Translating G protein subunit diversity into functional specificity
Janet D Robishaw1 and Catherine H Berlot
Historically, it has been assumed that the functional roles of
G proteins in receptor recognition and effector regulation are
specified by their diverse a subunits. However, the discovery of
similarly diverse bg subunits that participate in both of these
functional processes has called this assumption into question;
recent work suggests that G proteins function as heterotrimers
whose roles in particular receptor signaling pathways are
determined by their specific abg subunit combinations.
Although much remains to be learned, the assembly of
specific abg subunit combinations seems to involve both
structural and spatial factors.
Addresses
100 N. Academy Ave, Weis Center for Research, Geisinger Clinic,
Danville, PA 17822-2614, USA
1
e-mail: [email protected]
Current Opinion in Cell Biology 2004, 16:206–209
This review comes from a themed issue on
Cell regulation
Edited by Craig Montell and Peter Devreotes
0955-0674/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2004.02.007
Abbreviations
FRET fluorescence resonance energy transfer
PAK1 p21-activated kinase 1
Introduction
G proteins are composed of a, b, and g subunits.
Receptor activation of G proteins requires a dual mechanism involving guanine nucleotide exchange and changes
in subunit conformation. Early studies using nonhydrolyzable GTP analogs, cholera toxin and a mutants
demonstrated that the conformational changes include
actual dissociation into activated a and bg subunits [1].
This led to the subunit dissociation model of G protein
activation. However, more recent studies indicate that
dissociation into activated a and bg subunits may not be
required for downstream signaling. For example, crosslinked or chimerically fused G protein abg trimers are
fully functional [2]. Also, FRET studies of fluorescent
G protein subunits show that although conformational
change takes place upon activation, this may not necessarily include subunit dissociation [3,4]. These data
have led to the ‘clamshell’ model of G protein activation.
According to this model, receptor-induced changes in G
protein conformation expose previously buried interfaces
between the a and bg subunits that can then direct
Current Opinion in Cell Biology 2004, 16:206–209
interaction with their respective effectors. In contrast
to the subunit dissociation model, which allows a common
pool of bg dimers to be shared among several a subunits,
the ‘clamshell’ model predicts that each bg dimer remains
closely associated with the a subunit on the receptor. As a
corollary, it further predicts that the bg subunits are not
interchangeable and have unique functions in the context
of the cellular setting, for which there is increasing
experimental evidence [5]. Finally, it predicts that, by
remaining closely associated with their activated receptors, the G protein abg subunits may serve as scaffolds for
recruitment of other signaling molecules to the plasma
membrane. For instance, kinetic evidence [6] favors a
model in which the receptor, Gq, and phospholipase C
remain associated throughout multiple GTPase cycles. In
addition to effectors, numerous studies now indicate that
G proteins also recruit soluble proteins [7], such as
receptor kinases that regulate desensitization and internalization of G protein-coupled receptors, and scaffold
proteins that organize various kinase cascades. Association with such signaling complexes may contribute to
receptor/G protein specificity.
In this review, we will present evidence indicating that G
proteins function as heterotrimers whose roles in a broad
array of receptor signaling pathways are determined by
their specific complements of abg subunits.
G protein abc subunit diversity
There are at least sixteen a subunit genes in the human
and mouse genomes [8]. The structurally diverse a subunits are grouped into four functional subclasses: Gs, Gi,
Gq and G12. Representatives of each subclass are found in
Dictyostelium, Caenorhabditis and Drosophila. In addition,
there are at least 5 b and 12 g subunit genes in the human
and mouse genomes [8], while the zebrafish genome
contains at least 7 b and 17 g subunit genes [9]. Although
the b subunits are similar, the g subunits are quite
structurally diverse. The strict conservation of their
amino acid sequences across species strongly suggests
that the various g subunits have unique functions,
although this has been difficult to prove for reasons
discussed below. The diversity of the b and g subunit
families arose much later in the evolutionary process than
that of the a subunit family [9], with Dictyostelium
containing a single b and a single g subunit gene and
Caenorhabditis and Drosophila containing only two b and
two g subunit genes. The emergence of multiple b and g
isoforms suggests that chordates require additional complexity at the level of the G protein heterotrimer to
accommodate their diverse repertoire of signaling pathways.
