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Alternative Splicing of Rac1 Generates Rac1b, a
Self-activating GTPase*
Received for publication, September 16, 2003, and in revised form, October 24, 2003
Published, JBC Papers in Press, November 18, 2003, DOI 10.1074/jbc.M310281200
Dennis Fiegen‡, Lars-Christian Haeusler‡, Lars Blumenstein, Ulrike Herbrand,
Radovan Dvorsky, Ingrid R. Vetter, and Mohammad R. Ahmadian§
From the Max-Planck-Institut fu¨ r molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11,
44227 Dortmund, Germany
Rac1b was recently identified in malignant colorectal
tumors as an alternative splice variant of Rac1 contain-
ing a 19-amino acid insertion next to the switch II
region. The structures of Rac1b in the GDP- and the
GppNHp-bound forms, determined at a resolution of
1.75 Å, reveal that the insertion induces an open switch
I conformation and a highly mobile switch II. As a con-
sequence, Rac1b has an accelerated GEF-independent
GDP/GTP exchange and an impaired GTP hydrolysis,
which is restored partially by GTPase-activating pro-
teins. Interestingly, Rac1b is able to bind the GTPase-
binding domain of PAK but not full-length PAK in a
GTP-dependent manner, suggesting that the insertion
does not completely abolish effector interaction. The
presented study provides insights into the structural
and biochemical mechanism of a self-activating GTPase.
The small GTPase Rac acts as a binary molecular switch that
cycles between an inactive GDP-bound state and an active
GTP-bound state in response to a variety of extracellular stim-
uli. The interconversion between both states is controlled by
nucleotide exchange and GTP hydrolysis. The structures of
several GTPases in either state revealed that the switching
mechanism depends on the conformational change of two re-
gions, termed switch I and switch II (1, 2). The switch regions
consequently provide a surface that is in the GTP-bound state
specifically recognized by downstream effectors (1–3) and
GTPase-activating proteins (GAPs),
1
accelerating the slow in-
trinsic GTP hydrolysis reaction (1, 4–6). After GTP hydrolysis,
release of the cleaved
␥
-phosphate allows the switch regions to
relax into the GDP conformation. Guanine nucleotide exchange
factors (GEFs), stimulating the GDP/GTP exchange, bind in-
dependently of the nucleotide-bound state (1, 7), whereas gua-
nine nucleotide dissociation inhibitors (GDIs), which sequester
the GTPase from the membrane into the cytoplasm, interact
only with the GDP-bound state (8).
Rac1b was discovered in human tumors as an alternative
splice variant of Rac1 containing a 19-amino acid insertion
(between codons 75 and 76) at the end of the switch II region (9,
10). It has been suggested that the insertion may create a novel
effector-binding site in Rac1b and thus participate in signaling
pathways related to the neoplastic growth of the intestinal
mucosa (9). Most recently, it has been shown that Rac1b does
not interact with Rho-GDI and PAK1 and is not involved in
lamellipodia formation but able to activate the transcription
factor NF-
B (11). We tried to address what influence this
insertion might have on the structure and the biochemical
properties of Rac1b in comparison with Rac1. Therefore,
we solved the crystal structures of Rac1b in the GDP- and
GppNHp-bound conformations at 1.75 Å resolution. Further-
more we investigated nucleotide binding and hydrolysis of
Rac1b and studied its regulation by the RacGEF Tiam1 and
p50
GAP
and its interaction with the downstream effector PAK.
The Rac1b structures explain the drastic changes of the bio-
chemical properties of Rac1b, namely a dramatic decrease in
nucleotide affinity and GTP hydrolysis. The presented data
identify Rac1b as a predominantly GTP-bound form of Rac1.
EXPERIMENTAL PROCEDURES
Plasmids—The pcDNA3-FLAG constructs of human Rac1b, Rac1,
and the respective constitutive active Rac1(G12V) mutant were gener-
ated by PCR and cloned via BamHI and EcoRI restriction sites. Rac1,
Rac1⌬C (1–184), Rac1b, and Rac1b⌬C (1–201) were cloned in
pGEX4T1, using BamHI and EcoRI restriction sites. The DH-PH do-
main of Tiam1 (1033–1404) was cloned into pGEX4T1 using BamHI
and XhoI restriction sites. The coding region of Tiam1 contains an
internal BamHI site that was removed for the cloning procedure. pGEX-
PAK1-GBD was kindly provided by J. Collard (12). Full-length pGEX-
PAK was kindly provided by A. Wittinghofer (13).
Preparation of Recombinant Proteins—Rac1, Rac1⌬C, Rac1b, and
Rac1b⌬C, the catalytic domain of p50
GAP
(amino acids 198–439), the
Cdc42/Rac-interacting binding domain of PAK (amino acids 57–141),
full-length PAK, and the DH-PH domain of Tiam1 were produced as
glutathione S-transferase (GST) fusion proteins in Escherichia coli. All
of the proteins were purified as described previously for Rnd3 (14).
Nucleotide-free GTPases as well as fluorescent nucleotide-bound
GTPases were prepared, and concentration and quality were deter-
mined as described (15).
