From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611
A large number of hormones, neurotransmitters,
chemokines, local mediators, and sensory stimuli exert their effects on
cells and organisms by binding to G protein-coupled receptors. More than a thousand such receptors are known, and more are being discovered all the time. Heterotrimeric G proteins transduce ligand binding to
these receptors into intracellular responses, which underlie physiological responses of tissues and organisms. G proteins play important roles in determining the specificity and temporal
characteristics of the cellular responses to signals. They are made up
of G proteins are inactive in the GDP-bound, heterotrimeric state
and are activated by receptor-catalyzed guanine nucleotide exchange
resulting in GTP binding to the G
INTRODUCTION
Top
Introduction
References
,
, and
subunits, and although there are many gene
products encoding each subunit (20
, 6
, and 12
gene products
are known), four main classes of G proteins can be distinguished:
Gs, which activates adenylyl cyclase; Gi, which
inhibits adenylyl cyclase; Gq, which activates
phospholipase C; and G12 and G13, of unknown
function.
subunit. GTP binding leads to
dissociation of G
·GTP from G
subunits and activation of
downstream effectors by both G
·GTP and free G
subunits. G
protein deactivation is rate-limiting for turnoff of the cellular response and occurs when the G
subunit hydrolyzes GTP to GDP. The
recent resolution of crystal structures of heterotrimeric G proteins in
inactive and active conformations provides a structural framework for
understanding their role as conformational switches in signaling
pathways. As more and more novel pathways that use G proteins emerge,
recognition of the diversity of regulatory mechanisms of G protein
signaling is also increasing. The recent progress in the structure,
mechanisms, and regulation of G protein signaling pathways is the
subject of this review. Because of space considerations, I will
concentrate mainly on recent studies; readers are directed to a number
of excellent reviews that cover earlier studies.
G Protein Structure
subunits contain two domains, a domain involved in binding
and hydrolyzing GTP (the G domain) that is structurally identical to
the superfamily of GTPases including small G proteins and elongation factors (1) and a unique helical domain that buries the GTP in the core
of the protein (2, 3) (Fig. 1). The
subunit of heterotrimeric G proteins has a 7-membered
-propeller
structure based on its 7 WD-40 repeats (4-6). The
subunit
interacts with
through an N-terminal coiled coil and then all along
the base of
, making extensive contacts (Fig. 1). The
and
subunits form a functional unit that is not dissociable except by
denaturation. G protein activation by receptors leads to GTP binding on
the G
subunit. The structural nature of the GTP-mediated switch on the G
subunit is a change in conformation of three flexible regions designated Switches I, II, and III to a well ordered, GTP-bound activated conformation with lowered affinity for G
(7) (Fig. 1).
This leads to increased affinity of G
·GTP for effectors, subunit
dissociation, and generation of free G
that can activate a
number of effectors.
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Fig. 1.
Upon GTP binding to G , the G
-binding
site is rearranged and the subunits dissociate. Ribbon diagrams of
G protein subunits shown are the activated GTP
S-bound
G
t subunit (A) and the inactive GDP-bound
chimeric G
t/G
i subunit (B) (2,
5). Notice the N-terminal helix is visible only in the GDP-bound
structure. The G
subunit is silver, and the bound
nucleotides are magenta. The G
contact sites on G
are
indicated by space-filled residues. Polar residues are pink,
hydrophobic residues are yellow, basic residues are
blue, and acidic residues are red. The relative
orientations of the
contact sites in the switch interface of
G
t·GTP
S are very different from the G
·GDP and
result in decreased
binding. C, the
G
1
1 dimer (4). The G
subunit, in
metallic pink, forms a seven-bladed propeller structure that
contains a water-filled pore. The G
subunit, in blue, is
an
-helical structure that lies along the bottom of G
. The N
termini of G
and G
form a parallel coiled coil. When the subunits
dissociate, G
is free to activate a number of effectors as
discussed in the text.
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Mechanism of Activation of G Proteins by Receptors |
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G protein-coupled receptors have a common body plan with seven
transmembrane helices; the intracellular loops that connect these
helices form the G protein-binding domain (Fig.
