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and their downstream effectors has provided insight
into the regulation of the excitability and responsive-
ness of striatal neurons. Less is known about signaling
events that take place upstream of these secondary ef-
Communication in the dopamine system is particu-
larly important because a variety of neurological and
neuropsychiatric disorders, including schizophrenia,
attention deficit hyperactivity disorder, Tourette syn-
drome, obsessive-compulsive disorder, Parkinson’s dis-
ease, Huntington’s disease, and drug addiction, result
from impaired dopamine receptor signaling. Many of the
drugs used to treat these disorders target dopamine re-
ceptors. For example, D2 antagonists such as haloperi-
dol and risperidone are effective at reducing psychosis.
Work with these and other synthetic dopamine receptor
ligands indicates that there is more to blocking D2 re-
ceptor activity than reducing the intracellular cAMP
concentration, and that the signaling pathways that
make these drugs effective remain to be fully eluci-
There are several barriers to elucidating the signal-
ing pathways elicited by binding of dopamine to its D2
receptor. The receptors are polymorphic and are not
particularly abundant, and mono-specific antibodies
against D2 receptors have been difficult to obtain. D2
receptors are G protein-coupled receptors (GPCRs)
containing the characteristic seven transmembrane re-
gions and three N-linked glycosylation sites (see Figure
1). They form dimers with each other and occur as
either long (mostly postsynaptic) or short (mostly pre-
synaptic) isoforms that differ by 29 amino acids in the
third cytoplasmic loop.
A panoply of downstream effectors in addition to
cAMP and Ca2+are activated when dopamine binds to
D2 receptors. These include agents, such as Ca2+
channels, AMPA receptors, NMDA receptors, and
phospholipase C (PLC), that influence the Ca2+concen-
tration. Yet other D2 effectors are downstream of Ca2+,
such as calcineurin (PP2B) and the β and ? isoforms of
protein kinase C. Still other effector molecules may be
activated by D2 receptors independently of Ca2+: for
example, Na+channels, inwardly rectifying K+(GIRK)
channels, the Na+/H+exchanger, protein phospha-
tase-1 (PP1), glycogen synthase kinase 3 (GSK3), the
Raf/MEK/ERK1/2 pathway, phosphoinositol 3-kinase (PI
factor-κB (NF-κB), and serum-responsive element (Mis-
sale et al., 1998; Takeuchi and Fukunaga, 2004). Fur-
thermore, D2 receptors interact with the Ca2+-perme-
able GluR2 subunit of AMPA receptors and in this way
influence Ca2+signaling (Zou et al., 2005).
Some of these downstream effects depend on the
Gαi/o subunit of the D2 receptor, whereas others are
controlled through the Gβγ subunit. Other downstream
effectors are modulated by G protein-independent sig-
naling mechanisms, and some are controlled by both G
protein-dependent and -independent mechanisms. In-
teractions of G proteins with the D2 receptor are coor-
dinated by short peptide sequences near the amino
and carboxyl termini of the receptor’s third cytoplasmic
loop, leaving the remaining portion free to interact with
other proteins (see Figure 1). For example, the third cy-
toplasmic loop binds to the postsynaptic scaffolding
Decoding Dopamine Signaling
Dopamine is a key neurotransmitter that is important
for many physiological functions including motor
control, mood, and the reward pathway. In this issue
of Cell, the laboratories of Marc Caron and Li-Huei
Tsai identify two very different molecules—?-arrestin
2 and Par-4, respectively—that unexpectedly are in-
volved in dopamine signaling via the D2 receptor.
These two new signaling pathways mediate the ac-
tions of dopamine on behavior and facilitate crosstalk
between different signaling pathways that are acti-
vated by binding of dopamine to the D2 receptor.