Crystallization and Data Collection—Crystals of truncated Rac1b⌬C
(1–184) in complex with GDP and GppNHp (nonhydrolyzable GTP
analog) were grown at 20 °C using the hanging drop method by mixing
2
lofa0.5mMsolution of the Rac1b G domain in 20 mMTris/HCl, pH
7.5, 2 mMMgCl
2
,2mMdithioerythritol, 100
MGDP or GppNHp with
2
l of reservoir solution consisting of 100 mMHepes buffer, pH 7.5,
18–30% polyethylene glycol 3350, and 2–6% isopropanol. The crystals
of both complexes belonged to space group P2
1
2
1
2
1
(a⫽51.55 Å, b⫽
78.67 Å, c⫽96.88 Å). For data collection at 100 K, the crystals were
* This work was supported in part by a European Community Marie
Curie Fellowship (to R. D.), by funds from the Verband der Chemischen
Industrie and the Bundesministerium fu¨ r Bildung und Forschung (to
D. F.), and by funds from the Deutsche Forschungsgemeinschaft (to
L.-C. H. and L. B.). The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 1RYF and 1RYH)
have been deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
‡ These authors contributed equally to this work.
§ To whom correspondence should be addressed. E-mail: reza.
ahmadian@mpi-dortmund.mpg.de.
1
The abbreviations used are: GAP, GTPase-activating protein; GEF,
guanine nucleotide exchange factor; GDI, guanine nucleotide dissocia-
tion inhibitor; DH-PH, double homology-pleckstrin homology; GST, glu-
tathione S-transferase; HPLC, high pressure liquid chromatography;
GppNHp, quanosine 5⬘-

,
␥
-imidotriphosphate; GBD, GTPase-binding
domain.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 6, Issue of February 6, pp. 4743–4749, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 4743
by guest on November 18, 2016http://www.jbc.org/Downloaded from
transferred to a solution containing reservoir solution and 10% glycerol.
A cryo-protected crystal was then suspended in a rayon loop (Hampton
Research) and flash frozen in liquid nitrogen. X-ray diffraction data
were collected on an ADSC Q4 CCD detector at the beam line ID14–1
at the European Synchrotron Radiation Facility and were processed
using XDS (16). Details on data collection, structure determination, and
crystallographic refinement are summarized in Table I.
Structure Determination and Crystallographic Refinement—The ini-
tial phases were calculated by molecular replacement, using the pro-
gram AMoRe (17) and a Rac1 search model based on the Rac1䡠GppNHp
structure lacking the bound nucleotide and the switch regions (18)
(Protein Data Bank code 1mh1). After initial rigid body refinement, 20
cycles of simulated annealing and model building were performed. For
the last refinement steps REFMAC5 was used (19). For the detection of
crystallographic water molecules, ARP/wARP (20) and REFMAC5 were
used. The residue ranges that were included in the final model as well
as the corresponding R-factors are listed in Table I. For all four mole-
cules the two additional N-terminal Gly-Ser residues caused by the
thrombin cleavage site could be observed and were included in the
model.
Fluorescence Measurements—Long time fluorescence measurements
were monitored on a LS50B PerkinElmer Life Sciences spectrofluorom-
eter, and rapid kinetics were measured with a stopped flow apparatus
(Applied Photophysics SX16MV) as described (15). Nucleotide associa-
tion was performed with 0.1
Mfluorescent nucleotide and varying
concentrations of nucleotide-free Rac proteins at 20 °C as described
(15). The dissociation of the fluorescent nucleotide from Rac proteins
(0.1
M) was measured by the addition of 200-fold excess of nonfluores-
cent nucleotide in the absence and the presence of 5
MTiam1 DH-PH
at 20 °C. The equilibrium dissociation constant (K
d
) for the PAK-GBD
interaction with Rac1b was determined as previously described for the
Ras-Raf kinase interaction (21). The measurements were carried out
using 0.2
MmantGppNHp-bound GTPase, 40
MGppNHp, and in-
creasing concentrations of PAK-GBD at 25 °C for Rac1 and at 10 °Cin
the case of Rac1b because of its fast nucleotide dissociation rate. All of
the measurements were carried out in 30 mMTris/HCl, pH 7.5, 5 mM
MgCl
2
,10mMNa
2
HPO
4
/NaH
2
PO
4
pH 7.5, 5 mMdithioerythritol. The
observed rate constants were evaluated using Grafit (Erithacus
software).
GTPase Assay—The intrinsic and GAP-stimulated GTP hydrolysis
reactions were measured by HPLC on a C
18
reversed phase column
using a mixture of 80
Mnucleotide-free GTPase and 70
MGTP in the
presence and the absence of 8
MGAP at 25 °Cin30mMTris/HCl, pH
7.5, 5 mMdithioerythritol, 10 mMNa
2
HPO
4
/NaH
2
PO
4
,5mMMgCl
2
as
described (15). The relative GTP content was calculated by the ratio of
[GTP]/([GTP]⫹[GDP]). For exponential fitting of the data, the program
Grafit (Erithacus software) was used.
