14) or 252 (15) N-terminal residues of p115RhoGEF had specific GAP activity toward
the
subunits of the heterotrimeric G proteins, G12 and
G13, but not toward members of the Gs, Gi, or Gq subfamily
of G
proteins. In contrast to other RGS proteins, p115RhoGEF
requires elements outside of the RGS box to function as a GAP. A
construct that extends about 75 residues beyond the C terminus of the
RGS box is the smallest fragment of the holoprotein which could be
overexpressed as a soluble protein in Escherichia coli,
although as little as 50 additional C-terminal residues allowed
expression of a functional domain in eukaryotic cells. In addition to
an extended C terminus, 25 residues that precede the RGS box were also
required for full GAP activity (15). We refer to the N- and
C-terminally extended RGS box segment of p115RhoGEF and its homologs as
the rgRGS (RhoGEF RGS) domain. Two members of
the p115RhoGEF family, GTRAP48 (13, 15) and
PDZRhoGEF,2 bind to
G
13 but have little or no GAP activity; these form a distinct sequence subset with respect to both the RGS box domain and
the N-terminal segment.
and convey
GAP activity. These correspond to two surface polypeptide turns that
join helical segments, together with the surface of the C-terminal
helix of the RGS domain. These RGS elements directly contact the
catalytic site of G
, principally, the switch I and switch II
segments that undergo conformational rearrangement upon hydrolysis of
GTP (20). Together, these two segments in G
subunits contain
residues that participate directly in the catalytic mechanism of GTP
hydrolysis or bind the magnesium ion cofactor (21, 22). The complex
formed by GDP, Mg2+, and AlF
(23), also mimics the
pentacoordinate transition state for GTP hydrolysis (21, 22). RGS
proteins have been shown to bind more strongly to the
GDP·Mg2+·AlF
subunits than to those formed with GTP analogs (24). This, together
with structural evidence, indicates that RGS proteins accelerate GTP
hydrolysis by stabilizing a transition state-like conformation of G
or destabilize the GTP-bound ground state (25).
helices; the
N-terminal 7 helices form a bilobal fold similar to that in classic RGS
domains. The remaining 4 helices pack against the latter to generate a
single globular domain. The core of the rgRGS domain from p115RhoGEF,
which encompasses two of the three segments involved in G
binding,
shows high structural similarity to the corresponding segments in RGS
proteins of known structure. Accordingly, mutation of two p115RhoGEF
residues that correspond, in RGS4, to side chains that interact with
switch I and switch II, were found to diminish GAP activity (26). These observations led to the inference that the general features of the
protein-protein interface observed in the published structures of
RGS·G
complexes are likely be preserved in the interaction of
rgRGS with G
13. Although the N terminus of p115RhoGEF is
not present in the crystal structure, it is required for GAP activity (15), and structural modeling of the rgRGS·G
13 complex
suggests that N-terminal residues of the rgRGS domain might interact
with the helical domain and switch regions of G
13
(26).
13
and convey GAP activity. These studies focused on both the globular
helical domain of the rgRGS and the N-terminal residues that precede
the RGS box. Results indicate that there is global resemblance between
the interaction surface of the rgRGS domain with G
13 and
the interactions of RGS4 and RGS9 for their G
substrates. However,
there are distinct differences in the distribution of functionally
critical residues between these structurally similar surfaces and an
additional essential requirement for a cluster of negatively charged
residues at the N terminus of rgRGS. Placement of the same mutations in
the context of the p115RhoGEF molecule produces the same deficiencies
in GAP activity as observed with the rgRGS domain alone but shows no
attenuation of the regulation of Rho exchange activity by
G
13.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strains
of E. coli or in cultured Sf9 cells infected with
recombinant baculoviruses. The rgRGS domains were produced in E. coli as fusion proteins with glutathione S-transferase
(GST). Cells were grown in LB medium at 37 °C to
A600 ~0.6 and induced for 3 h at
30 °C with 0.2 mM isopropyl-1-thiogalactopyranoside for
expression of mutant proteins. Bacteria were then pelleted by
centrifugation and lysed for 30 min in solution A (25 mM
NaHepes, pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol, 50 mM NaCl, and protease inhibitors (2.5 µg/ml leupeptin, 1 µg/ml pepstatin A, 21 µg/ml phenylmethylsulfonyl fluoride, 21 µg/ml
Na-p-tosyl-L-lysine
chloromethyl ketone, 21 µg/ml tosylphenylalanyl ketone, and 21 µg/ml
Na-p-tosyl-L-arginine
methyl ester)) containing 2 mg/ml lysozyme, 1 mg/ml DNase I, and 5 mM MgCl2. GST-tagged rgRGS proteins were purified by chromatography through glutathione-Sepharose 4B (Amersham Biosciences) in solution A and elution with solution A containing 15 mM reduced glutathione.