The neurotransmitter dopamine is important for many
physiological functions including motor control, mood,
and the reward pathway. Many of these functions are
integrated by the medium spiny neurons of the stria-
tum, which lie below the cortex in the brain and re-
spond to dopamine. Dopamine exerts its effects on
neurons through five known subtypes of dopamine re-
ceptor (D1, 2, 3, 4, and 5). When dopamine binds to
Gαs-coupled D1 and D5 receptors, the enzyme adenyl-
ate cyclase is activated and the secondary messenger
cAMP is produced. In contrast, when dopamine binds
to the Gαi/o-coupled D2, D3, and D4 receptors, adenyl-
ate cyclase activity is blocked and cAMP production is
reduced (Neve and Neve, 1997). Neurons in the mid-
brain project their axons to the striatum and release
dopamine, which modulates cAMP production by acti-
vating D1 and D2 receptors expressed by striatal neu-
rons. These receptors work antagonistically to modu-
late synthesis of cAMP. Striatal neurons also receive
input from neurons in the cortex that release the excit-
atory neurotransmitter glutamate. This results in stimu-
lation of AMPA and NMDA ligand-gated ion channels
and increases the intracellular concentration of Ca2+,
leading to activation of signaling pathways dependent
upon this second messenger. Among the downstream
effectors of cAMP and Ca2+are DARPP-32 and RCS
(regulator of calmodulin signaling), which integrate sig-
nals from both of these second messengers (Rakhilin et
al., 2004). Characterization of these signaling pathways
D2 receptor that interacts with Par-4 contains a calmo-
dulin binding domain, and Ca2+-activated calmodulin
competes with Par-4 for this site. This discovery is im-
portant, as the authors demonstrate that Gαi/o-depen-
dent D2 regulation of gene expression dictated by the
transcription factor CREB depends on an equilibrium
between binding of Par-4 and of calmodulin to the D2
receptor. Increases in the intracellular Ca2+concentra-
tion, possibly in response to activation of the D2 recep-
tor, could result in displacement of Par-4 and uncou-
pling of the D2 receptor from Gαi/o, thereby providing
negative feedback on D2-mediated cAMP attenuation.
In turn, the resulting elevation of cAMP levels could
provide a second round of feedback by attenuating
some of the effects of Ca2+signaling. This group also
demonstrates that mice deficient in Par-4 exhibit spe-
cific behavioral phenotypes reminiscent of depression;
these depression-like behaviors are responsive to anti-
depressant drugs of the selective serotonin reuptake
inhibitor (SSRI) class. Thus, this signaling pathway may
serve as a critical point for integrating serotonin and
dopamine inputs on striatal neurons expressing D2 re-
Meanwhile, Beaulieu and colleagues had discovered
that prolonged activation of D2 receptors in mice lack-
ing the dopamine transporter (DAT)—which prevents
dopamine removal from the synaptic cleft—inactivates
protein kinase B (Akt) in a cAMP-independent fashion
(Beaulieu et al., 2004). Akt is activated by phosphoryla-
tion of its serine residues 308 and 473. Akt binds to
phosphatidylinositol-3,4,5-P3(PIP3) at the plasma mem-
brane and its activation is associated with PI3-kinase
activity. Once bound to the membrane, Akt can be acti-
vated by phosphoinositide-dependent kinase 1 (PDK1),
which also binds to PI3-K. Activated Akt phosphoryl-
ates a number of target proteins including GSK3, result-
ing in its inhibition.
Beaulieu et al. (2005) realized that there must be a
connection between D2-receptor signaling and Akt be-
cause raclopride, a D2 receptor inhibitor, disrupted Akt
inactivation. Furthermore, they observed that inhibition
of GSK3 or activation of Akt in response to lithium or
other GSK3 inhibitors reduced dopamine-associated
increases in locomotor behavior in DAT-deficient mice
or in wild-type mice treated with amphetamines, which
elevate synaptic dopamine for prolonged periods.
Moreover lithium, a GSK3 inhibitor that also activates
Akt, could antagonize dopamine-dependent locomotor
hyperactivity in these same mice. The investigators
have now identified β-arrestin 2 as the biochemical
connection between D2 receptors and Akt (Beaulieu et
al., 2005). Using animal models coupled with behavioral
and biochemical assays, they show that activation of
D2 receptors induces the formation of a signaling com-
plex involving β-arrestin 2, Akt, and protein phospha-
tase-2A (PP2A) that mediates the effects of dopamine.