Transfection and GTPase Pull-down Assay—COS-7 cells were trans-
fected using DEAE-dextran as described (12). Pull-down assay for the
active GTP-bound Rac proteins was carried out using GST-PAK-GBD
(glutathione S-transferase-fused Rac-binding domain of PAK) conju-
gated with glutathione beads as described (12). The interaction of
full-length GST-PAK with the Rac proteins was examined under the
same conditions using purified proteins. The beads were washed four
times and subjected to SDS-PAGE (15% polyacrylamide). Bound Rac
proteins were detected by Western blot using a monoclonal antibody
against Rac (Upstate Biotechnologies, Inc.).
RESULTS AND DISCUSSION
Rapid GEF-independent Nucleotide Dissociation Reaction of
Rac1b—To investigate the influence of the 19-amino acid in-
sertion on the nucleotide binding affinity, we first determined
kinetic constants for the association of fluorescently (methyl-
anthraniloyl- or mant-) labeled nucleotides to nucleotide-free
Rac1b protein. This allowed us to monitor nucleotide associa-
tion kinetics at increasing protein concentrations. As shown in
Fig. 1A, the formation of the binary complex is not affected by
the 19-amino acid insertion. The association rate constants
(k
on
) for the binding of mantGDP and mantGTP to Rac1b were
obtained by linear fitting of the observed rate constants at the
given protein concentrations. They are only marginally slower
than those of Rac1 (Table II).
To determine the intrinsic and GEF-accelerated nucleotide
dissociation rates, Rac1b and Rac1 were loaded with the re-
spective fluorescently labeled guanine nucleotides. The dis-
placement of the fluorescent nucleotides was initiated by the
addition of excess amounts of nonfluorescent nucleotides in the
presence and absence of the DH-PH domain of Tiam1 (a Rac-
specific GEF). Drastic increases in the intrinsic dissociation of
mantGDP (26-fold), mantGTP (27-fold), and mantGppNHp
(250-fold) from Rac1b compared with the very slow dissociation
rates of the respective nucleotides from Rac1 were observed
(Fig. 1Band Table II). Accordingly, the calculated K
d
for nu-
cleotide binding revealed that the 19-residue insertion dramat-
TABLE I
Data collection and refinement statistics of Rac1b
GDP GppNHp
Intensity data processing
Resolution (Å) 24.92–1.75 Å26.26–1.75 Å
Number of reflections 130,220 138,306
Number of independent reflections 38,560 38,875
R
sym
(%)
a
5.4 (40.9)
b
5.1 (40.6)
b
Completeness of data (%) 95.1 (88.3)
b
96.0 (90.1)
b
Mean 具I/
(I)典15.21 (3.37)
b
16.62 (3.75)
b
Molecular replacement statistics
Resolution range rotation translation 10.0–4.0 A/8.0–4.0 Å10.0–4.0 A/8.0–4.0 Å
Rotation (°)
c
152.02, 70.81, 163.02 (151.49, 70.92, 162.12)
d
152.21, 70.87, 163.31 (151.46, 70.96, 161.47)
d
Translation (Å)
c
⫺4.99, ⫺7.34, ⫺8.63 (⫺1.80, 32.33, ⫺9.06)
d
⫺4.54, ⫺7.50, ⫺8.47 (⫺1.89, 32.30, ⫺9.40)
d
Correlation coefficient 61.5 (22.6)
e
62.6 (25.3)
e
R
cryst
(%)
f
47.9 (64.1)
e
45.3 (60.7)
e
Refinement Statistics
R
cryst
(%)
f
18.8 18.1
R
free
(%)
f
22.3 21.9
Root mean square bond lengths (Å) 0.021 0.021
Root mean square bond angles (°) 1.87 1.88
Total number of residues 614 (273)
g
638 (296)
g
Residue ranges 1–59 and 93–201 (1–58 and 93–199)
h
1–60 and 93–201 (1–58 and 93–199)
h
a
R
sym
⫽100䡠⌺兩I⫺具I典兩/⌺I.
b
Brackets are quantities calculated in the highest resolution bin at 1.85–1.75 Å.
c
Eulerian angles (
␣
,

,
␥
) are as defined in AMoRe, and translation is given in the orthogonal system.
d
Brackets show the solution for the second molecule in the asymmetric unit.
e
Brackets are quantities of the second best solution.
f
R
cryst
⫽100䡠⌺兩F
o
⫺F
c
兩/⌺F
o
.R
free
is R
cryst
that was calculated using 5% of the data, chosen randomly, and omitted from the subsequent
structure refinement.
g
Brackets show the number of included water molecules.
h
Brackets show the residue range for the second molecule in the asymmetric unit.
Consequences of Alternative Rac1 Splicing4744
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ically affects the overall affinity for GDP, GTP, and particularly
the GTP analog GppNHp (Table II). The reason for the reduced
affinity of GppNHp compared with that of GTP is the disrupted
hydrogen bond of the GTP-

,
␥
-bridging imino group to the
main chain NH group of the P-loop residue Ala
13
. A similar
observation has been reported for Ras䡠mantGppNHp (22).
