Departments of Pharmacology and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032
GS was identified over 20 years ago as a heterotrimeric protein required for hormone-dependent
activation of adenylyl cyclase in liver membranes (10).
Since those original studies, Gs has come to be recognized
as a ubiquitously expressed G protein family member that relays signals
from a diverse array of heptahelical receptors to adenylyl cyclase as
well as other effectors. Gs is composed of an
Several clinical disorders have been linked to aberrant Gs
signaling. The pathogenic exotoxins of Vibrio cholerae and
exotoxins of certain strains of Escherichia coli catalyze
the ADP-ribosylation of a key arginine residue at position 201 in the
GTPase domain of Mutations that inhibit the intrinsic GTPase activity of
Transgenic mice that overexpress normal or mutationally activated
G In the current article in focus (Ref. 5; see p. C386 in
this issue) Huang et al. have used a similar molecular approach to
target the constitutively activated Q227L G Like most molecular models, transgenic mice with PEPCK-driven Q227L
G Huang et al. (5) have developed a model that should reveal
clinically important G Direct consequences of Q227L G
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s-subunit (which contains the guanine nucleotide binding
site) complexed with a tightly associated dimer of
- and
-subunits (2). The basic model of G protein activation involves two independent cycles, with both guanine nucleotide exchange
and subunit dissociation regulating Gs activity. In the basal state, Gs exists as an inactive GDP-liganded
s-
heterotrimer. G protein activation promotes a
conformational change that catalyzes the exchange of GDP for GTP on the
s-subunit. This results in the dissociation of
s from
-dimers, allowing both the freed GTP-liganded
s-subunit and the liberated
-dimers
to regulate effectors. Signal termination is via hydrolysis of GTP to
GDP by the intrinsic GTPase of
s. The GDP-liganded
s reassociates with
-dimers to reform inactive
heterotrimers (thereby preventing further
-dimer modulation of
effectors). However, recent studies identify RGS-PX1 as a bifunctional
protein that acts both as a "regulator of G protein signaling"
(RGS) to accelerate GTP hydrolysis on Gs and a regulator of
endocytic trafficking to lysosomes. The link between G protein
signaling and membrane trafficking events may be a common theme that
introduces spatial and temporal constraints on signaling protein
interactions in native cells (15).
s. Mutagenesis and X-ray
crystallographic studies identify this residue as critical for the
proper orientation of the
-phosphate of GTP and efficient GTP
hydrolysis (12). Covalent modification of
Arg201 slows the inherent GTPase activity of
s and impairs signal termination. This results in a
constitutively activated
s, which persistently stimulates adenylyl cyclase (even in the absence of ligand). The rise
in cAMP levels in intestinal mucosal cells promotes salt and water
secretion, leading to profuse watery diarrhea.
s also have been identified in many endocrine tumors,
where cAMP is a mitogenic signal (13). Oncogenic
G
s mutations have been mapped to Arg201 (the
site for ADP-ribosylation by cholera toxin) as well as an adjacent
glutamine (which also contributes to optimal GTP
-phosphate alignment and efficient GTP hydrolysis). The
Arg201-activating
s mutation has also been
identified in affected tissues of patients with McCune-Albright
syndrome (MAS; Ref. 7). This is a sporadic congenital
disorder caused by a dominant somatic mutation in early development;
the clinical features of MAS are dictated by the distribution of cells
bearing the mutation. MAS generally is characterized by the triad of
scattered areas of hyperpigmentation (café au lait spots),
polyostotic fibrous dysplasia, and autonomous hyperfunction of one or
more endocrine glands (leading to precocious puberty, hyperthyroidism,
Cushing syndrome, or acromegaly). When MAS abnormalities are restricted
to bone, skin, and endocrine organs, there is little effect on
mortality. However, some patients with MAS manifest a more severe form
of the disorder, with more widespread abnormalities beyond the
traditional target tissues. This presumably reflects a more widespread
distribution of the G
s mutation. For example, the
constellation of cardiac abnormalities identified in some patients with
MAS (cardiomegaly, arrhythmias, and sudden death in infancy) is
consistent with elevated G
s activity in myocardial tissue.
s in specific tissues provide models to explore the
functional role of G
s in human disease.