2). Although no high resolution structure
of a G protein-coupled receptor has yet been determined, recently a low
resolution electron diffraction structure of rhodopsin, a model G
protein-coupled receptor, shows the position and orientation of the
seven transmembrane -helices (8, 9). Both mutagenesis and
biochemical experiments with a variety of G protein-coupled receptors
suggest that receptor activation by ligand binding causes changes in
the relative orientations of transmembrane helices 3 and 6. These
changes then affect the conformation of G protein-interacting
intracellular loops of the receptor and thus uncover previously masked
G protein-binding sites (10, 11) (reviewed in Ref. 12). When an
activated receptor interacts with a heterotrimeric G protein, it
induces GDP release from the G protein. It is thought that the receptor contact sites on the G protein are distant from the GDP-binding pocket,
so the receptor must work "at a distance" to change the conformation of the protein (13). Because GDP is buried within the
protein between the two domains of G
, this must necessarily involve
changing some interdomain interactions. Upon GDP release and in the
absence of GTP a stable complex between the activated receptor and the
heterotrimer is formed. This so-called "empty pocket" conformation
is of great interest, but its structure is as yet unknown.
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What are the regions on G proteins that contact receptors, and how does
G protein activation occur? The conformation of the GDP-bound
heterotrimeric G proteins Gt and Gi (5, 6)
shows the overall shape of the GDP-bound heterotrimer and the residues on the surface that can interact with other proteins and provides the
structural context for understanding a variety of biochemical and
mutagenesis studies of receptor-interacting regions on G proteins. The
N-terminal region of the subunit and the C-terminal region of the
subunit are both sites of lipid modification (reviewed in Ref. 14).
These lipidated regions are relatively close together in the
heterotrimer, suggesting a site of membrane attachment. There is good
evidence for receptor contact surfaces on all three subunits.
On the subunit, the best characterized receptor contact region is
at the C terminus (reviewed in Refs. 13 and 15). The last 7 amino acids
of the
subunit are disordered in the heterotrimer crystal
structures, and analysis of receptor-binding peptides selected from a
combinatorial peptide library shows that these 7 residues are the most
critical (16). Studies using chimeric G
subunits confirm that in
fact the last 5 residues contribute importantly to specificity of
receptor G protein interaction. Elegant mutagenesis studies have shown
that the C terminus of the third intracellular loop of receptors binds
to this C-terminal region on G
subunits. In the case of
M2 muscarinic receptor coupling to Gi, the
exact residues of the receptor that are critical for recognizing the C
terminus of G
i/o have been elucidated (Val-385, Thr-386,
Ile-389, and Leu-390) (17).
A larger region of the C-terminal region of G subunits, as well as
the N-terminal helix, has been implicated in receptor contact.
Alanine-scanning mutagenesis of G
t (18) and analysis of
residues conserved in subclasses of G protein
subunits (19) both
identify a number of residues in the C-terminal 50 amino acids of
G
t that contact rhodopsin. Arg-310 located at the
4-
6 loop of G
t is completely blocked from tryptic
proteolysis in the presence of light-activated rhodopsin, suggesting
that the
4-
6 loop region contributes to receptor contact (20).
The
4-
6 loop has also been implicated in specific interaction of the 5HT1B serotonin receptor with G
i1 as
well as in receptor-catalyzed Gi activation
(21).
It is clear that the subunits of heterotrimeric G proteins
enhance receptor interaction with
subunits (reviewed in Ref. 15).
Single Ala mutations in residues of the
subunit that contact the
subunit block receptor-mediated GTP/GDP
exchange.1 This suggests that
the
subunit must hold the
subunit rigidly in place for GDP
release to occur. Direct binding interactions between receptor and
subunit have been reported (24-26). A cross-linking study
demonstrated that the C-terminal 60-amino acid region of G
can be
cross-linked to an
2-adrenergic receptor peptide
corresponding to the intracellular third loop of the receptor (24). In
addition, the C-terminal region of the
subunit of G proteins has
been shown to be involved in receptor coupling and specificity (25, 26).
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Mechanisms of Effector Activation |
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Upon GTP binding to the subunit, the
·GTP (a*) and
subunits dissociate (5, 7). In the GTP-bound, active conformation, a
new surface is formed on G
* subunits (27), and they interact with
effectors with 20-100-fold higher affinity than in their GDP-bound
state. The various G
*s interact in a highly specific manner with the
well studied, classical effector enzymes through this surface.
G
*s activates (and G
*i inhibits) adenylyl
cyclase, G
*t activates photoreceptor cGMP
phosphodiesterase, and G
*q activates phospholipase
C-
. However, this conserved switch surface on G
subunits does not
explain the exquisite specificity of G protein
subunit effector
interaction. A chimeric G
t/G
i approach identified two other regions that underlie the specific interaction of
G
t with phosphodiesterase
(27). Similar regions are
involved in effector interaction of G
q with
PLC2 (80) and
G
s with adenylyl cyclase (AC) (reviewed in Ref. 15).