Moreover, in contrast to the slow nucleotide dissociation of
Rac1, which could be 50-fold accelerated in the presence of the
DH-PH domain of Tiam1, the fast intrinsic mantGDP dissoci-
ation of Rac1b could not be further increased by the DH-PH
domain of Tiam1 (Fig. 1Cand Table II).
These in vitro results show that Rac1b does not require any
GEF to get activated. It rather activates itself by a very fast
nucleotide dissociation and by the subsequent binding of the
cellular abundant GTP. Despite the drastically increased nu-
cleotide dissociation, Rac1b exhibits a nucleotide binding affin-
ity in the low nanomolar range, which is still high enough to act
as a GTP-binding protein in cells. However, because the
DH-PH domain of Tiam1 is a weak exchange factor in vitro (51)
and displays a 10 times higher activity on prenylated Rac1
bound to liposomes than on soluble unprenylated Rac1 (23), we
cannot exclude the possibility that Rac1b can in principle in-
teract with Tiam1 under cellular conditions as shown with
constitutive active Tiam1, overexpressed in NIH3T3 cells (11).
Impaired Intrinsic GTP Hydrolysis Reaction of Rac1b—A
second crucial function of small GTPases is their slow intrin-
sic GTP hydrolysis reaction, which needs to be stimulated by
GAPs to switch off downstream signaling. Therefore, we
measured the GTP hydrolysis reaction of Rac1b in direct
comparison with that of Rac1 using a HPLC-based technique.
Interestingly, we found that the intrinsic GTP hydrolysis
reaction of Rac1b (0.0035 min
⫺1
) was about 30-fold reduced
compared with that of Rac1 (0.11 min
⫺1
) (Fig. 1Dand Table
II). In contrast to our results it has been previously published
that Rac1 and Rac1b show the same GTPase activity (10).
This discrepancy can be explained by the method this group
employed. The filter binding assay seems to be inappropriate
for a protein with a fast nucleotide dissociation such as
Rac1b. Unlike the constitutive active mutants of Rac1 (G12V
in the P-loop and Q61L in the switch II region) that also have
an impaired intrinsic GTP hydrolysis (24), the defective
GTPase reaction of Rac1b can be restored by GAP proteins.
As shown in Fig. 1D, the catalytic domain of p50
GAP
stimu-
lated the GTPase reaction of Rac1b up to 55-fold (21-fold
for Rac1), showing that GAP is able to stabilize the catalytic
elements of Rac1b and thus accelerate the GTPase
reaction.
High Level of Rac1b䡠GTP in COS-7 Cells—Considering the
FIG.1.Biochemical properties of Rac1b. A, nucleotide association. The binding of increasing concentrations of nucleotide-free Rac1 (filled
symbols) and Rac1b (open symbols) to mantGDP (circles), mantGTP (squares), and mantGppNHp (triangles) was measured, and the dependence
of the observed rate constant on the protein concentration was linearly fitted to determine association constants (k
on
). B, nucleotide dissociation.
The release of mantGDP (open circles), mantGTP (open squares) and mantGppNHp (open triangles) from Rac1b and mantGDP from Rac1 (filled
circles) was measured after addition of excess of the respective nonfluorescent nucleotides and exponentially fitted to determine dissociation
constants (k
off
). C, Tiam1-catalyzed nucleotide dissociation. Dissociation of bound mantGDP from 0.1
MRac1 (circles) and Rac1b (squares) was
monitored in the presence (filled symbols) and absence (open symbols)of5
MTiam1 DH/PH. D, GTP hydrolysis reaction. HPLC measurements
of the GTP hydrolysis of Rac1 (filled symbols) and Rac1b (open symbols) were performed in the presence (squares) and absence (circles)of
p50RhoGAP.
Consequences of Alternative Rac1 Splicing 4745
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increased nucleotide dissociation and the decreased GTP hy-
drolysis, it was tempting to assume that Rac1b is GTP-bound in
cells. To prove this assumption, Rac1, its constitutive active
mutant Rac1(G12V) and Rac1b were overexpressed in COS-7
cells under serum-starved conditions for 48 h. The fact that
wild-type Rac1b could be pulled down with GST-PAK-GBD
verifies our hypothesis that Rac1b exists in an active confor-
mation in serum-starved cells. Thereby it resembles the con-
stitutive active Rac1(G12V) mutant (Fig. 2A). As expected wild-
type Rac1 could not be detected under these conditions and
obviously needs GEF proteins to be activated. A GTP-depend-
ent Rac1b-PAK interaction was demonstrated by performing
the pull-down assay with purified GDP- and GppNHp-bound
Rac1b. As shown in Fig. 2B, GST-PAK-GBD selectively binds
Rac1b䡠GppNHp but not Rac1b䡠GDP, similar to the Rac1 control
experiment. These results reveal that Rac1b is, independent of
external stimuli, GTP-bound in cells and can selectively inter-
act with Rac effector proteins.
Rac1 involvement in transcription and growth control and its
requirement for Ras-induced malignant transformation is
widely known (25–29). This knowledge is based on experiments
using the expression of a constitutively active Rac1(G12V) mu-
tant. A fast cycling mutant of Cdc42, Cdc42(F28L), has been
shown to undergo spontaneous nucleotide exchange in the ab-
sence of a GEF while maintaining full GTPase activity (30).