13 was expressed in Sf9 cells and purified as
described previously (28). RhoA was expressed in Sf9 cells with
an N-terminal hexahistidine (His6) tag and purified from
lysates by isolation with Ni2+-nitriloacetic acid resin
(Qiagen) in 25 mM NaHepes, pH 7.5, 2.5 mM
-mercaptoethanol, 50 mM NaCl, and protease inhibitors.
Elution was facilitated by a step gradient of increasing imidazole
concentration, with His6-RhoA being released at ~75
mM.
13--
2 µM
G
13 was loaded with 5 µM
[
-32P]GTP (50-100 cpm/fmol) for 15 min at 30 °C in
solution B (20 mM NaHepes, pH 8.0, 5 mM EDTA, 1 mM dithiothreitol, and 0.05% polyoxyethylene 10 lauryl
ether (Lubrol)). Free [
-32P]GTP and
32Pi were removed rapidly by centrifugation of
the samples at 4 °C through Sephadex G-50 that had been equilibrated
with solution B. Hydrolysis of GTP was initiated by adding
G
13 loaded with [
-32P]GTP (final
concentration of 1-5 nM) to solution B containing 10 mM MgSO4, 1 mM GTP, and 1-100
nM p115RhoGEF rgRGS domain (wild type or mutant) in a
50-µl reaction volume. After incubation for the indicated times at
4 °C, reactions were quenched with 750 µl of 5% (w/v) NoritA in
50 mM NaH2PO4. The mixtures were
then centrifuged at 3,000 rpm for 5 min, and 400 µl of supernatant containing 32Pi was counted by liquid
scintillation spectrometry.
13 was diluted to 2 µM
in solution D (20 mM NaHepes, pH 8.0, 1 mM
EDTA, 1 mM dithiothreitol, 0.1 mM GDP, and
0.05% Lubrol) and then gel filtered rapidly by centrifugation through
Sephadex G-50 that had been equilibrated with the same solution. This
removes residual activating ligands in the preparation. Samples of
glutathione-Sepharose 4B containing bound GST-tagged rgRGS proteins
were then pelleted in a microfuge for 30 s and washed with 200 µl of solution D to remove unbound protein. 200-µl binding
reactions were prepared by mixing the immobilized rgRGS proteins and
gel-filtered G
13 in solution D either with or without
AMF (30 µM AlCl3, 5 mM
MgCl2, and 5 mM NaF). Samples were mixed gently
for 30 min at 4 °C, and unbound G
13 was then removed
by two 200-µl washes of solution D either with or without AMF. The
washed beads were finally resuspended with 40 µl of solution D and
then boiled in SDS sample buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
13--
Structural modeling of the
rgRGS·G
13 complex predicted that residues of loops L3,
L5, helix
8, and loop L11 would interact with the switch regions of
G
13 (Fig. 1A)
(26). To test this hypothesis, we mutated residues located within this
predicted interaction surface of the p115RhoGEF rgRGS domain and
measured the GAP activity of the mutant domains toward
G
13. Unless stated otherwise, "p115rgRGS" refers to
the protein fragment encompassing residues 1-252 of p115RhoGEF.
Mutations are identified by the one-letter code for the residue in wild
type p115rgRGS, followed by the position of the residue in the amino
acid sequence and the one-letter code for the residue to which it was
mutated (e.g. E71A has glutamic acid in position 71 mutated
to an alanine). Residues chosen for mutagenesis (Fig. 1B)
fall into three categories. The first group includes residues predicted
to form direct and specific contacts with the switch regions of
G
13, according to structural modeling studies of the
rgRGS·G
13 complex (Glu-71, Arg-111, Pro-113, Pro-115,
Pro-116, Glu-155, and Lys-214). The second group of residues in the
predicted interface region includes those that are charged, have
solvent accessible surface areas greater than 25 Å2, and
are conserved in the rgRGS domains but not in the classical RGS
proteins (Gln-69, Asp-156, Lys-160, Arg-161, Glu-212, and Glu-213). A
third group of residues includes surface residues that are predicted to
project toward, but not necessarily contact, switch regions of
G
13 (Phe-70, Arg-152, and Ser-159). In most instances,
residues were mutated to alanine or residues with opposite charge.