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Novel ![]() |
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The major classes of Gs, the Gs, Gi,
and Gq families of
subunits, have well known cellular
targets. More recently yeast two-hybrid screening has uncovered new
targets. GAIP, a G
-interacting protein and a member of the RGS
family of GTPase-activating proteins (reviewed in Ref. 28), was first
identified in this way; and recently two more putative
targets,
nucleobindin (29) and a novel LGN repeat protein (30), were described
by Insel and co-workers. So far, no physiological role of the latter
two G
targets has been determined.
Other effectors of G protein subunits are being discovered. For
example, G
q directly stimulates the activity of
Bruton's tyrosine kinase in vitro as well as in
vivo in lymphoma cells (31). Two G
subunits without known
effectors are G
12 and G
13. They are
reported to couple to thrombin, thromboxane, and angiotensin receptors
(32). The cellular effects of mutant constitutively activated forms of
these G proteins have been studied, and it is well established that
they can regulate Na+/H+ exchange (33). They
are involved in bradykinin activation of voltage-dependent
Ca2+ channels via activation of Rac and Cdc42 (34). To
understand the biological roles of G
13, knockout mice
were produced (35). Homozygous G
13 (
/
) mice were
never found, and although embryos were normal at embryonic day 8.5, they were resorbed before embryonic day 10.5. It appeared that lack of
G
13 led to an impaired angiogenesis of endothelial cells
and caused inability to develop an organized vascular system. In
addition, G
13 (
/
) embryonic fibroblasts showed
greatly impaired migratory responses to thrombin, suggesting that
chemotaxis was impaired. Interestingly, although G
12
shares 67% amino acid identity to G
13, it cannot
substitute for G
13.
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Once G·GTP has dissociated from G
, free
is an
activator of a dizzying array of proteins, and the list continues to
increase (see Ref. 36 for review). Significantly, the conformation of free G
is identical to G
in the heterotrimer (4),
suggesting that G
inhibits G
interactions with its effectors
through the G
-binding site on G
.1 Evidence for this
comes from the laboratory of Iyengar and co-workers (37), who found a
peptide from ACII that bound to G
and blocked its activation of
various effectors, suggesting that part of the effector binding site is
shared between ACII, G protein-activated inward rectifier
K+ channel (GIRK), and PLC
. Cross-linking and docking
experiments localized the binding site to a part of the G
-binding
region (38). Besides the G
-binding region, other regions of G
subunits that have been implicated in effector interaction include the N-terminal coiled coil (39, 40) and blades 1 and 7 of the
-propeller
of G
(41, 42).
G has well defined effects on some isoforms of the classical
second messenger enzymes, PLC
2 and -
3 (reviewed by Ref. 43) and
AC (G
stimulates G
s-activated ACII, -IV, and -VII
whereas it inhibits ACI (44)). It also recruits the
-adrenergic
receptor kinase to the membrane where the kinase phosphorylates
activated
-adrenergic receptors. It binds to the phosphoprotein
phosducin, which is thought to sequester
and thereby regulate
its availability via a cAMP-dependent protein
kinase-regulated mechanism. Phosducin-like proteins have also been
shown to bind to G
(45). Elucidation of the crystal structure of
the phosducin-G
complex showed that there is a shared surface on
the top of G
for interaction with G
and phosducin but that a
second site of interaction occurs between phosducin's C terminus and
-propeller blades 1 and 7 at the side of G
(46).
Interestingly, the phosphorylation site on phosducin, which regulates
its affinity with G
, is far from the protein-protein
interface.
In addition, G serves as the direct activator of certain G
protein-responsive K+, Ca2+, and perhaps also
Na+ channels (for reviews, see Refs. 36, 47, and 48).
IKACh is the inwardly rectifying K+ channel
responsible for slowing heart beat in response to the parasympathetic
transmitter acetylcholine. It is a homo- or heteromultimer of GIRK (49)
monomers found in the heart and brain. G
subunits bind the N- and
C-terminal intracellular domains of GIRKs and directly activate them
(49-51). The G
subunit similarly plays an important modulatory
role in certain presynaptic Ca2+ channels (52, 53),
especially
1A,
1B, and to a lesser extent
1E but not
1C,
1D, or
1S isoforms (47). It has been shown that G
inhibits
Ca2+ channel current by directly contacting two regions on
Ca2+ channel
1 subunits: the intracellular I-II loop
(55, 56) and the C terminus (57, 58).