This mutant has an even greater cell-transforming potential in
fibroblasts compared with the constitutively active Cdc42(G12V)
mutant. However, our biochemical data clearly shows that
Rac1b behaves as a self-activating GTPase that is predomi-
nantly GTP-bound in cells.
Low Affinity Binding of Rac1b to PAK-GBD—To characterize
the effect of the insertion on effector interaction, we determined
equilibrium dissociation constants (K
d
) of PAK-GBD binding to
GppNHp-bound Rac1b and Rac1 using the GDI assay (31). As
shown in Fig. 3, increasing concentrations of PAK-GBD re-
sulted in incremental inhibition of the mantGppNHp dissocia-
tion from Rac1b and Rac1. We obtained a K
d
value of 0.49
M
for the PAK-GBD interaction with Rac1, which nicely corre-
sponds to previous reports (32). For Rac1b we observed a 7-fold
reduced binding affinity of PAK-GBD. The lower K
d
of 3.55
M
can be most likely attributed to the 19-amino acid insertion.
MantGDP dissociation from Rac1b was not inhibited under
these conditions (data not shown), confirming the GTP-depend-
ent interaction of Rac1b with PAK-GBD.
Furthermore, we examined the interaction of Rac1 and
Rac1b with full-length PAK using a GST pull-down assay. Fig.
2Bshows that we could not detect binding of Rac1b to full-
length PAK, and hence Rac1b stands in contrast to Rac1.
Compared with the high affinity binding of the isolated PAK-
GBD domains to Cdc42, it has been recently shown that full-
length PAK has a much lower binding affinity for Cdc42 (13,
32, 33). Assuming that this is also true for Rac1, our biochem-
ical data suggest an extremely low affinity of full-length PAK
for Rac1b.
Conserved Overall Structure of Rac1b䡠GDP and Rac1b䡠
GppNHp—To gain insight into the structural impact of the
19-amino acid insertion of Rac1b, we determined the crystal
structures of Rac1b in the GDP- and GppNHp-bound states.
The crystals diffracted to 1.75 Åresolution (Table I). Size
exclusion chromatography showed that Rac1b is monomeric in
solution (data not shown), but it crystallized as a dimer with
two molecules/asymmetric unit in a head to head fashion. The
contact surface has a size of 1347 Å
2
and is build up by

1to

3,
␣
1, and the switch I of both molecules. Both molecules in the
asymmetric unit are very similar as can be derived from the
low root mean square deviation of 0.45 Åfor 165 common C
␣
atoms.
The ribbon representation in Fig. 4 shows the secondary
structure elements of Rac1b䡠GDP and Rac1b䡠GppNHp super-
imposed on the GppNHp-bound Rac1 (18). The overall struc-
tures of both nucleotide-bound forms of Rac1b are remarkably
similar to each other and conserved as compared with the
structures of Rac1䡠GppNHp (18), RhoA䡠GTP
␥
S (34), and
Cdc42䡠GDP (35) with root mean square deviations of 0.67 ⌭
(156 common C
␣
atoms), 0.80 ⌭(155 common C
␣
atoms), and
TABLE II
Biochemical properties of Rac1 and Rac1b
The dissociation constant (K
d
) for the nucleotide binding values has
been calculated from the association and the dissociation rate constants
of the respective nucleotides (K
d
⫽k
off
/k
on
).
Rac1 Rac1b
mantGDP
k
on
(s
⫺1
M
⫺1
)2.47 ⫻10
6
1.12 ⫻10
6
k
off
(s
⫺1
)7.0 ⫻10
⫺5
1.8 ⫻10
⫺3
Tiam1-catalyzed k
off
(s
⫺1
)
a
3.6 ⫻10
⫺3
1.9 ⫻10
⫺3
K
d
(nM)0.028 1.6
mantGppNHp
k
on
(s
⫺1
M
⫺1
)1.05 ⫻10
6
k
off
(s
⫺1
)1.1 ⫻10
⫺4
2.9 ⫻10
⫺2
K
d
(nM)27.6
mantGTP
k
on
(s
⫺1
M
⫺1
)1.49 ⫻10
6
1.05 ⫻10
6
k
off
(s
⫺1
)1.0 ⫻10
⫺4
2.7 ⫻10
⫺3
K
d
(nM)0.067 2.6
GTP hydrolysis
Intrinsic (min
⫺1
)0.11 0.0035
GAP-stimulated (min
⫺1
)
b
2.36 0.194
PAK binding
K
d
(
M)0.49 3.55
a
5
MTiam1 was applied in the reaction of 0.1
MGTPase.
b
The GAP concentration (8
M) was 10-fold below the GTPase con-
centration (80
M). FIG.2.GTP-dependent binding to PAK. A, high Rac1b䡠GTP level
in serum-starved COS-7 cells. FLAG-tagged wild-type (wt) Rac1, con-
stitutive active Rac1(G12V), and wild-type Rac1b were pulled down
with GST-PAK-GBD and the respective Rac-proteins were detected by
Western blot. The upper panel shows the expression control (total Rac),
and the lower panel shows the GTP-bound active Rac. The arrows
indicate the endogenous (open) and transfected (filled) Rac proteins. B,
Rac1b binds PAK-GBD but not full-length PAK. GDP- and GppNHp-
bound Rac1 and Rac1b (C-terminal truncated) were employed in pull-
down (PD) assay using GST-PAK-GBD and full-length GST-PAK, re-
spectively. PAK-GBD selectively binds the GTPase in a GTP-dependent
manner, whereas full-length GST-PAK only binds GppNHp-bound
Rac1 but not Rac1b.