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Translating G protein subunit diversity into functional specificity Robishaw and Berlot 207
Functional significance of G protein abc
subunit diversity
There are now known to be several hundred receptors
that require G proteins to mediate their functions. If a
receptor recognizes a specific G protein abg subunit
combination, then combinatorial association of the known
number of these three subunits could provide the level of
selectivity that is needed to interact with this vast number
of receptors. Although such a scenario was first proposed
more than a decade ago, the demonstration that bg
diversity contributes to the formation of functionally
distinct G protein trimers has been difficult. Biochemical
approaches have revealed only modest functional differences among various G protein abg subunit combinations
[10]. This may reflect the high homology between certain members of the a, b and g subunit families that allows
them to readily substitute for one another and to compensate for any disruption of the physical organization of
signaling systems [11].
However, genetic approaches have begun to identify
important functional differences between different abg
subunit combinations. Although tractable model systems such as Saccharomyces provided the first genetic
evidence that a bg dimer can act as an active signaling
moiety in its own right [12], the relative lack of b and g
subunits in lower eukaryotes has limited their usefulness when addressing the issue of diversity. However,
antisense and ribozyme approaches have offered the
first experimental evidence indicating that certain
receptors require G proteins with particular bg subunit
compositions in higher eukaryotes. In a seminal series of
papers, Schultz and colleagues used an antisense approach to demonstrate that the somatostatin and muscarinic receptors utilize G protein trimers of different
abg subunit composition to modulate a calcium channel
in pituitary cells [13]. Over the following decade, several
groups sought to extend these findings; their success was
variable as a result of the frequently poor specificity of
antisense oligonucleotides [10]. By acting as sequencespecific nucleases, ribozymes provide greater specificity.
Recently, a ribozyme approach demonstrated that the
b-adrenergic and prostaglandin receptors require Gs
proteins of varying bg subunit composition to stimulate
adenylyl cyclase activity in kidney cells [14,15]. More
recently, a gene-targeting approach showed that the D1
dopamine receptor requires a G protein containing the
g7 subtype to stimulate adenylyl cyclase activity in a
particular region of the brain [16]. Although it provides
the first conclusive proof that a receptor recognizes a
specific complement of G protein abg subunits in the
context of the organism, this result also highlights a
large gap in our knowledge about signaling: in the vast
majority of cases, we do not know which G protein
abg subunit combinations actually exist in vivo, nor
do we understand the factors controlling their selective
assembly.
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Differential interaction of G protein subunits
Just how b and g subunits are brought together to form
specific bg dimers remains unresolved. Biochemical studies indicate that structural constraints preclude the formation of certain bg dimers, most notably those involving
the b2, b3 and g1 subtypes. Nevertheless, the vast majority
of b and g subtypes are able to form distinct bg dimers
[17]. As yet, it is not clear to what extent the specificity of
a subunits for particular bg dimers may limit the number
of possible G protein trimers. Biochemical studies suggest
that the a, b and g subunits may form preferred G protein
trimers. When bovine brain G protein trimers are separated on the basis of their a subtypes, the different groups
have very different g subtypes associated with them [18].
Intriguingly, biochemical studies revealed a direct interaction between the a and g subunits [19], raising the
possibility that the interaction of these more highly
divergent components may provide a basis for the assembly of specific G protein heterotrimers. Unfortunately,
crystallographic studies neither confirm nor reject this
possibility, as potentially interacting regions of the a and g
proteins are either truncated or disordered in the crystal
structure [20].
Recently, a model has been proposed in which an interaction between the N terminus of the g subunit and the
helical domain of the a subunit plays an important role in
receptor activation [21]. Although experimental evidence is sparse, the conformational changes proposed
in this model are supported by FRET studies of the G
protein ai1b1g2 trimer [4] and by mutagenesis studies
that reveal a role for interactions at the domain interface
of as in receptor-mediated activation [22]. In this regard,
it is noteworthy that RGS14, which interacts with the
helical domain of the a subunit to modulate GDP release,
contains a GoLoco domain showing substantial sequence
homology to the N terminus of the g1 subunit [21].