Three of the mutations, F70A, P113K, and R152E, were also designed to
reflect the differences in sequence between p115RhoGEF and two
rgRGS-containing proteins with little or no GAP activity, GTRAP48 and
PDZRhoGEF. None of the residues chosen for mutagenesis is involved in
extensive packing interactions. Hence, alterations of these residues
are therefore not expected to disrupt the tertiary structure of
p115rgRGS.
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Fig. 1.
Tertiary and primary structure of
p115rgRGS. A, the tertiary structure of
p115rgRGS (26) is depicted using the coloring scheme published for RGS4
(5); the N-terminal 42 residues and C-terminal 19 residues of the rgRGS
domain are not present in the structure. Helices that comprise
structural elements of the RGS box are colored orange,
green, and blue. L3, L5, and helix 8 in RGS4
and RGS9 form contacts with G
i1 and G
t,
respectively (5, 7) (see "Results"). The C-terminal helical
bundle (colored red) is unique to rgRGS domains.
B, the amino acid sequences of rgRGS domains were aligned
with two members of the RGS family for which crystal structures have
been determined. Sequence alignments performed using ClustalW (31) were
modified on the basis of structural superposition (26). Colored
bars set above the sequences represent helices with color codes
that match the ribbon diagram shown in A. Gray
bars represent helices in the structure of RGS4 (5). Residues in
RGS4 or RGS9 (7) which are involved in contacts (closer than 4.0 Å)
with G
i1 or G
t, respectively, are colored
in red. Residues in rgRGS proteins which are conserved and
have solvent-accessible surface areas greater than 25 Å2
in the structure of p115rgRGS are colored in purple, and
those conserved with solvent-accessible surface areas less than 25 Å2 are colored in blue. Residues marked with
asterisks were targeted for site-directed mutagenesis.
13 (see "Materials
and Methods"). Examples of these assays, which assessed the effect of
10 nM mutant p115rgRGS protein on the time dependence at
4 °C of hydrolysis of GTP bound to G
13, are shown in
Fig. 2, A and B. At
this concentration and conditions, the wild type rgRGS domain
stimulated the GTPase activity of G
13 by ~5-10-fold
and caused almost complete hydrolysis of the bound GTP by 2 min. The total amount of GTP hydrolyzed differed from assay to assay, largely because of variations in GTP loading caused by weak nucleotide affinity
and the high intrinsic GTPase rate of G
13 (29); thus, the final concentration of [
-32P]GTP-loaded
G
13 used in the assays ranged from 1 to 10 nM.
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Fig. 2.
Effect of mutations within the RGS box of
p115rgRGS on GAP activity. A, G 13 was
loaded with [
-32P]GTP (final concentration, 1-5
nM), and hydrolysis of the bound GTP was initiated by
mixing with buffer containing 10 mM MgSO4 and 1 mM GTP (basal, closed circles). Samples were
incubated for the indicated times at 4 °C and then quenched as
described under "Materials and Methods."
32Pi evolved from the reactions was counted by
liquid scintillation spectrometry. As indicated, 10 nM
p115rgRGS domains were included in the reaction: wild type p115rgRGS
(closed diamonds), D156A mutant (open circles),
K160A mutant (closed inverted triangles), R161A mutant
(open inverted triangles), E212A mutant (closed
squares), or E213A mutant (open squares). B,
the time course of GTP hydrolysis, as described in A, is
shown for basal (closed circles), wild type p115rgRGS
(open diamonds), Q69A mutant (open circles), F70A
mutant (closed inverted triangles), F70Y mutant (open
inverted triangles), R152A mutant (closed squares),
R152E mutant (open squares), or S159A mutant (closed
diamonds). C, the apparent initial rate of GTP
hydrolysis catalyzed by G
13 in the presence of each
p115rgRGS mutant (10 nM) is represented as a percentage of
the initial rate of GTP hydrolysis by G
13 in the
presence of 10 nM wild type p115rgRGS. D, the
quantity of 32Pi released in the assay as
described in A after a 2-min incubation was determined in
the presence of the indicated concentration of mutant or wild type
p115rgRGS·wild type p115rgRGS (closed circles), Q69A
mutant (open circles), F70A mutant (closed inverted
triangles), E71K mutant (open inverted triangles),
D156A mutant (closed squares), or E71K/P113K mutant
(open squares).