Consequences of Alternative Rac1 Splicing4746
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0.81 ⌭(151 common C
␣
atoms), respectively, except for the loop
regions as described below.
Nucleotide binding requires five conserved sequence ele-
ments (36), of which three are well ordered in the Rac1b䡠GDP
and Rac1b䡠GppNHp structures. The P-loop (
13
AVGKT motif)
plays a crucial role in phosphate binding and strongly con-
tributes to magnesium (Mg
2⫹
) coordination by the side chain
of the Thr
17
residue. The
115
TKLD and
157
CSAL motifs (Rac
numbering), on the other hand, directly interact with the
guanine base and thereby ensure specificity of guanine
nucleotide binding.
The most critical motifs, the central region of switch I
(
32
YIPT) and the N-terminal part of switch II (
57
DTAGQ),
that govern the Mg
2⫹
and
␥
-phosphate binding display the
major structural difference between Rac1b and Rac1. The
switch I region (amino acids 30–38) is drastically displaced
from the nucleotide-binding site in the GDP- and GppNHp-
bound Rac1b structures compared with the Rac1 structure
(see below). We did not observe any electron density for the
switch II region and the proximate 19-amino acid insertion
(amino acids 76–94), indicating that this insert is highly
mobile and also leads to a higher mobility of the switch II
region (see below).
Highly Mobile Switch II and the 19-Amino Acid Insertion—
The switch II region and the adjacent 19-amino acid insertion
(amino acids 59–92 in the GDP-bound and 61–93 in the Gp-
pNHp-bound structures) are not resolved and therefore not
included in the crystal structures (Fig. 4). In most structures of
GppNHp-bound GTPases, the switch II region is well ordered
(37), indicating that the insertion in Rac1b contributes to a
higher mobility of the switch II region and thus leads to an
impaired GTPase reaction of Rac1b (Fig. 1D). The catalytic
residue Gln61 in switch II (
57
DTAGQ motif) is crucial in this
context, and its high flexibility is most likely the reason for the
impaired GTPase reaction of Rac1b (Fig. 1D). This is due to the
missing stabilization of the nucleophilic water leading to a
decreased GTP hydrolysis rate of Rac1b. It has been shown
before that the mutation of this key residue (Gln
61
) signifi-
cantly affects the intrinsic and the GAP-stimulated GTP hy-
drolysis reactions (5, 24, 38, 39). Interestingly, the fact that
GAP is able to stimulate the GTP hydrolysis of Rac1b strongly
indicates that the switch regions, naturally providing the GAP-
binding site, can be stabilized in a GTPase-competent confor-
mation. It has been proposed previously that the 19-amino acid
insertion could form a new functional domain built of two

-strands connected by a turn (9), but our results make it
unlikely that this region, because of its flexibility, has any
secondary structure.
Displacement of the Switch I Region in Rac1b—The most
obvious structural difference of Rac1b in comparison with Rac1
is observed in the region between Ala
28
and Asn
39
encompass-
ing the switch I of Rac1b. The drastic displacement of the
switch I region from the nucleotide-binding site is similar in
the Rac1b䡠GDP and Rac1b䡠GppNHp structures (Fig. 5, Aand
B). Switch I exhibits a maximum distance of 6.5 Åto the
nucleotide-binding site compared with 3.2 Åin Rac1, where it
completely covers the nucleotide (Fig. 5C).
The reason for this open switch I conformation is most likely
the disruption of the interaction between switches I and II. In
the Rac1䡠GppNHp structure Phe
37
of switch I lies in a hydro-
phobic cleft that is composed by the side chains of Thr
58
, Tyr
64
,
Leu
67
, and Arg
68
of switch II. Additionally, Val
36
makes a
hydrophobic interaction with Tyr
64
. Because of the high mobil-
ity of the switch II in Rac1b, this interaction cannot take place,
resulting in a destabilization of the switch I region. In the
crystal, the open conformation of switch I is stabilized by a
hydrophobic interaction of the Phe
37
side chains of the adjacent
molecules. Although switch I is stabilized by the crystal pack-
ing, it still exhibits an increased B-factor of about 45 compared
with 30 for residues in the core of the protein. This suggests
FIG.3.Quantitative measurement of the PAK-GBD interaction
with Rac1 (A) and Rac1b (B). Dissociation of mantGppNHp from
Rac1b was inhibited by increasing concentration of the PAK-GBD (2–50
M). The observed rate constants were plotted against the concentra-
tion of the PAK-GBD to obtain equilibrium dissociation constants of
0.49
Mfor Rac1 and 3.55
Mfor Rac1b.
