1040 Biochemical Society Transactions (2004) Volume 32, part 6

Small GTP-binding protein-coupled receptors

M. Bhattacharya, A.V. Babwah and S.S.G. Ferguson1
Cell Biology Research Group, Robarts Research Institute, 100 Perth Drive, P.O. Box 5015, London, ON, Canada N6A 5K8, and Department of Physiology
and Pharmacology, the University of Western Ontario, London, ON, Canada N6A 5C1

Abstract
Heterotrimeric GPCRs (G-protein-coupled receptors) form the largest group of integral membrane receptor
proteins and mediate diverse physiological processes. In addition to signalling via heterotrimeric G-proteins,
GPCRs can also signal by interacting with various small G-proteins to regulate downstream effector pathways.
The small G-protein superfamily is structurally classified into at least five families: the Ras, Rho/Rac/cdc42,
Rab, Sar1/Arf and Ran families. They are monomeric G-proteins with molecular masses over the range
20–30 kDa, which function as molecular switches to control many eukaryotic cell functions. Several studies
have provided evidence of crosstalk between GPCRs and small G-proteins. It is well documented that GPCR
signalling through heterotrimeric G-proteins can lead to the activation of Ras and Rho GTPases. In addition,
RhoA, Rabs, ARFs and ARF GEFs (guanine nucleotide-exchange factors) can associate directly with GPCRs,
and GPCRs may also function as GEFs for small GTPases. In this review, we summarize the recent progress
made in understanding the interaction between GPCRs and small GTPases, focusing on understanding how
the association of small G-proteins with GPCRs and GPCR-regulatory proteins may influence GPCR signalling
and intracellular trafficking.

Introduction In particular, we will emphasize how the association of small

Heterotrimeric GPCRs (G-protein-coupled receptors) form GTPases with both GPCRs and GPCR-regulatory proteins
the largest group of integral membrane receptor proteins may influence GPCR signalling and intracellular trafficking.
and mediate diverse physiological processes, including vision,
olfaction, inflammation, immunity, cognition, pain percep- Small GTPases
tion, cardiac function and neurotransmission [1–4]. These The small G-protein superfamily contains over 100 members
receptors represent a primary target, either directly or in- that are generally classified, by structural similarity, into five
directly, for most pharmaceuticals and drugs of abuse. subfamilies: Ras family GTPases (e.g. Ras, Rap and Ral), Rho
GPCRs transduce the information provided by a diverse family GTPases (Rho, Rac and cdc42), Arf family GTPases
assortment of stimuli into intracellular second messengers by (Arf1–Arf6, Arl1–Arl7 and Sar), Rab family GTPases (>60
functioning as ligand-regulated GEFs (guanine nucleotide- members, e.g. Rab5) and Ran family GTPases [7]. The small
exchange factors) for a family of heterotrimeric G-proteins. GTPases are monomeric G-proteins with molecular masses
Agonist binding promotes the ability of a GPCR to adopt an over the range 20–30 kDa. The functions of the many small
active conformation that facilitates the exchange of GDP for G-proteins are still being elucidated and are reviewed in detail
GTP on the G-protein Gα subunit, leading to dissociation [7–9]. In general, Ras family GTPases regulate cell signalling
of the Gα and Gβγ subunits. The activated proteins Gα events that lead to alterations in gene transcription; Rho
and Gβγ can positively or negatively regulate various down- family GTPases function as regulators of the actin cytoskel-
stream effectors such as phospholipases, adenylate cyclases eton and can also influence gene transcription; Rab and Arf
and ion channels (Figure 1A). More recently, it has also family GTPases control the formation, fusion and movement
become apparent that GPCR signalling through hetero- of vesicular traffic between different membrane compart-
trimeric G-proteins leads to the activation of both Ras and ments of the cell; and Ran GTPases regulate both microtubule
Rho family GTPases [5,6] (Figure 1A). In the present review, organization and nucleocytoplasmic protein transport. All of
we outline newly discovered mechanisms by which GPCRs these GTPases function as molecular switches that control
are coupled with the regulation of activities of small GTPases. eukaryotic cell function by cycling between two intercon-
vertible forms, a GDP-bound ‘inactive’ form and a GTP-
bound ‘active’ form [7,8] (Figure 1B). The rate-limiting step
Key words: cell signalling, endocytosis, G-protein-coupled receptor, small G-protein, trafficking.
of the GDP/GTP exchange, which is the dissociation of
Abbreviations used: AT1A R, angiotensin II type 1A receptor; β 2 AR, β 2 -adrenergic receptor;
ARNO, ADP ribosylation factor nucleotide-binding site opener; EGF, epidermal growth factor; GDP, is promoted by the association of a GEF, the activity
ER, endoplasmic reticulum; fMLP, N-formylmethionyl-leucylphenylalanine; GEF, guanine of which may be regulated by an upstream signal such as
nucleotide-exchange factor; GPCR, G-protein-coupled receptor; MAPK, mitogen-activated protein
the activation of a heterotrimeric G-protein. The binding
kinase; MEF, embryonic fibroblast; MMP, matrix metalloproteinase; PLD, phospholipase D.
1
To whom correspondence should be addressed, at Cell Biology Research Group, Robarts of GTP eventually leads to a conformational change in the
Research Institute (email [email protected]). downstream effector-binding domain of the G-protein such