Cardiac-selective overexpression of wild-type G
s has
been accomplished with the
-myosin heavy chain (
-MHC) promoter.
This model was conceived as a paradigm to decipher the consequences of
chronic catecholamine stimulation of Gs-coupled
-adrenergic receptors in heart failure. Transgenic mice with a
three- to fourfold increase in G
s protein expression (and approximately a doubling in G
s activity) display
augmented inotropic and chronotropic responses to sympathomimetic
amines with normal cardiac architecture at 10 mo of age. However, this phenotype gives way to a dilated cardiomyopathy, with reduced left
ventricular ejection fraction, interstitial fibrosis, cellular hypertrophy and apoptosis, ventricular arrhythmias, and sudden death as the mice age (6). These studies argue that
G
s plays a complex role, both to provide critical
inotropic support and to promote tissue remodeling during the evolution
of heart failure.
s
mutant to fat, liver, and skeletal muscle tissues [using
the phosphoenolpyruvate carboxykinase (PEPCK)
promoter]. Like the
-MHC promoter, the PEPCK
promoter drives expression only in postnatal tissues, obviating the
concern that G
s overexpression in utero would reduce
fetal viability. Biochemical characterization of this mouse reveals a
40-50% increase in G
s protein expression in
targeted tissues, a surprisingly modest increase in basal (but not
agonist or forskolin activated) cAMP levels and no obvious gross
macroscopic phenotype. The very modest changes in cAMP in affected
tissues led these authors to consider compensatory mechanisms that
might mitigate cAMP accumulation during unabated Q227L
G
s stimulation. Indeed, modest increases in
G
i2 and cAMP-specific phosphodiesterase, which would
decrease cAMP accumulation, were detected in affected tissues. Reduced
in vitro measurements of cAMP-regulated protein kinase (PKA) activity
were also detected in the affected tissues and were interpreted as a
mechanism to diminish cAMP actions. However, immunoblot analyses of PKA
regulatory and catalytic subunit expression identified increases in PKA
regulatory subunit expression with no change in PKA catalytic subunit
expression. These types of changes in PKA protein subunit expression
actually might serve to reroute (rather than reduce) PKA
phosphorylation pathways in intact cells, and they deserve further
study in more physiological models. To the extent that changes in G
proteins, phosphodiesterases, and PKA mimic adaptive responses observed
in other conditions associated with increased cAMP signaling (MAS,
heart failure), the changes identified in this model might represent a
compensatory response to chronically enhanced cAMP signaling in the
affected tissue. However, the Q227L G
s transgenic mice
display delayed rectification of blood glucose during glucose
challenge. To the extent that Q227L G
s overexpression in
metabolically active tissues (such as liver, adipose, and skeletal
muscle) leads to a generalized metabolic disorder, alternative
mechanism(s) whereby Q227L G
s overexpression can induce
generalized changes in signaling protein expression/action in both
affected and bystander tissues also must be considered in future studies.
s expression are likely to be best suited to
investigate certain aspects of G
s signaling. For
example, the molecular model developed by Huang et al. (5)
uses a Q227L G
s mutant identified in certain endocrine
tumors and a promoter that drives expression in adipose, liver, and
skeletal muscle. Although neither the mutant construct nor the tissue
distribution mimics the abnormalities identified in MAS, this model is
likely to provide a generally useful strategy to explore the
consequences of G
s activation, particularly in the
context of disorders associated with constitutively activated mutant
proteins (endocrine adenomas, MAS) or in disorders affecting
metabolically active tissues. Other promoters that selectively drive
expression in other tissues might induce a very different phenotype and
identify tissue-specific differences in the consequences of
Gs activation. However, to the extent that physiological
G
s interactions with
-dimers critically influence
the fidelity and specificity of heterotrimeric G protein interactions
with G protein-coupled receptors and effectors, models of Q227L
G
s overexpression (which might bind but not be regulated
in a traditional manner by RGS proteins and
-dimers) eventually
are likely to prove inadequate for studies of the intricacies of
physiological Gs signaling. The very nature of the
results obtained in molecular models of G
s
overexpression, where substantial increases in G
s expression lead to only relatively modest increases in cAMP levels, suggests that the stoichiometry of individual elements in the signaling
cascade (receptor, G protein, effector) and potential compartmentation
of signaling pathways also critically influence functional phenotypes
and deserve attention.