FIG.4.Comparison of the Rac1b structures with Rac1. Ribbon
representation of Rac1b䡠GDP (purple) and Rac1b䡠GppNHp (red) were
superimposed on the structure of Rac1䡠GppNHp (brown) (17). The
switch II region of Rac1 is highlighted in orange. The nucleotide (Gp-
pNHp of Rac1b) and the Mg
2⫹
ion are shown as ball-and-stick.
Consequences of Alternative Rac1 Splicing 4747
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that switch I is probably very flexible in solution.
High affinity nucleotide binding and GTPase activity of
small GTPases are crucially dependent on the presence of an
Mg
2⫹
ion. The octahedral Mg
2⫹
coordination is highly con-
served throughout small GTPases (40–42) but shows signifi-
cant differences in Rac1b. In the GDP-bound state, the magne-
sium is directly coordinated by an oxygen of the

-phosphate,
the side chain of T17 (P-loop) and three water molecules (Wat2,
116 and 117), but lacks the coordination of Thr
35
. This key
interaction is replaced by a fourth water molecule (Wat1; Fig.
5A). A similar arrangement is observed for Rac1b䡠GppNHp,
except for the replacement of one water molecule (Wat2) by the
␥
-phosphate oxygen of GppNHp (Fig. 5B).
As a consequence of the open switch I, contacts of the invari-
ant Thr
35
with the
␥
-phosphate (main chain NH-group) and the
Mg
2⫹
ion (side chain OH group) are disrupted and provide an
explanation for the rapid nucleotide dissociation of Rac1b. This
situation is comparable with the T35A mutation in Ras, which
drastically reduces the nucleotide affinity, because of the loss
in Mg
2⫹
coordination (41, 43) and in particular to the mecha-
nism of Tiam1-catalyzed nucleotide exchange (44). The crystal
structure of the nucleotide-free Rac1䡠Tiam1 complex has shown
that the interaction of the DH domain of Tiam1 with Rac1 has
shifted switch I and

2 (amino acids 27–45) up to 2.7 Åalong
the nucleotide-binding cleft (44). Thereby, Thr
35
in Rac1 is
displaced, and the Thr
35
-Mg
2⫹
interaction is disrupted (Fig.
5C). Thus, Tiam1 binding to Rac1 prevents Thr
35
from binding
to the Mg
2⫹
ion and allows GDP release from Rac1 (1, 44).
Because the P-loop contacts with either GDP or GppNHp are
well conserved and the increased dissociation rates of GDP and
GTP are rather similar, we propose that the 19-amino acid
insertion in Rac1b induces similar effects on the switch I region
as Tiam1.
Interaction with Regulators and Effectors—For comparative
structural analyses of Rac1b interaction with regulators and
effectors, we used the following structures: the nucleotide-free
Rac1䡠Tiam1 complex (44); RhoGDI in complex with Rac1, Rac2,
and Cdc42 (45–47); the transition state complex of RhoA䡠GAP
(48); Rac1䡠GppNHp䡠PAK (49); and Rac1(Q61L)䡠GTP䡠p67
PHOX
(50). In addition to

2/

3/

4 (Tiam1),

4/
␣
3 (RhoGDI), or
␣
1/
␣
5
(PAK-GBD), these proteins basically interact with the switch
regions of the GTPase, except for p67
PHOX
, which contacts
␣
1,

2, and
␣
5.
A superposition of GDI on Rac1b revealed that GDI binding
to switch II and the
␣
3 helix of Rac1b could accommodate the
insertion. However, it has been recently shown that GDI does
not bind Rac1b (11). The proper contact of GDI to the
␣
3 helix
of Rac1 has been shown to be essential because a H103A
mutation abolishes its interaction with Rho-GDI (51). Because
the structure of the
␣
3 helix is not changed comparing Rac1b to
Rac1, we suggest that the insertion may sterically interfere
with GDI binding.
The association of Tiam1 with Rac1b has recently been
shown in immunoprecipitation experiments (11), indicating
that the structural requirements for the Rac1b-Tiam1 interac-
tion are not affected by the 19-amino acid insertion. However,
in contrast to the Rac1b-GAP interaction, which resulted in
stimulation of the GTP hydrolysis reaction of Rac1b (Fig. 1D),
no further increase in the nucleotide dissociation rate in the
presence of a 50-fold molar excess of the Tiam1 DH-PH domain
was observed (Fig. 1C). It is generally accepted that both GEFs
and GAPs require activation and membrane recruitment to
FIG.6.Self-activating mechanism of Rac1b. Rac1b䡠GTP accumu-
lates at the membrane because of the missing GDI regulation, the rapid
intrinsic nucleotide exchange, and the impaired GTPase reaction. A
GEF (e.g. Tiam1) may further accelerate the activation process,
whereas a GAP (e.g. p50
GAP
) can effectively catalyze the inactivation,
presuming both regulators are activated and recruited to the mem-
brane. Downstream signaling is PAK independent but possibly involves
p67
PHOX
and other yet unknown effectors (?). The arrows highlight
effective (solid,thick), ineffective (solid,thin), possible (dashed), or no
interaction (crossed).