G also directly activates more than one phosphatidylinositol
3-kinase isoform (59). There is a unique G
-responsive
phosphatidylinositol 3-kinase, P110
, that does not have a p85
subunit or the N-terminal p85-binding region on the catalytic subunit
(60, 61). G
has also been reported to activate a number of
kinases as well, for example, the Raf1 protein kinase (62) and Bruton
and Tsk tyrosine kinases (63).
In yeast, G is the activator of a pheromone-stimulated MAP kinase
pathway. It is known to bind to the N-terminal region of the scaffold
protein Ste5 in yeast (64). Recently, Thorner and co-workers (65)
showed that Ste5 contains a homodimerization domain, which is required
for
binding. They demonstrated that G
interaction leads to
oligomerization of this domain on Ste5. Most interestingly,
dimerization of this domain by making a glutathione S-transferase fusion protein of Ste5 leads to
G
-independent activation of the MAP kinase cascade. Recently,
yeast G
was also shown to activate Cdc24, the exchange factor for
the Rho-type GTPase Cdc42 (66). G
has also been reported to bind
to other members of the Rho family of GTPases, Rho and Rac (67), as
well as to the small G protein Arf (ADP-ribosylation factor), which is
involved in coat formation and vesicular trafficking (68).
Given this very rich and expanding list of G effectors and
effector activation mechanisms, a number of key questions are posed for
future investigation. Under what physiological situations are the
various effectors activated, and what are the constraints that keep all
of these effectors from being activated at the same time? Does more
than one G protein-coupled signaling pathway need to be activated for
enough G
to be generated to cause activation of these effectors?
What is the specificity of G
effector interactions and what is
the mechanism by which effector activation occurs? And finally what is
the turnoff mechanism?
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Determinants for G![]() ![]() |
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There are multiple genes for G and G
, and most G
pairs
can form functional G
s (reviewed in Ref. 36). One of the first questions that was posed was whether different G
s regulated different effectors. The answer from a large number of biochemical experiments was: not much. G
1
1 is better
than the others at interacting with rhodopsin and phosducin in
photoreceptor cells and somewhat worse than all the other G
pairs
at interacting with other effectors. One series of studies that showed
selectivity of G
pairs at interacting with receptors and
effectors was done using antisense oligonucleotides to suppress the
translation of particular proteins, and these studies showed a very
high degree of selectivity (see below). Other evidence of specificity,
using different techniques, is slowly emerging. G
5, a
recently discovered G
subunit found in the central nervous system
(69), differentially couples to two MAP kinase pathways (54).
Because G can inhibit all the actions of G
, the
G
-binding residues are candidate effector activation determinants.
We have tested this idea by singly mutating the 15 G
-binding
residues of G
to alanines, and in all effectors that have been
examined, some of the mutants no longer activate the
effector.2 In each effector interaction, however, different
residues clustered on the surface of G
are critical, suggesting a
mechanism whereby a unique contact surface of G
can make specific
interactions with a number of different effectors. Interestingly, in
some cases, removing the side chain increases the potency of the mutant
G
to activate an effector.
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Specificity of Signaling Defined by Molecular Interactions |
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The complexity of signal transduction events in cells that are receiving and processing multiple signals is the subject of intense research. Some of the key questions are: 1) how much specificity is encoded in the direct protein-protein interactions; 2) are there other levels of cellular organization that impart specificity; and 3) what are the mechanisms of cross-regulation resulting in the final integrated cellular response?
It is well known that multiple receptors can converge on a single G protein, and in many cases a single receptor can activate more than one G protein and thereby modulate multiple intracellular signals. In other cases, it seems that interaction of a single receptor with a given G protein is regulated by a high degree of selectivity imparted by specific heterotrimers. A number of excellent reviews describing the determinants of specific receptor-G protein interaction have recently appeared (12, 13, 70-72). Earlier in vitro studies of receptor-G protein interaction were often characterized by high promiscuity of receptor-G protein interaction, but a number of recent studies demonstrate that some receptors discriminate even between related G proteins within the same family.