s effectors. Although
G
s-dependent activation of adenylyl cyclase and
enhanced signaling via the traditional cAMP/PKA pathway largely can
explain the contractile phenotype induced by cardiac G
s
overexpression, the mechanism(s) whereby increased G
s
signaling leads to cellular remodeling and changes in the regulation of
growth is less straightforward. p38-mitogen-activated protein kinase
(MAPK) has been identified downstream from PKA in cardiomyocytes, where
it provides a potential mechanism to explain the more chronic
deleterious changes induced by G
s overexpression in the
heart (16). In other tissues, G
s/cAMP
modulation of growth results from complex and highly tissue-specific
regulatory controls. In vascular smooth muscle cells, cAMP antagonizes
growth factor-dependent activation of the Raf/extracellular
signal-regulated kinase (ERK) cascade and proliferation. The inhibitory
effects of cAMP are sufficiently robust in this cell type that
adenovirus-mediated delivery of constitutively activated
G
s has been evaluated as a strategy to inhibit vascular
smooth muscle cell proliferation and neointimal hyperplasia in models
of vessel injury (4). Inhibitory modulation of ERK by cAMP
has been attributed to PKA-dependent phosphorylation of c-Raf (at
Ser43, Ser259, and Ser259), which
inhibits signaling via the MAPK kinase (MEK)/ERK cascade at least in
part by preventing c-Raf-Ras interactions (1, 14). Constitutively activated G
s (and elevated cAMP)
activates ERK and stimulates proliferation in cell types (including
endocrine tumors) that express B-Raf, a different Raf isoform that
lacks a PKA site corresponding to Ser43 and displays a more
restricted tissue distribution. Here, the effects of cAMP/PKA to
activate ERK and stimulate proliferation have been attributed to a
parallel pathway involving Rap-1 (a member of the Ras superfamily
of small GTP-binding proteins that shares structural homology with Ras
in its effector domain) and B-Raf. Both PKA-dependent pathway and
PKA-independent/cAMP-dependent mechanisms (involving the cAMP-activated
guanine nucleotide exchange factor, Epac I) promote GTP loading of
Rap-1 and activation of the B-Raf/ERK phosphorylation cascade (8,
11). Finally, recent studies identify Src family tyrosine
kinases as additional nontraditional alternative targets of
G
s that could initiate signaling via the ERK cascade as
well as other pathways (3, 9).
s transgene overexpression
are not always easily distinguished from secondary compensatory changes
that develop over the life of the animal (and with the development of a
diseased phenotype). Nevertheless, the mouse model developed by Huang
et al. (5) is well suited for studies that delve into
mechanisms activated by Gs and their role in
biochemical/structural remodeling and growth control. The more
formidable challenge of future studies will be to integrate concepts of
signaling protein stoichiometry, targeting, and protein-protein
interactions into this and other models of G
s function,
to fully understand the exquisite (and seemingly contradictory) high
level of specificity and interdependence on other signaling molecules
achieved by the G
s protein.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-28958.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. F. Steinberg, Dept. of Pharmacology, College of Physicians and Surgeons, Columbia Univ., 630 West 168 St., New York, NY 10032 (E-mail: sfs1{at}columbia.edu).
10.1152/ajpcell.00198.2002
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