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Figure 1 Crosstalk between GPCRs and small GTPases

(A) The binding of agonist to a GPCR promotes the exchange of GDP for GTP on the G-protein Gα subunit, which leads to
dissociation of the Gα and Gβγ subunits. The activated proteins Gα and Gβγ can positively or negatively regulate various
downstream effectors. In addition to this ‘classical’ method of signalling, GPCRs can also signal via heterotrimeric G-proteins
to Ras (via Gβγ ) and Rho GTPases (via G12 /G13 ). Gβγ proteins stimulate Src-dependent activation of MMPs that release
heparin-binding EGF (HB-EGF), which can activate receptor tyrosine kinases (RTK), resulting in Ras activation. G12 /G13 -
mediated RhoA activation involves RhoGEFs, e.g. PDZ-RhoGEF, LARG and p115-RhoGEF. (B) Before fMLP receptor activ-
ation, the Ral GEF RalGDS is localized to the cytosol and maintained in an inactive complex with β-arrestins. fMLP receptor
activation results in the membrane translocation of the β-arrestin–RalGDS complex. β-Arrestin (Arr) receptor binding is
proposed to dissociate the β-arrestin–RalGDS complex freeing RalGDS to activate membrane-bound Ral. Ral activation results
in actin cytoskeleton reorganization. (C) Rab5 regulates the endocytosis of AT1A R into clathrin-coated vesicles and mediates
the fusion with early endosomes. Rab7 regulates the targeting of AT1A R to lysosomes for degradation, whereas Rab4 and
Rab11 regulate the rapid or slow recycling of receptors from early endosomes.

that this region interacts with either one or many downstream GPCR signalling via Ras family GTPases
effector(s), which function to either activate or inactivate
a variety of signalling pathways. The GTP-bound form of The discovery that extracellular signals that activate GPCRs
the small G-protein is converted into the GDP-bound form cause the activation of small GTPases occurred relatively
due to its intrinsic GTPase activity, which is stimulated by recently, and it provided a mechanism to explain how
GTPase-activating proteins. agonist stimulation of GPCRs resulted in alterations in

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1042 Biochemical Society Transactions (2004) Volume 32, part 6