13 interaction sites are
consistent with the involvement of the structurally conserved L3 and L5
loops and also the less well conserved helix,
8. However, the lack
of effects by mutations in L11 from the C-terminal helical region does
not support a role for this structural element in the GAP activity of
the rgRGS domain.
13, yet its conformation differs from that of RGS4. The
group 1 residue in this loop, Glu-71, is proposed to engage in
electrostatic and van der Waals interactions with Lys-204 of G
13 and is conserved among rgRGS proteins (Fig.
1B). Accordingly, mutants in which Glu-71 was replaced by
either alanine or lysine have 50% of the GAP activity toward
G
13 compared with the wild type rgRGS domain (Fig.
2C). Nonconservative mutations of the group 2 and group 3 residues in L3, Q69A, and F70A, respectively, caused large reductions
in GAP activity. Both residues are exposed to solvent and are in close
proximity to switch I of G
13 in the model of the
rgRGS·G
13 complex, although their interaction partners cannot be predicted. Gln-69 is absolutely conserved among known rgRGS
proteins (Fig. 1B), whereas Phe-70 is conserved only in p115RhoGEF, Lsc, and LARG, but replaced by alanine in PDZRhoGEF and
GTRAP48. The potential interactive role of this residue is supported by
the retention of about 50% of GAP activity by the conservative
mutation of Phe-70 to tyrosine.
8 regions. Other mutations of group 1 residues in L5, including R111A and P115G/P116G, had little effect on
GAP activity (Fig. 2C).
3-L5 core region of the rgRGS domain align
poorly in tertiary structure with RGS4. Therefore, although
8 is a
probable interaction partner for switch I of G
13, only one residue in this helix, Glu-155, could be included in group 1. Mutation of this residue had little effect on GAP activity. In
contrast, mutation of one of the two neighboring group 2 residues, Arg-152, reduced GAP activity by 50%. Arg-152 is replaced by glutamate in PDZRhoGEF and GTRAP48, two rgRGS proteins with very weak GAP activity toward G
13. Replacing Arg-152 with glutamate in
p115rgRGS resulted in only a modest reduction in activity similar to
replacement with alanine. Three charged group 3 residues were selected
for mutagenesis in
8. Of these, substitution of Asp-156, a residue in
8 which is conserved among rgRGS proteins (Fig. 1B),
severely reduced GAP activity. However, structure-based modeling
reveals no residues in G
13 poised to interact directly
with Asp-156 in the rgRGS domain. Mutation of other charged residues in
8, Glu-155, Ser-159, and Arg-161, had little effect.
13 within a 2-min period as a function of rgRGS concentration (Fig. 2D). At a sufficiently high
concentration, all of the mutated rgRGS domains were capable of
stimulating G
13 to the extent observed for the wild type
domain. Thus, the D156A, Q69A, and F70A mutations do not substantially
alter the efficacy of rgRGS as a GAP for G
13, but
strongly reduce the potency of the domain for this function. Even the
most debilitated mutant, p115rgRGS (D156A), exhibited GAP activity that
was comparable with that of the wild type protein at concentrations
that were 100-1,000-fold greater than G
13 in the assay.
13--
In Fig.
3A, the solvent-accessible
surface of rgRGS is color-coded according to the relative loss of GAP
activity that results from mutation of the underlying residues. The
relative GAP activity of a mutant p115rgRGS domain is quantified as the
apparent initial velocity of the GTPase reaction catalyzed by
G
13 in the presence of the mutant normalized by the rate
in the presence of wild type p115rgRGS (% V0WT) (for examples, see Fig. 2,
A and B). The mutagenesis data identify a
functionally critical surface on the rgRGS domain which is centered on
three key residues from the RGS box: Gln-69 and Phe-70 from L3, and
Asp-156 from
8. The functional map of rgRGS is grossly similar to
that of RGS4 as charted by Srinivasa and colleagues (16) (Fig.
