Rho-family GTPases comprise a main branch of the
Ras superfamily of small (~21 kDa) GTPases .So far,
22 human members of the Rho family have been
identified and can be subdivided into 10 groups on
the basis of their identity to Cdc42, Rac1, RhoA,
mitochondrial Rho (Miro1/RhoT1) or Rho-related BTB-
domain-containing protein (RhoBTB).Like Ras,Rho
proteins function as bi-molecular switches by adopt-
ing different conformational states in response to
binding GDP or GTP. In contrast to Rho•GDP,
Rho•GTP actively transduces signals by interacting
with downstream effectors1,2.The activation of Rho-
family proteins is often mediated through various
cell-surface receptors,including the cytokine,tyro-
sine kinase and adhesion receptors, as well as the
G-PROTEIN-COUPLED RECEPTORS (GPCRs)3,4. Rho-family
proteins are regulators of the actin cytoskeleton,
cell-cycle progression and gene transcription, and
have been implicated in cellular processes such as
adhesion and migration, PHAGOCYTOSIS, CYTOKINESIS,
neurite extension and retraction, cellular morpho-
genesis and polarization,growth and cell survival5–8.
Furthermore, aberrant regulation of Rho-family
GTPases promotes malignant transformation and is
essential for the oncogenic properties of Ras and
Cycling between GDP- and GTP-bound states is
controlled primarily by two classes of regulatory mole-
cule (FIG.1):GTPase-activating proteins (GAPs),which
enhance the relatively slow intrinsic GTPase activity of
Rho proteins;and guanine nucleotide-exchange factors
(GEFs),which catalyse the exchange of GDP for GTP
in vivo.GAPs suppress Rho activity,whereas GEFs pro-
mote Rho activity.A third set ofregulatory proteins,the
guanine nucleotide-dissociation inhibitors (GDIs),
sequester GTPases in the cytosol in a GDP-bound state.
The mechanism of release of Rho GTPases from GDIs
remains to be fully elucidated.
The first mammalian Rho GEF was identified as a
transforming gene from diffuse B-cell-lymphoma cells,
and was therefore designated Dbl10,11.Dbl contains a
region of ~240 residues that is homologous to a region
in Saccharomyces cerevisiae Cdc24,which cooperates
with Cdc42 during S.cerevisiaebudding and polarity.
Dbl was subsequently shown to function as a GEF for
human Cdc42.So Dbl and Cdc24 represented the initial
members ofa new family ofGEFs12that specifically acti-
vate Rho GTPases.Since then,69 distinct members of
this Dbl family have been identified in humans (FIG.2).
The region of homology between Dbl and Cdc24
contains a ~200-residue Dbl homology (DH) domain
and an adjacent,C-terminal,~100-residue PLECKSTRIN
HOMOLOGY (PH) DOMAIN.Small GTPases have well-defined
GEF MEANS GO:TURNING ON
RHO GTPasesWITH GUANINE
Kent L.Rossman*‡,Channing J.Der*‡and John Sondek*‡§
Abstract | Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation
of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate
numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct
homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in
humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure
to do so can have significant consequences and is reflected in the aberrant function of Dbl-family
GEFs in some human diseases.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 6 | FEBRUARY 2005 | 167
and Biophysics,University of
North Carolina at Chapel
Correspondence to: C.J.D.
A seven-helix membrane-
spanning cell-surface receptor
that signals through
and -hydrolysing G proteins to
stimulate or inhibit the activity
of a downstream enzyme.
An actin-dependent process by
which cells engulf external
particulate material by extension
and fusion of pseudopods.
R E V I E W S
The separation of a cell into two,
which is marked by ingression of
the cleavage ‘furrow’between
two segregated masses of
PLECKSTRIN HOMOLOGY (PH)
A sequence of ~100 amino acids
that is present in many signalling
molecules and that binds to lipid
products of phosphatidylinositol
3-kinase.Pleckstrin is a protein
of unknown function that was
originally identified in platelets.
It is a principal substrate of
protein kinase C.
Regions of nucleotide-binding
proteins that have different
conformations in the
to the diphosphate-bound,state.
An element of protein secondary
structure in which hydrogen
bonds that lie along the
backbone of a single polypeptide
cause the chain to form a right-
168 | FEBRUARY 2005 | VOLUME 6
R E V I E W S
Structures ofDH domains.The three dimensional (3D)
structures of several DH domains, both free and in
complex with Rho GTPases,have been determined13–23.
The DH-domain fold is structurally distinct relative to the
domains of other GEFs and is consistently shown to be
helical (FIG.3).It comprises 10–15 α-HELICESand 310-HELICES
that are roughly arranged along six main axes to form an
oblong helical bundle that has been compared in appear-
ance to a chaise longue13,with the ‘seat back’created by a
U-shaped arrangement ofα-helices.
DH domains have three conserved regions
(CR1–CR3), which pack to form the domain core.
CR1 and CR3,along with conserved residues within
the C terminus of the domain (helix α6),form a con-
tiguous patch that constitutes the bulk of the GTPase-
binding surface. Amino-acid substitutions within
these regions typically adversely affect nucleotide-
exchange activity14,16 . The largest conformational
differences among different DH domains occur in the
length and orientation of the C-terminal helix;subtler
differences are found in the relative positions of the
seat backs.For example,the seat back within the struc-
ture of T-cell-lymphoma invasion and metastasis-1
(Tiam1) bound to Rac1 is more upright relative to the
DH domains of Son-of-sevenless-1 (Sos1) and β-Pix
(Pak-interacting exchange factor β; also known as
COOL1 and ARHGEF6)13and this difference might
be functionally important.