cell morphology and tyrosine phosphorylation. The best- of the β-arrestin–RalGDS complex. Since Ral is localized
characterized mechanism by which GPCRs activate small to the inner leaflet of the plasma membrane, redistribution
GTPases is activation of the Ras/MAPK (mitogen-activated of RalGDS to the plasma membrane overcomes the spatial
protein kinase) cascade. The mechanism by which GPCRs paradox for RalGDS localization, allowing RalGDS to be
engage the Ras/MAPK signalling cascade varies between appropriately localized to activate Ral-dependent reorganiz-
different cell types and the GPCR activating the cascade (re- ation of the cytoskeleton. This molecular mechanism may
viewed in [6,7]). There are two primary mechanisms by which explain the impairment in chemotaxis observed for lymph-
GPCRs activate Ras/MAPK signalling: (i) via signals initiated ocytes obtained from β-arrestin2 null mice [21]. For Gq/11 -
by classical heterotrimeric G-protein effectors, such as the coupled receptors, Ral may be activated by the direct binding
second-messenger-dependent protein kinases, and (ii) via of Ca2+ -calmodulin to the C-terminus of RalA [22].
the transactivation of receptor tyrosine kinases [10]. In neur-
ons, Gq/11 -coupled receptors activate the Ca2+ - and protein
kinase C-regulated FAK family kinase Pyk2, which promotes GPCR signalling via Rho family GTPases
the Ras-dependent activation of the MAPK cascade [11]. It is now well established that many GPCRs for ligands
For Gi -coupled GPCRs, Gβγ subunits stimulate the Src- like acetylcholine, lysophosphatidic acid, thrombin and en-
dependent activation of MMPs (matrix metalloproteinases), dothelin induce cytoskeletal reorganization, such as filopodia
which release substances such as heparin-binding EGF formation and stress fibre formation, as well as cell prolifer-
(epidermal growth factor) from the cell surface, which in turn ation (reviewed in [5]). Although many of these GPCRs are
stimulate either the autocrine or paracrine activation of cell- coupled with Gi and Gq/11 signalling pathways and exhibit
surface EGF receptors leading to Ras-dependent activation oncogenic activity when expressed in NIH3T3 cells, the
of MAPK [12]. For receptors coupled with Gq/11 , activation transforming ability of constitutively active Gi and Gq/11
of the MMPs is mediated by Gα q/11 [13]. The activity of the proteins is several orders of magnitude less effective com-
Ras family GTPase Rap1 is increased by the activation of pared with GPCR activation (reviewed in [23]). Thus the
Gs -coupled receptors by the direct phosphorylation of Rap1 transforming abilities of many GPCRs are linked to their
by cAMP-dependent protein kinase as well as by the direct coupling with a novel G12/13 family of G-proteins. The
binding of cAMP to the Rap1 GEF EPAC [14–16]. G12/13 family of G-protein α subunits are ubiquitously
Activation of the Gi -coupled chemoattractant, fMLP expressed and exhibit 67% sequence homology with one
(N-formylmethionyl-leucylphenylalanine) receptor, leads to another, but have limited sequence homology with other