3B) but differs in the extent to which mutations at
corresponding positions affect GAP activity. For example, mutation of
Asn-128 in the L5 loop of RGS4 severely impairs GAP activity, whereas
the corresponding P113A mutation is only modestly debilitating in
rgRGS. Conversely, the Q69A and F70A substitutions generate severe
effects in rgRGS, whereas mutation of the structurally equivalent
residues in RGS4 only reduce GAP activity to ~30% of wild type. On
the other hand, Asp-156 in rgRGS and Arg-167 in RGS4 occupy equivalent
positions in
8, and both are critical for GAP activity of the
respective domains.
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Fig. 3.
Relative GAP activity of p115rgRGS and RGS4
mutants mapped on the G binding surface.
A, the solvent accessible surface of p115rgRGS (excluding
residues 1-42, which are not present in the crystal structure) was
computed using the program GRASP (32). The surface of the protein
presumed to interact with G
13 faces the viewer. Residues
that were mutated are labeled. The surface corresponding to
each solvent accessible residue is color-coded from light
pink to deep red, in proportion to the loss of GAP
activity as measured by % V0WT (Fig.
2C) upon mutation (see "Results"). The surfaces
of residues that were not mutated, or where mutation had no significant
affect on GAP activity (% V0WT > 80%), are
rendered in light gray. B, the corresponding
surface representation of RGS4, color-coded according to GAP activity
as in A, using relative activity data obtained for mutants
of RGS4 reported by Srinivasa et al. in Table I of Ref. 16.
The activity data obtained in the presence of 200 nM RGS4
were used to color code this surface diagram. Only those
solvent-accessible residues mutated to alanine are color-coded in this
diagram. In both panels, circles labeled and
color-coded according to the scheme used in Fig. 1A enclose
structural elements that form the RGS4·G
i1 binding
site or the proposed p115rgRGS·G
13 binding site.
13 (15). This region contains a hydrophobic
sequence followed by a 16-residue segment containing 9 acidic residues (Figs. 1B and 8A). Within this region, sequence
conservation between p115RhoGEF and GTRAP48, which possesses only weak
GAP activity, is low, and fewer negatively charged side chains are
present in GTRAP48. To define the contribution of residues within the
acidic sequences to the GAP activity of rgRGS, a series of single site mutations was generated within the segment extending from residue 27 to
residue 45. Also included for mutagenesis were 2 amino acids at the
N-terminal border of the functional rgRGS domain, Ser-14 and Arg-15.
Within this set, negatively charged glutamic acid residues were mutated
to lysine; the remaining amino acids were mutated to alanine. All of
the mutant p115rgRGS domains could be expressed as soluble proteins in
E. coli and were assayed for GAP activity toward
G
13 as described above. Residues 27-31 were identified
as crucial to the GAP activity of the rgRGS domain. Mutation of Glu-27
or Glu-29 to lysine severely impaired GAP activity (Fig. 4,
A and B) as did the
mutation of Asp-28 or Phe-31 to alanine. Mutations of the charged
residues immediately flanking this segment reduced GAP activity to
~50% that of wild type, whereas mutations of residues 40-45 had no
effect. Substitution of Ser-14 and Arg-15 did not reduce GAP activity
appreciably. The concentration dependence of GAP activity was also
measured for p115rgRGS bearing single mutations in residues 27-31
(Fig. 4D). In contrast to mutations within the RGS box
region, mutation of these residues appeared to reduce efficacy as well
as potency severely.
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Fig. 4.
Effect of mutations within the N terminus of
p115rgRGS on GAP activity. A, the time course of GTP
hydrolysis by G 13, as described in Fig. 2A,
is shown for the basal condition (closed circles) or
inclusion of the 10 nM wild type (open squares),
E32K mutant (open circles), E34K mutant (closed
inverted triangles), E27K mutant (open inverted
triangles), or E29K mutant (closed squares) form of the
p115rgRGS domain. B, the time course of GTP hydrolysis by
G
13, as described in Fig. 2A, is shown for
basal conditions (closed circles) or inclusion of the 10 nM wild type (open squares), S44A mutant
(open circles), Q45A mutant (closed inverted
triangles), D28A mutant (open inverted triangles), or
F31A mutant (closed squares) form of the p115rgRGS domain.