In situ there can sometimes be high specificity. How is it
achieved? The most exquisite specificity of receptor coupling to intracellular pathways by G proteins in vivo has been
demonstrated using antisense oligonucleotides to suppress translation
of specific G protein subunits. This technique allows suppression of
distinct components involved in the signal transduction pathway and
examination of any subsequent impaired cellular responses. Kleuss
et al. (73) showed that inhibition of calcium channels by
somatostatin receptors in the GH3 cell is mediated by
Go2
1
3, whereas inhibition
by M4 muscarinic receptors is mediated by
G
o1
1
4. The elimination of
G
o by antisense technique abolishes somatostatin,
M4 muscarinic, or D2 dopamine receptor-mediated
inhibition of calcium entry in rat pituitary
GH4C1 cells (74). By contrast, depletion of
G
i2 selectively impairs receptor-mediated inhibition of
cAMP accumulation in the same system. Another antisense study indicates
that the M1 muscarinic receptor utilizes a specific G
protein complex composed of
G
q/11
1/4
4 to activate
phospholipase C (75). A recent study showed coupling of angiotensin II
AT1A receptors to regulation of Ca2+ channels,
calcium-induced calcium release channels, and
Na+/H+ exchange is via
13
1
3 (76). This level of
specificity is not seen in vitro or in transfection studies
using overexpressed proteins. This raises the question of how targeting
proteins or other cellular mechanisms can achieve high specificity.
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Limiting the Repertoire of Signaling Outcomes |
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A number of organizing and targeting proteins and cellular
structures are candidates for a role in specifying protein interactions in G protein signaling cascades. Another potential regulator of G
protein specificity is targeted inactivation of a G protein by a
GTPase-activating protein (discussed in Ref. 28). In yeast, Ste5 is a
scaffold protein that organizes the MAP kinase sequential enzyme
cascade and contributes to specificity and fidelity of signaling (77).
No mammalian homolog of Ste5 has been found. A particularly interesting
possible scaffold for G protein-coupled signal transduction molecules
is the growing family of PDZ domain-containing proteins, so named for
the three proteins that contain them, postsynaptic density protein 95 (P), Drosophila discs large tumor suppressor (D), and zona
occludens protein (Z) (for review see Ref. 23). An unusual PDZ domain
containing protein in Drosophila photoreceptors called InaD
has 5 PDZ domains, each of which bind different signaling molecules of
the Gq-regulated visual cascade including rhodopsin, PLC, protein kinase C, and the transient receptor potential protein (Trp), a homologue of the calcium-induced calcium release channel (23,
78, 79). Notably, Gq was missing from the complex. Another
unusual PDZ domain-containing protein, Homer, contains a single PDZ
domain, which binds to certain G protein-coupled metabotropic glutamate
receptors in the brain (22). Other scaffold proteins are characterized
by having multiple conserved domains such as
phosphotyrosine-recognizing Src homology 2 (SH2) domains, SH3 domains,
pleckstrin homology domains, Dbl homology domains, and domains with
enzymatic activities, particularly activity controlling the GTP binding
state of small G proteins such as guanine nucleotide exchange and
GTPase-activating protein activity. Future studies may reveal more
scaffolding or clustering mechanisms that may greatly increase the
specificity of in vivo signal transduction by heterotrimeric
G proteins.
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Summary |
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Progress in areas of research that once might have seemed distant from the field of G protein signaling now shows that G proteins are involved in a broad range of cellular regulatory activities. The understanding of how the proteins interact (receptors, G proteins, and effectors, as well as other regulatory proteins) thus has enormous implications for physiology. The rapid progress in determining three-dimensional structures of G proteins, and more recently their regulators and effectors, has illuminated the search for mechanisms of activation and regulation and has allowed structure-based mutagenesis to test these ideas. The structural and mechanistic studies will in the future also hopefully provide opportunities to alter those interactions in pathological situations.
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ACKNOWLEDGEMENT |
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I acknowledge Carolyn Ford for careful reading of the manuscript and help preparing the figures.
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FOOTNOTES |
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* This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. This is the first article of three in the "Signaling by Heterotrimeric G Proteins Minireview Series."
To whom correspondence should be addressed: 5-555 Searle,
Northwestern University Medical School, 320 E. Superior, Chicago, IL
60611. Tel.: 312-503-1109; Fax: 312-503-7345; E-mail:
h-hamm{at}nwu.edu.
1 C. E. Ford, N. P. Skiba, H. Bae, Y. Daaka, E. Reuveny, L. R. Shekter, R. Rosal, G. Weng, C. S. Yang, R. Iyengar, R. J. Miller, L. Y. Jan, R. J. Lefkowitz, and H. E. Hamm, submitted for publication.
2
The abbreviations used are: PLC, phospholipase
C; AC, adenylyl cyclase; GIRK, G protein-activated inward rectifier
K+ channel; MAP, mitogen-activated protein; GTPS,
guanosine 5
-3-O-(thio)triphosphate.
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REFERENCES |
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