both Ras-dependent and Ras-independent activation of the Gα subunits such as Gi and Gq/11 [24]. G12/13 -mediated
Ras family GTPase, Ral [17]. Ral is required for the initiation RhoA activation involves direct interactions with RhoGEF
of a number of fMLP receptor-stimulated neutrophil res- proteins, e.g. PDZ-RhoGEF, LARG and p115-RhoGEF [25].
ponses including cytoskeletal reorganization required for For example, the association of RhoGEFs with G12/13 α
chemotaxis [9]. Yeast two-hybrid studies revealed that the Ral subunits is facilitated by the RGS (regulators of G-pro-
GEF (RalGDS) interacts with the GTP-bound form of Ras, tein signalling) domain localized within the N-terminus of
suggesting that Ral activity is Ras-dependent [18]. Consistent PDZ-RhoGEF, LARG and p115-RhoGEF [26,27]. Vogt et al.
with this observation, EGF receptor activation of Ral is [25] used Gα q /Gα 11 - and Gα 12 /Gα 13 -double-deficient MEFs
inhibited by dominant-negative Ras mutants, suggesting (embryonic fibroblasts) to determine a role for Gα q/11 sig-
that Ral activity is downstream of Ras [19]. However, for nalling in the activation of Rho via endogenous receptors.
GPCRs such as the fMLP receptor, Ral activation is mediated In these studies, it was determined that Gα q/11 -coupled
by both Ras-dependent and Ras-independent mechanisms receptors can couple with activation of Rho in Gα 12 /Gα 13 -
[17]. Recently, the Ral GEF, RalGDS, was identified as a double-deficient MEFs, albeit with less efficacy when
β-arrestin-binding partner, suggesting that β-arrestin might compared with Gα q /Gα 11 -double-deficient MEFs. Gq/11
mediate the Ras-independent mechanism for GPCR-medi- proteins may also modulate activated Rho signalling by
ated Ral activation [20] (Figure 1B). β-Arrestins were first enhancing responses downstream of Rho activation through a
identified as proteins that function to uncouple GPCRs direct association of Gq/11 with the Rho GEF, lymphoid blast
from heterotrimeric G-proteins and are instrumental in crisis [28]. Thus many GPCRs can influence Rho activity
attenuating GPCR signalling (reviewed in [3,4]). More via signalling through both Gα q/11 and Gα 12/13 families of
recently, β-arrestins have been shown to mediate the internal- heterotrimeric G-proteins.
ization of GPCRs and to function as scaffolds for the organiz-
ation of signalling complexes (reviewed in [4,10]). In the
absence of receptor activation, RalGDS exists as a complex Arf family GTPases and GPCR endocytosis
with β-arrestin in the cytosol [20]. However, in response Arf GTPases play an essential role in regulating the
to fMLP receptor activation, both β-arrestin and RalGDS membrane-trafficking events involved in endocytosis (re-
rapidly translocate to the plasma membrane, where β-arrestin viewed in [7]). Arf6 is involved in regulating both recycling
binds to the fMLP receptor to uncouple the receptor from of endosomal vesicles and receptor-mediated endocytosis.
heterotrimeric G-proteins. Translocation and binding of The activity of Arf6 is regulated by two Arf GEFs, ARNO
β-arrestin to the receptor is correlated with the dissociation (ADP ribosylation factor nucleotide-binding site opener) and