C, the apparent initial rate of GTP hydrolysis catalyzed by
G
13 in the presence of each p115rgRGS mutant (10 nM) is represented as a percentage of the initial rate of
GTP hydrolysis by G
13 in the presence of wild type
p115rgRGS domain. D, the quantity of
32Pi released in the assay described in
A after a 2-min incubation was determined in the presence of
the indicated concentration of mutant or wild type p115rgRGS: wild type
p115rgRGS (closed squares), E27K (closed
circles), D28A mutant (open circles), E29K mutant
(closed inverted triangles), or F31A mutant (open
inverted triangles).
13--
Recombinant p115rgRGS proteins,
which possess single mutations from the N terminus (E27K, D28A, E29K,
F31A, E32K, or E34K) of the rgRGS domain or from the RGS box region
(Q69A, F70A, F70Y, E71K, or D156A), or the double mutation
(E71K/P113K), were tested for their ability to bind G
13
in a pull-down assay. Immobilized p115rgRGS proteins were incubated
with G
13 in the presence of GDP and Mg2+ or
GDP with Mg2+ and AlF
13·GDP in
the absence of Mg2+ and AlF
subunit
(G
13·GDP·Mg2+·AlF
13·GDP·Mg2+·AlF
13·GDP·Mg2+·AlF
13; in no
case do we observe an inactive rgRGS mutant that retains its full
ability to bind G
13.
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Fig. 5.
Binding of
G 13 to rgRGS domain mutants.
Purified GST-tagged rgRGS domains (20 pmol) were bound to
glutathione-Sepharose 4B (see "Materials and Methods"). Washed
beads were incubated with gel-filtered GDP-bound G
13 (20 pmol) prepared in the presence or absence of AMF and mixed
gently for 30 min at 4 °C. The beads were then washed, and the
relative amount of G
13 that remained bound to the
immobilized rgRGS domains was determined by immunoblot with B860
anti-G
13 antiserum after separation by SDS-PAGE.
13 (Fig.
6B). This indicates that the rgRGS domain confers the
dominant elements required for binding of p115RhoGEF to
G
13.
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Fig. 6.
GAP and
G 13 binding activities of
p115RhoGEF containing mutations in the rgRGS domain. A,
G
13 was loaded with [
-32P]GTP (final
concentration, 1-5 nM), and hydrolysis of the bound GTP
was initiated as described in Fig. 2 either in the absence or presence
of the indicated concentrations of wild type (WT) p115RhoGEF
or the exchange factor containing the indicated mutations in its rgRGS
domain: wild type (closed circles), D156A mutant (open
circles), E27K mutant (closed inverted triangles), D28A
mutant (open inverted triangles), E29K mutant (closed
squares), or F31A mutant (open squares). Hydrolysis of
GTP was measured for 2 min at 4 °C. B, binding of
G
13 to the mutant proteins was done as described in Fig.
5 except the Glu-tagged p115RhoGEFs were bound to Sepharose containing
immobilized anti-Glutag IgG.
13 to stimulate the Rho exchange activity of p115RhoGEF (for examples, see Fig. 7). Among the
mutants tested, stimulation of exchange activity by either the wild
type or mutant proteins ranged from 3- to 5-fold with an
EC50 for G
13 between 5 and 15 nM. There is no apparent decline in potency which
correlates with the observed effects on activities of the rgRGS domain.
Thus, although the rgRGS domain is required for regulation of Rho
exchange by G
13 (15), binding of the domain to the
subunit does not appear to be coupled to this effect.
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Fig. 7.
Stimulation of p115RhoGEF mutants by
G 13. Exchange of guanine
nucleotide on Rho was assessed by binding of [35S]GTP
S
(30) either in the absence or presence of p115RhoGEF and the addition
of increasing concentrations of activated G
13 as
indicated. Reactions contained 2 µM His6-RhoA
and 20 nM p115RhoGEF when indicated: wild type (open
circles), F31A mutant (closed inverted triangles), D28A
(open inverted triangles), D156A (closed
squares), no GEF (closed circles). Reactions were
incubated at 30 °C for 3 min. Activation of G
13 was
accomplished by preincubation with AMF at 4 °C for 30 min and
inclusion of AMF in the exchange assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
13 interaction surface of the RGS box region
in p115RhoGEF is roughly similar to that observed in the structures of
RGS4 and RGS9 bound to their G
substrates (5, 7). However, the set
of rgRGS residues that emerges from the analysis as "hot spots" for
GAP activity are not all cognates of the functionally critical residues
in RGS4 (16).