Although selectivity of a g interactions may be one
factor, it is clear that structural constraints alone cannot
account for the assembly of specific G protein trimers in
the vast majority of cases. Biochemical studies show that
most a subtypes can associate with most bg dimers, albeit
with varying affinities [10]. This suggests that cellspecific factors must be involved.
Differential expression of G protein subunit isoforms
Another mechanism for controlling assembly of specific
G protein abg trimers could be achieved by differentially expressing these subunits in specific cell types.
Many studies indicate that the a, b and g subtypes show
cell-type-specific patterns of expression [23,24], raising
the possibility that each cell contains only a subset of all
possible G protein abg combinations, which therefore
act downstream of receptors in a specific fashion. For
instance, retinal rod cells express a G protein containing
the at1b1g1 subunits to carry out phototransduction,
whereas retinal cone cells express a G protein containing
Current Opinion in Cell Biology 2004, 16:206–209
208 Cell regulation
the at2b3g8 subunits to carry out the equivalent role
[23]. The a, b and g subtypes also show specific temporal patterns of expression that can have profound
consequences on developmental processes and particular signaling cascades. For example, in HL-60 cells,
retinoic-acid-induced differentiation into neutrophillike cells involves induction of g2 expression, which
appears to mediate fMLP stimulation of PLC via Gi
[25]. The mechanisms governing G protein subunit
expression have not been defined, but may include
differences in promoter function, mRNA stability,
mRNA localization, translation efficiency, protein stability or protein localization.
Conclusions
Despite a clear role for differential G protein subunit
expression in regulating the formation of specific heterotrimers, it is clear that most cell types express multiple a,
b and g subtypes. However, it is also clear that G protein
trimers do not assemble simply through random associations. For example, ribozyme-mediated suppression of
the g7 subtype coordinately reduces the level of the b1
subtype but has no effect on the other three b subtypes
expressed in these cells [15]. A more recent study
showed that gene-targeted loss of the g7 subtype produces a striking reduction in the level of the aolf subtype,
but has no effect on the levels of the other a subtypes
[16]. As preferential interactions among a, b and g7
subunits have not been observed in vitro [10], these
results suggest that differential expression and localization must contribute to the assembly of the Golf protein in
the striatum.
Acknowledgements
Differential subcellular localization of G protein
subunits
4.
Subcellular compartmentalization of G proteins and
receptors in membrane microdomains, such as caveolae
and focal adhesions, may facilitate or impair interactions
between proteins expressed in the same cell [11,26,27].
This may account for the diverse cellular effects of
receptors that apparently couple to the same G proteins.
Such differences in subunit localization patterns may
direct the assembly of preferred G protein trimers in a
cell-specific fashion [28]. These unique localization
patterns may result from targeting events that occur
shortly after synthesis, because the a, b and g subunits
appear to play roles in mutually targeting each other to
the plasma membrane. For example, specific bg isoforms
have differing abilities to restore plasma membrane
targeting to as and aq mutants [29], and a g subunit
that is mislocalized to the mitochondria can cause a and
b to mistarget [30]. Conversely, co-expression of as is
required to target b1g2 to the plasma membrane under
certain conditions, and when as is targeted to the mitochondria, b1g2 follows [31]. Finally, fluorescently
tagged a and g subunits co-localize on the Golgi and
plasma membrane, suggesting that association of a and
bg takes place on the Golgi [32].
Current Opinion in Cell Biology 2004, 16:206–209
Recent genetic studies indicate that receptors recognize
specific G protein heterotrimers in a tissue- and cellspecific fashion. Additional studies are now needed to
identify which G protein abg subunit combinations exist
in particular cell types, to understand the mechanics of
their assembly, and to dissect their functional roles in
particular receptor signaling pathways.
Update
A recent review reveals the growing use of gene-targeted
mouse models to identify physiological functions for the
various G-protein a and bg subunits [33].
This work was supported by NIH grants GM39867 and GM58191
awarded to JDR and GM050369 awarded to CHB.
References and recommended reading
Papers of particular interest, published within the annual period of
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of special interest
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Current Opinion in Cell Biology 2004, 16:206–209