DƯỢC LÍ Goodman & Gilman's The Pharmacological Basis of Therapeutics 12th, 2010

56 Second Messengers
SECTION I
GENERAL PRINCIPLES
Cyclic AMP. Cyclic AMP is synthesized by adenylyl
cyclase under the control of many GPCRs; stimulation
is mediated by the G s
α subunit, inhibition by the G i
α
subunit. The cyclic AMP pathway provides a good basis
for understanding the architecture and regulation of
many second messenger signaling systems (for an
overview of cyclic nucleotide action, see Beavo and
Brunton, 2002).
There are nine membrane-bound isoforms of
adenylyl cyclase (AC) and one soluble isoform found
in mammals (Hanoune and Defer, 2001). The membrane-bound
ACs are glycoproteins of ~120 kDa with
considerable sequence homology: a small cytoplasmic
domain; two hydrophobic transmembrane domains,
each with six membrane-spanning helices; and two
large cytoplasmic domains. Membrane-bound ACs
exhibit basal enzymatic activity that is modulated by
binding of GTP-liganded α subunits of the stimulatory
and inhibitory G proteins (G s
and G i
). Numerous
other regulatory interactions are possible, and these
enzymes are catalogued based on their structural
homology and their distinct regulation by G protein α
and βγ subunits, Ca 2+ , protein kinases, and the actions
of the diterpene forskolin. Cyclic AMP generated by
adenylyl cyclases has three major targets in most cells,
the cyclic AMP dependent protein kinase (PKA),
cAMP-regulated guanine nucleotide exchange factors
termed EPACs (exchange factors directly activated by
cAMP), and via PKA phosphorylation, a transcription
factor termed CREB (cAMP response element binding
protein). In cells with specialized functions, cAMP can
have additional targets such as cyclic nucleotide-gated
ion channels (Wahl-Schott and Biel, 2009), cyclic
nucleotide-regulated phosphodiesterases (PDEs), and
several ABC transporters (MRP4 and MRP5) for
which it is a substrate (see Chapter 7).
PKA. The best understood target of cyclic AMP is the
PKA holoenzyme consisting of two catalytic (C) subunits
reversibly bound to a regulatory (R) subunit dimer
to form a heterotetramer complex (R 2
C 2
). At low concentrations
of cAMP, the R subunits inhibit the C subunits;
thus the holoenzyme is inactive. When AC is
activated and cAMP concentrations are increased, four
cyclic AMP molecules bind to the R 2
C 2
complex, two
to each R subunit, causing a conformational change in
the R subunits that lowers their affinity for the C subunits,
causing their activation. The active C subunits
phosphorylate serine and threonine residues on specific
protein substrates.
There are multiple isoforms of PKA; molecular cloning has
revealed α and β isoforms of both the regulatory subunits (RI and
RII), as well as three C subunit isoforms Cα, Cβ, and Cγ. The R subunits
exhibit different subcellular localization and binding affinities
for cAMP, giving rise to PKA holoenzymes with different thresholds
for activation (Taylor et al., 2008). Both the R and C subunits
interact with other proteins within the cell, particularly the R subunits,
and these interactions can be isoform-specific. For instance,
the RII isoforms are highly localized near their substrates in cells
through interactions with a variety of A kinase anchoring proteins
(AKAPs) (Carnegie et al., 2009; Wong and Scott, 2004).
PKA can phosphorylate a diverse array of physiological targets
such as metabolic enzymes and transport proteins, and numerous
regulatory proteins including other protein kinases, ion
channels, and transcription factors. For instance, phosphorylation
of the cAMP response element–binding protein, CREB, on serine
133 recruits CREB-binding protein (CBP), a histone acetyltransferase
that interacts with RNA polymerase II (POLII) and leads to
enhanced transcription of ~105 genes containing the cAMP
response element motif (CRE) in their promoter regions (e.g., tyrosine
hydroxylase, iNOS, AhR, angiotensinogen, insulin, the glucocorticoid
receptor, BC12, and CFTR) (Mayr and Montminy, 2001;
Sands and Palmer, 2008).
Cyclic AMP–Regulated Guanine Nucleotide Exchange
Factors (GEFs). The small GTP-binding proteins are
monomeric GTPases and key regulators of cell function.
The small GTPases operate as binary switches that
exist in GTP- or GDP-liganded conformations. They
integrate extracellular signals from membrane receptors
with cytoskeletal changes and activation of diverse
signaling pathways, regulating such processes as
phagocytosis, progression through the cell cycle, cell
adhesion, gene expression, and apoptosis (Etienne-
Manneville and Hall, 2002). A number of extracellular
stimuli signal to the small GTPases directly or through
second messengers such as cyclic AMP.
For example, many small GTPases are regulated by GEFs.
GEFs act by binding to the GDP-liganded GTPase and catalyzing the
exchange of GDP for GTP. The two GEFs regulated by cAMP are
able to activate members of the Ras small GTPase family, Rap1 and
Rap2; these GEFs are termed exchange proteins activated by cyclic
AMP (EPAC-1 and EPAC-2). The EPAC pathway provides an additional
effector system for cAMP signaling and drug action that can act
independently or cooperatively with PKA (Cheng et al., 2008;
Roscioni et al., 2008).
PKG. Stimulation of receptors that raise intracellular
cyclic GMP concentrations (Figure 3-11) leads to the
activation of the cyclic GMP-dependent protein kinase
(PKG) that phosphorylates some of the same substrates
as PKA and some that are PKG-specific. In
some tissues, PKG can also be activated by cAMP.
Unlike the heterotetramer (R 2
C 2
) structure of the
PKA holoenzyme, the catalytic domain and cyclic