7-
8 in RGS4, which
folds as a single helix,
8, in rgRGS. Residues from both segments
are important for GAP activity in both the RGS and rgRGS domains. A
triad of residues, Gln-69, Phe-70, and Asp-156, appears to form a
functional core in rgRGS. Their counterparts in RGS4 are Glu-83,
Tyr-84, and Arg-167, respectively. In the complex with
G
i1, Arg-167 is involved in a weak or indirect
electrostatic interaction with the main chain of Thr-182 in switch I,
near the center of the RGS4·G
i1 contact region. Glu-83
plays a supporting role through formation of an ion pair with Arg-167
but does not directly contact the G protein. Tyr-84 forms a stacking
interaction with His-213 at the C terminus of the switch II helix in
G
i1. As is true for its counterpart, Asp-156 in
p115rgRGS, mutation of Arg-167 causes a severe loss of GAP activity
(17), whereas the effects of mutations at positions 83 and 84 are less
damaging (16). However, if we create a model of the
rgRGS·G
13 complex by superposing both components onto
the corresponding structures in RGS4·G
i1 (26), none of
the core triad residues directly contacts G
13. We
therefore suppose that the rgRGS·G
13 complex differs
in significant molecular detail from that of
RGS4·G
i1.
13. Pro-113, and possibly the L5 loop, therefore appear
to be less important to the function of rgRGS than equivalent regions
of RGS proteins.
8, which, of the residues mutated, has the least solvent-accessible (~25 Å2)
surface area in the structure of p115rgRGS. Thus, residues within the
RGS box and a small cluster in the preceding N-terminal region are both
necessary for the GAP activity of rgRGS.
View larger version (26K):
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Fig. 8.
Putative
G 13 binding site for the N
terminus of p115RhoGEF. A, sequence of the N-terminal
41 residues of p115RhoGEF. Acidic residues are colored red,
and basic residues colored blue. B, the complex
of p115rgRGS (colored as in Fig. 1A) with G
13
(gray except for switch segments in purple) was
modeled as described previously (26). The position of the most
N-terminal residue that is defined in the structure of the p115rgRGS
domain (residue 44) is labeled. C, the putative binding site
for the N terminus of p115RhoGEF (a detailed view of the area shown
enclosed by a dotted box in B) is formed by the
helical domain and the switch regions of Ga13, and helix
8 of the rgRGS domain.
In the absence of structural information, we speculate that residues
N-terminal to the RGS box might function in GAP activity through an
electrostatic binding mechanism. A model of the
rgRGS·G13 complex based on that of previously
determined RGS·G
complexes shows a cluster of positively charged
residues contributed by helix
8 of the rgRGS domain and the switch
regions and central helix (residues 79-105) of the helical domain of
G
13. The latter form an electropositive trough that
could provide a binding site for the negatively charged residues that
precede the N terminus of the rgRGS domain (Fig. 8, B and
C). The electropositive character of helical domain of
G
13 appears to be a conserved feature of the
G12 class of G
subunits. In G
i,
G
o, G
q, and G
z, which are
not substrates for p115RhoGEF, many of the positively charged residues
in the helical domain are replaced by neutral or acidic residues. The
most critical residues within the RGS domain of rgRGS: Gln-69, Phe-70,
and Asp-156, might be supposed to form, with the critical EDEDF
segment, a functional core of the rgRGS·G
13 interface.
Phe-31, which flanks this highly charged segment, might be accommodated
in a hydrophobic pocket, possibly within the helical domain of
G
13.
The rgRGS subfamily represented by GTRAP48 and PDZRhoGEF have little or
no GAP activity toward G13. The absence of GAP activity for these two rgRGS proteins may stem from specific sequence
differences in either the RGS box or the preceding N-terminal sequence.
The overall sequence similarity between p115RhoGEF and GTRAP48 is high
(57%) in the RGS box region (Fig. 1B), and many of the
substitutions are conservative. Three mutations, F70A, P113K, and
R152E, were designed to reflect nonconserved differences in sequence
between p115RhoGEF and GTRAP48 in regions hypothesized to form contact sites with G
13. Of these, F70A is sufficient to reduce
catalytic efficiency and binding affinity of the rgRGS domain toward
G
13 severely. In contrast, differences between the
N-terminal sequences of p115RhoGEF and GTRAP48 are so great that it is
difficult to align them. Although both active and inactive RhoGEFs
possess a sequence consisting of 3 or 4 acidic residues preceding a
phenylalanine (residues 27-31 in p115RhoGEF) or tyrosine, the
positions of this sequence relative to the N terminus differ.