FIG.5.The nucleotide-binding site. Shown are stereo views of the
final 2F
o
⫺F
c
electron density map around the nucleotide-binding site
of Rac1b䡠GDP (A) and Rac1b䡠GppNHp (B) contoured at 1
. The nucle-
otide is shown as a ball-and-stick representation. Water molecules
inside the nucleotide binding cleft are colored in orange, and the Mg
2⫹
ion is in turquoise.C, structural comparison of the P-loop and switch I
regions of Rac1b䡠GppNHp (red), Rac1䡠GppNHp (brown) (17), and the
nucleotide-free Rac1 in complex with the DH-PH domain of Tiam1
(green) (42). The side chains of Tyr
32
, Ile
33
, and Pro
34
are shown in stick
representation. Thr
35
, GppNHp, and the Mg
2⫹
ion (turquoise) are
shown as ball-and-stick.
Consequences of Alternative Rac1 Splicing4748
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fulfill their regulatory function in the cell. This and the fact
that Rac1b is, without external stimuli, GTP-bound in COS7
cells (Fig. 2A) strongly indicate that, independent of its regu-
lators, Rac1b intrinsically persists in the activated state.
Our biochemical data lead us to the assumption that PAK-
GBD has to stabilize the highly mobile switch regions of Rac1b
by the expense of a 7-fold lower affinity (Table II). Because of
an even more reduced affinity, we could not observe binding of
Rac1b to full-length PAK. This suggests that Rac1b is not able
to interact with PAK in the cellular context. An interaction of
Rac1b with the other major Rac effector, p67
PHOX
has not been
tested and cannot be excluded, because the p67
PHOX
-binding
site on Rac1b is largely conserved.
The fact that Rac1b does interact with GAP and PAK-GBD
(Figs. 1Dand 3) indicates (i) that the highly mobile switch
regions of Rac1b can in principle be recognized by different
binding domains and (ii) that the insertion does not neces-
sarily abolish binding. However, a proper interaction re-
quires a stabilization of the Rac1b switch regions that obvi-
ously causes an overall reduction of binding affinity, as shown
here for PAK-GBD.
Conclusions—The current study clearly demonstrates how
the 19-amino acid insertion affects two fundamental biochem-
ical properties of small GTP-binding proteins. An impaired
GTP hydrolysis coupled with an accelerated nucleotide disso-
ciation lead to a predominantly GTP-bound protein in the cel-
lular context. The insertion seems to resemble an intrinsic GEF
function by modifying the structure and dynamics of the switch
regions. The fact that Rac1b binds GDP and GTP with compa-
rable affinities and that the P-loop contacts with the phosphate
groups of both nucleotides are unaffected strongly supports the
notion that the missing stabilization of Mg
2⫹
and
␥
-phosphate
binding as well as the highly mobile switch II are the reasons
for the changed properties.
Our data together with the recent report of Matos et al. (11)
enabled us to suggest a concept of Rac1b regulation and its
interaction with downstream targets (Fig. 6). The missing reg-
ulation by GDI most likely keeps Rac1b constitutively mem-
brane-bound. In consideration of the aberrant intrinsic activi-
ties of Rac1b presented in this study, it is tempting to assume
that in contrast to Rac1, Rac1b regulation by GEFs and GAPs
seems to be redundant. Activated GAPs can effectively down-
regulate Rac1b, but it enters immediately a new activation
cycle by a self-activating mechanism. GEFs may contribute to
an even faster exchange of the bound nucleotide. We conclude
that the self-activation, the impaired GTPase reaction, and the
GDI insensitivity are the reasons for the accumulation of GTP-
bound Rac1b in cells. Although the presented overall struc-
tures of both nucleotide-bound forms of Rac1b are remarkably
similar, we cannot exclude the possibility that the missing
switch II region exhibits conformational differences and there-
fore may be responsible for the exclusive recognition and bind-
ing of GppNHp-bound Rac1b by PAK-GBD. These observations
strongly suggest that the very flexible switch regions of Rac1b
may adopt a GTP-bound conformation upon effector binding. In
this state Rac1b may interact with p67
PHOX
and certainly other
yet unknown effectors. However, important questions concern-
ing the involvement of Rac1b in signal transduction and in
tumor progression remain to be elucidated.
Acknowledgments—We thank A. Wittinghofer for continuous sup-
port and E. Lengyel for providing the cDNA of human Rac1b. We
thank I. Schlichting, W. Blankenfeld and the staff of the European
Synchrotron Radiation Facility for data collection at the ID14 beam-
line, as well as O. Daumke and A. Ghosh for critical reading of the
manuscript.
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Consequences of Alternative Rac1 Splicing 4749
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Dvorsky, Ingrid R. Vetter and Mohammad R. Ahmadian
Dennis Fiegen, Lars-Christian Haeusler, Lars Blumenstein, Ulrike Herbrand, Radovan
Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase
doi: 10.1074/jbc.M310281200 originally published online November 18, 2003
2004, 279:4743-4749.J. Biol. Chem.
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