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EFA6. Several recent studies have linked Arf6 and ARNO (reviewed in [7,35–37]). The 60 different Rab GTPases
to the regulation of GPCR signalling, desensitization and constitute the largest and most diverse group of Ras-like
endocytosis [29–33]. The effects of Arf6 and ARNO on GTPases and, although these proteins are ubiquitous and
GPCR activity involve both the direct association of Arf6 highly conserved in their structure, each Rab has a distinct
with the receptors themselves as well as the association of intracellular localization [35,37]. Multiple Rab GTPases, such
both Arf6 and ARNO with β-arrestin [29–33]. The first as Rab1, Rab4, Rab5, Rab7 and Rab11, have been identified
indication that small GTPases may directly associate with to regulate ER (endoplasmic reticulum)–Golgi transport as
GPCRs comes from a study by Mitchell et al. [29], where well as the endocytosis and trafficking of GPCRs between
both Arf1/3 and Rho were demonstrated to co-immuno- early, late and recycling endosomes and lysosomes.
precipitate with M3 muscarinic acetylcholine receptors in GPCRs are synthesized and modified in the ER, and they
response to agonist activation. One functional consequence are then transported to the Golgi apparatus for additional
of this association is an enhanced coupling of receptors with post-translational modification and, then, to the cell surface.
PLD (phospholipase D) activation, which is independent of Rab1 has been demonstrated to regulate the trafficking
the heterotrimeric G-proteins Gq/11 and Gi/o . Subsequently, of both AT1A Rs (angiotensin II type 1A receptors) and
studies by the Hunzicker-Dunn laboratory demonstrated β 2 ARs from the ER–Golgi to the plasma membrane [38].
that the desensitization of luteinizing hormone receptors Several groups have investigated the role of Rab GTPases in
is influenced by the association of ARNO with β-arrestin regulating the endocytosis and recycling of GPCRs (reviewed
[31,32]. Specifically, ARNO promotes the release of mem- in [36,37]). The internalization and intracellular trafficking of
brane-bound β-arrestin such that it could bind to and de- GPCRs is a highly regulated and dynamic process that is not
sensitize the luteinizing hormone receptor, and receptor only essential for GPCR desensitization, but is also required
desensitization is blocked by the expression of a catalytically to allow the dephosphorylation and resensitization of many
inactive ARNO. GPCRs [4]. For many GPCRs, endocytosis to the early endo-
ARF1 and ARF6 have recently been shown to regulate somal compartment is required to allow dephosphorylation
GPCR endocytosis [30,33]. Specifically, Claing et al. [30] of the GPCR kinase-phosphorylated GPCRs. For example,
have revealed that ARF6 regulates the internalization of the treatments that block β 2 AR internalization prevent both
β 2 AR (β 2 -adrenergic receptor). Expression of constitutively receptor dephosphorylation and resensitization [39,40]. For
active and dominant-negative Arf6 mutants significantly the β 2 AR, dephosphorylation occurs as the receptor transits
attenuates β 2 AR internalization. β 2 AR activation promotes between the early Rab5-regulated endosomal compartment
the formation of a complex between β-arrestin, GDP-Arf6 and Rab4-regulated recycling endosomes [41]. The recycling
and ARNO. It is proposed that β-arrestin functions as of other GPCRs such as the M4 muscarinic acetylcholine
a scaffold to promote ARNO-dependent Arf6 activation receptor is mediated by Rab11-regulated slow-recycling
to facilitate β 2 AR endocytosis. Whereas Arf1 dominant- endosomes [42]. For some GPCRs, such as the protease
negative mutants have no effect on the internalization of the receptor family, targeting to the lysosomal compartment is the
β 2 AR, Arf1 may contribute to regulating the endocytosis only mechanism by which receptor signalling can be termi-
of the µ-opioid receptor [33]. Yeast two-hybrid screens nated [43], whereas the AT1A R is internalized to the early
with the C-terminal tail of the µ-opioid receptor identified endosomal compartment, where it remains [44] (Figure 1C).
PLD2 as a µ-opioid receptor-interacting protein [33]. The RabGTPases may also associate directly with GPCRs
activity of PLD2 and Arf plays a key role in regulating (Figure 1C). Yeast two-hybrid screens with the AT1A R
the endocytosis of the µ-opioid receptor [33]. PLD1/2 has C-terminal tail identified Rab5 as an AT1A R-binding partner
been implicated as a key regulator of vesicular trafficking, [44]. Rab5 not only binds to the C-terminal tail of the AT1A R,
cytoskeletal organization and endocytosis and exocytosis but agonist activation of the AT1A R also stimulates Rab5
(reviewed in [34]). Many GPCRs, including metabotropic GDP/GTP exchange, suggesting the possibility that GPCRs
glutamate receptors, can activate PLD1/2. Recently, we might function as GEFs for small GTPases. AT1A R activity
have demonstrated that metabotropic glutamate receptors leads to the homotypic fusion of early endosomes into large
function as scaffolds for the recruitment of Ral, RalGDS and hollow cored vesicular structures, the formation of which can
PLD2, and that Ral and PLD2 activity are essential for the be prevented by the expression of a dominant-negative Rab5
constitutive endocytosis of these receptors in both fibroblasts mutant. The association of Rab5 with the AT1A R prevents
and neurons (M. Bhattacharya, A.V. Babwah and S.S.G. both AT1A R recycling and targeting to lysosomes, but the
Ferguson, unpublished work). Thus PLD2 may represent removal of the last ten amino acids of the AT1A R C-terminal
a novel endocytic adaptor mediating the internalization tail to disrupt Rab5 binding results in the targeting of the
of many GPCRs either independently or in concert with AT1A R to lysosomes [45]. Overexpression of Rab11 and
β-arrestin activity. Rab7 promotes AT1A R recycling and AT1A R degradation
respectively. These observations suggest that additional Rab
GTPases may compete with Rab5 to bind the AT1A R and
Rab family GTPases and GPCR trafficking that the AT1A R may exhibit diverse desensitization and re-
Rab GTPases regulate vesicular protein transport in sensitization profiles in different cell types depending on the
endocytosis, trafficking, endosome fusion and exocytosis Rab expression profiles in the cells.