Therefore, spatial mismatch of the N-terminal acidic cluster with
respect to its potential binding site on G
13 might
explain the reduced GAP activity of GTRAP48.
All of the mutations that diminish the GAP activity of p115rgRGS also
reduce its affinity for G13. A quantitative relationship between affinity and activity cannot be deduced from the data presented
here, as is the case for RGS4 (18); however, no mutant capable of
binding G
13 is devoid of GAP activity. It therefore appears that the GAP activity of p115rgRGS arises from its ability to
bind and stabilize a conformational state of G
13 which
is conducive to transition state formation. The mechanism by which rgRGS acts in this role may be in part elucidated by structural studies
now in progress. The requirement for specific N-terminal residues
outside of the conserved RGS box indicates that the rgRGS domains have
developed alternate mechanisms to stabilize such a state in the
G12 subfamily of heterotrimeric G proteins.
The rgRGS domain contributes to the intrinsic nucleotide exchange
activity of p115RhoGEF as demonstrated by the 60% reduction of this
activity when the domain is deleted (30). However the most notable
effect that results from this truncation is a total loss of the ability
of p115RhoGEF to be stimulated by G13 (15). One
hypothesis is that it is the binding of the rgRGS to G
13 which facilitates this regulation. This is consistent with the observation that the regulation of exchange activity by
G
13 was retained when the rgRGS region of GTRAP48
replaced the endogenous rgRGS domain of p115RhoGEF (15). Yet, as we
show here, mutations that compromise the ability of the rgRGS domain to
bind to G
13 do not affect the susceptibility of
p115RhoGEF to activation by G
13. This finding is
consistent with a model in which the rgRGS domain plays a structural or
allosteric role, for example, by conferring stability upon
conformational states of other segments of p115RhoGEF which are
required for G
13-mediated stimulation of GEF activity.
The reduction of inherent GEF activity upon deletion of the rgRGS
domain (30) is evidence for direct intramolecular coupling between the
rgRGS and Dbl homology/pleckstrin homology domains. This raises the
intriguing possibility that the ability of the rgRGS domain to affect
the efficacy of G
13 might itself be subject to
regulatory mechanisms such as covalent modification, which have yet to
be discovered.
The data presented here also suggest that stimulation of p115RhoGEF by
G13 results from interaction of the
subunit at a surface on p115RhoGEF different from the rgRGS domain. Previous binding
studies of G
13 to p115RhoGEF detected a second site for interaction between residues 288 and 760, which includes the Dbl homology and pleckstrin homology domains (15). The surface of G
13 which binds the rgRGS domain and that which engages
the second site are most likely distinct. This could resemble the
interaction demonstrated by Slep et al. (7), that
G
t binds the
subunit of cyclic GMP phosphodiesterase
and RGS9 simultaneously at separate yet interacting interfaces (7). The
mechanisms by which G
13 interacts with regions beyond
the rgRGS domain of p115RhoGEF and exploitation of the coupling between
this and other domains of the molecule remain to be explored.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Stephen Gutowski and Abiola Badejo for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM31954 (to P. C. S.), DK46371 (to S. R. S.), and GM07062 (to C. D. W.), by the Robert A. Welch foundation (to P. C. S. and S. R. S.), the Alfred and Mabel Gilman chair in molecular pharmacology (to P. C. S.), and the John W. and Rhonda K. Pate professorship in biochemistry (to S. R. S.).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.
§ These authors contributed equally to this work.
Current address: Program in Molecular Biology and Cancer,
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON
M5G 1X5, Canada.
To whom correspondence may be addressed: Dept. of
Biochemistry, Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX
75390-9050. Tel.: 214-648-5008; Fax: 214-648-6336; E-mail:
stephen.sprang@utsouthwestern.edu.
§§ To whom correspondence may be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9041. Tel.: 214-648-2835; Fax: 214-648-2971; E-mail: paul.sternweis@utsouthwestern.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212695200
2 T. Kozasa, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RGS, regulator of G protein signaling; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; rg, RhoGEF; Rho, Ras homology; % V0WT, initial velocity of a reaction in the presence of the mutant normalized by the rate in the presence of wild type p115rgRGS.
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