The interface between DH domains and Rho GTPases.
DH domains interact extensively with the switch
regions of Rho GTPases (FIG. 3). Switch 1 (residues
25–39 in Cdc42) interacts with CR1 and CR3;a highly
conserved glutamate (Glu639 in Dbl’s big sister
(Dbs)) in CR1 is crucial for complex formation and
nucleotide-exchange activity.Switch 2 (residues 57–75
in Cdc42) predominantly contacts CR3 and portions
of the C-terminal helix (α6) of the DH domain.
Conserved hydrophobic residues within switch 2
anchor it into a hydrophobic cleft on the surface of the
DH domain.Two residues in DH domains — a con-
served basic residue (Lys774 in Dbs) in CR3 and a
semi-conserved Asn (Asn810 in Dbs) — also make
significant interactions with switch 2 to contribute to
the exchange potential.
Furthermore, a significant portion of the Rho-
GEF–GTPase interface is mediated by interactions
between the seat-back region of the DH domain and
structural elements between the switch regions of the
GTPases — mainly within the β2- and β3-strands of
the GTPase. These interactions are highly variable
among different DH domains and GTPases,and they
mediate Dbl selectivity among the Rho-GTPase family.
Mechanism ofnucleotide exchange.DH domains cause
the remodelling of the switch regions to significantly
alter the nucleotide-binding pocket,while leaving the
remainder of the GTPase unperturbed (FIG. 4). The
switch regions are reconfigured into essentially identical
conformations between different Rho-GEF–GTPase
nucleotide- and Mg2+-binding pockets. The form of
nucleotide (GDP or GTP) that is bound modulates the
conformation of the SWITCH REGIONS, whereas Mg2+is
required for high-affinity binding ofguanine nucleotides
in Rho GTPases. DH domains are responsible for
catalysing the exchange of GDP for GTP within Rho
GTPases by promoting GTPase intermediates that are
devoid of nucleotide and Mg2+(FIG.1).In cells,GTP is
preferentially loaded into Rho GTPases during nucleotide
exchange because GTP is found at substantially higher
concentrations than GDP.
DH-associated PH domains,by binding phospho-
inositides,have been proposed to localize Dbl proteins
to plasma membranes,and to regulate their GEF activity
through allosteric mechanisms.Outside the DH–PH
domains,Dbl-family proteins show significant diver-
gence and typically contain other protein domains that
underlie the unique cellular functions of the different
In this review,the molecular details that control the
guanine nucleotide-exchange activity and selectivity of
Dbl-family proteins for Rho GTPases are outlined.In
addition,special attention is paid to intra- and intermol-
ecular mechanisms that regulate this exchange,with
particular emphasis on roles for DH-associated PH
domains. The review concludes by describing the
expanding set ofdiseases that arise from malfunctioning
Figure 1 | Regulating Rho-GTPase activity. Rho GTPases are considered functionally ‘primed’
when they are bound to GTP and essentially non-functional when they are GDP-bound. These two
nucleotide-bound states are tightly regulated. Guanine nucleotide-dissociation inhibitors (GDIs)
mainly bind the switch regions and the C-terminal isoprenyl moiety (orange wavy line) of Rho
GTPases to sequester them in the cytosol. The functional importance of GDI sequestration is
poorly understood, but it might be used to provide a large, stable pool of Rho GTPases that can be
easily mobilized on extensive Rho activation. Our knowledge of GDI release is also incomplete, but
this process is probably regulated, and is necessary before the engagement of guanine nucleotide-
exchange factors (GEFs), which also bind the switch regions. GEFs stabilize nucleotide-depleted
GTPases. However, owing to the relatively high concentration of intracellular GTP, nucleotide-
depleted complexes rapidly dissociate into GTP-bound GTPases and free GEFs. When they are
GTP-bound, Rho GTPases regulate the activity of their binding partners, or effectors (E), to
promote a host of cellular responses that usually influence the organization of the actin
cytoskeleton or the expression levels of various genes. GTPase-activating proteins (GAPs)
stimulate the intrinsic hydrolytic capacity of Rho GTPases to promote GDP-bound forms and
terminate signalling. Pi, inorganic phosphate.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 6 | FEBRUARY 2005 | 179
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We would like to apologize for not being able to cite original work of
many colleagues due to space constraints. Our studies are sup-
ported by grants to C.J.D. and to J.S from the National Institutes of
Health. K.L.R. was supported by a fellowship from the National
Competing interests statement
The authors declare no competing financial interests.
The following terms in this article are linked online to:
SopE | SopE2
BAR domain | DH domain | PH domain
Saccharomyces genome database:
Cdc24 | Cdc25
Cdc42 | Dbl | Dbs | ITSN-L | LARG | Miro1 | p115-RhoGEF | PDZ-
RhoGEF | α-Pix | β-Pix | Rac1 | Ras | Rcc1 | RhoA | RhoBTB1 |
RhoD | RhoE | RhoF | RhoG | RhoH | RhoV | Sos1 | Tara | Tiam1 |
Trio | Vav1
SMART database: http://smart.embl-heidelberg.de
Protein Data Bank: http://www.rcsb.org/pdb/
Access to this links box is available online.