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1044 Biochemical Society Transactions (2004) Volume 32, part 6

Concluding remarks 15 DeRooij, J., Zwartkruis, F.L., Verheijen, M.H., Cool, R.H., Nijman, S.M.,
Wittinghofer, A. and Bos, J.L. (1998) Nature (London) 396, 474–477
In the last few years, considerable progress has been made 16 de Rooij, J., Rehmann, H., van Triest, M., Cool, R.H., Wittinghofer, A. and
towards understanding the downstream pathways of GPCRs. Bos, J.L. (2000) J. Biol. Chem. 275, 20829–20836
It is becoming increasingly apparent that small G-proteins 17 M’Rabet, L., Coffer, P.J., Wolthuis, R.M., Zwartkruis, F., Koenderman, L.
and Bos, J.L. (1999) J. Biol. Chem. 274, 21847–21852
play important roles in regulating a variety of fundamental 18 Hofer, F., Fields, S., Schneider, C. and Martin, G.S. (1994) Proc. Natl.
cellular processes regulated by GPCRs. The precise details of Acad. Sci. U.S.A. 91, 11089–11093
many of the molecular mechanisms governing the crosstalk 19 Wolthuis, R.M., Zwartkruis, F., Moen, T.C. and Bos, J.L. (1998) Curr. Biol. 8,
471–474
and signalling cascades among small G-proteins and various 20 Bhattacharya, M., Anborgh, P.H., Babwah, A.V., Dale, L.B., Dobransky, T.,
GPCRs remain to be elucidated. Further studies are clearly Benovic, J.L., Feldman, R.D., Verdi, J.M., Rylett, R.J. and Ferguson, S.S.G.
required to determine and clarify these signalling networks (2002) Nat. Cell Biol. 4, 547–555
21 Fong, A.M., Premont, R.T., Richardson, R.M., Yu, Y.R., Lefkowitz, R.J. and
and their biological significance. However, what is clear Patel, D.D. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7478–7483
is that GPCRs can activate small GTPases both through 22 Wang, K.L., Khan, M.T. and Roufogalis, B.D. (1997) J. Biol. Chem. 272,
classical intracellular signalling cascades and through direct 16002–16009
23 Dhanasekaran, N., Heasley, L.E. and Johnson, G.L. (1995) Endocr. Rev.
association with small G-proteins. One unresolved issue 16, 259–270
is whether GPCRs function as GEFs for small GTPases 24 Strathman, M.P. and Simon, M.I. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,
or whether they recruit protein complexes that also contain 5582–5586
25 Vogt, S., Grosse, R., Schultz, G. and Offermanns, S. (2003) J. Biol. Chem.
small GTPase GEFs and effector proteins. In conclusion, 278, 28743–28749
GPCRs not only modulate the activity of small GTPases, but 26 Fukuhara, S., Murga, C., Zohar, M., Igishi, T. and Gutkind, J.S. (1999)
small GTPase activity has a profound effect on the regulation J. Biol. Chem. 274, 5868–5879
27 Fukuhara, S., Chikumi, H. and Gutkind, J.S. (2000) FEBS Lett. 485,
of GPCR function. 183–188
28 Sagi, S.A., Seasholtz, T.M., Kobiashvili, M., Wilson, B.A., Toksoz, D. and
Brown, J.H. (2001) J. Biol. Chem. 276, 15445–15452
M.B. is the recipient of a CIHR (Canadian Institutes of Health 29 Mitchell, R., McCulloch, D., Lutz, E., Johnson, M., MacKenzie, C.,
Research) fellowship and A.V.B. is supported by a Canadian Fennell, M., Fink, G., Zhou, W. and Sealfon, S.C. (1998) Nature (London)
392, 411–414
Hypertension Society/CIHR fellowship. S.S.G.F. is the recipient of 30 Claing, A., Chen, W., Miller, W.E., Vitale, N., Moss, J., Premont, R.T. and
a Premier’s Research Excellence Award and Canada Research Chair Lefkowitz, R.J. (2001) J. Biol. Chem. 276, 42509–42513
in Molecular Neuroscience. This work is supported by an HSFO (Heart 31 Mukherjee, S., Gurevich, V.V., Jones, J.C., Casanova, J.E., Frank, S.R.,
Maizels, E.T., Bader, M.F., Kahn, R.A., Palczewski, K., Aktories, K. et al.
and Stroke Foundation of Ontario) grant T4987 and the CIHR grants (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 5901–5906
MA 15506 and MOP 62738 to S.S.G.F. 32 Mukherjee, S., Casanova, J.E. and Hunzicker-Dunn, M. (2001)
J. Biol. Chem. 276, 6524–6528
33 Koch, T., Brandenburg, L.O., Schulz, S., Liang, Y., Klein, J. and Hollt, V.
(2003) J. Biol. Chem. 278, 9979–9985
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