Dopamine-related behaviors were consistently attenu-
ated in mice lacking β-arrestin 2, despite the fact that
cAMP-dependent signaling remained intact. Further-
more, in animals lacking β-arrestin 2, amphetamine was
unable to dephosphorylate and inactivate Akt. These
results precisely identify β-arrestin as the bridge be-
tween D2 receptors and downstream effectors of Akt
and show the importance of this new pathway in medi-
Figure 1. The D2 Dopamine Receptor and Its Downstream Signal-
Binding of dopamine to the D2 receptor results in activation of the
Gαi/o protein coupled to its third cytoplasmic loop. This inhibits
the ability of adenylate cyclase to synthesize cAMP and thereby
activates PKA (left). Park et al. (2005) report that D2-dependent
regulation of cAMP depends on the association of the D2 receptor
with Par-4, an interaction that is competitively inhibited by acti-
vated Ca2+-associated calmodulin. Beaulieu et al. (2005) describe
a complex containing β-arrestin 2, PP-2A, and Akt (right) that medi-
ates the effects of D2-receptor activation independently of Gαi/o-
coupled mechanisms. Components of this signaling complex are
regulated by phosphorylation/dephosphorylation events. The Akt
substrate GSK3 may also be a part of this complex. Interactions
between the D2 receptor, spinophilin, and PP1, as well as between
Gαi/o and Gβ/γ, are also depicted.
protein, spinophilin, linking it to PP1, the actin cytoskel-
eton, and possibly other transmembrane proteins. Like
other GPCRs, D2 receptors undergo desensitization,
clathrin-mediated sequestration, and resensitization,
presumably via the actions of G protein-coupled recep-
tor kinases, arrestins, and components of the endocytic
pathway, such as dynamin-2, which bind to the recep-
tor (Macey et al., 2004). β-arrestin also acts as a scaf-
fold for signaling complexes, coupling the D2 receptor
to the Raf/MEK/ERK2 signaling pathway.
As more pathways radiating from activated D2 recep-
tors are revealed, it is clear that these receptors encode
an incredibly complex program of signaling cascades.
In this issue of Cell, Park et al. (2005) and Beaulieu et
al. (2005) reveal interactions of D2 receptors with two
new and unexpected signaling pathway components.
In their study, Park and colleagues demonstrate that
the proapoptotic protein Par-4 (prostate apoptosis re-
sponse 4) interacts with the third cytoplasmic loop of
the D2 receptor. They demonstrate that this interaction
is physiological and that Par-4 and D2 receptors colo-
calize in the synaptic membranes of striatal neurons.
Furthermore, this interaction, which involves the leu-
cine zipper domain of Par-4, is essential for Gαi/o-
mediated inhibition of cAMP activity. The region of the
ating dopamine-dependent functions. Interestingly, in
cultured neurons, D1 receptors can also activate Akt
via a cAMP-dependent pathway (Brami-Cherrier et al.,
2002). It will be important for future work to delineate
which aspects of this pathway are under the control of
D1 or D2 receptors, or both.
The two new studies expose hidden insights into do-
pamine signaling via D2 receptors, with their revela-
tions of new functions for Par-4 and Akt. These proteins
are associated with quite different cellular processes:
Par-4 is a proapoptotic factor implicated in neurode-
generative disorders such as Alzheimer’s disease (Xie
and Guo, 2005), whereas Akt activity prevents cell
death (Brunet et al., 2001). An emerging theme seems
to be that components of the programmed cell death
pathway are commandeered as mediators of neuro-
transmission and synaptic plasticity in the brain.
I thank J.P. Albanesi, D.C. Cooper, and K.A. Neve for helpful com-
ments. J.A.B. is supported by the National Institute of Drug Abuse
and the Ella McFadden Charitable Trust Fund at the Southwestern
James A. Bibb
Department of Psychiatry
The University of Texas Southwestern Medical Center
Dallas, Texas 75390
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