Dopamine Receptor Signaling

Abstract

The D1-like (D1, D5) and D2-like (D2, D3, D4) classes of dopamine receptors each has shared signaling properties that contribute to the definition of the receptor class, although some differences among subtypes within a class have been identified. D1-like receptor signaling is mediated chiefly by the heterotrimeric G proteins Gα_s and Gα_olf, which cause sequential activation of adenylate cyclase, cylic AMP-dependent protein kinase, and the protein phosphatase-1 inhibitor DARPP-32. The increased phosphorylation that results from the combined effects of activating cyclic AMP-dependent protein kinase and inhibiting protein phosphatase 1 regulates the activity of many receptors, enzymes, ion channels, and transcription factors. D1 or a novel D1-like receptor also signals via phospholipase C-dependent and cyclic AMP-independent mobilization of intracellular calcium. D2-like receptor signaling is mediated by the heterotrimeric G proteins Gα_i and Gα_o. These pertussis toxin-sensitive G proteins regulate some effectors, such as adenylate cyclase, via their Gα subunits, but regulate many more effectors such as ion channels, phospholipases, protein kinases, and receptor tyrosine kinases as a result of the receptor-induced liberation of Gβγ subunits. In addition to interactions between dopamine receptors and G proteins, other protein:protein interactions such as receptor oligomerization or receptor interactions with scaffolding and signal-switching proteins are critical for regulation of dopamine receptor signaling.

• Adenylate cyclase
• G protein
• Phospholipase
• Glutamate
• Protein kinase
• Extracellular signal-regulated kinase
• Potassium channel
• Sodium channel
• Calcium channel

Introduction

Five subtypes of mammalian dopamine receptors are grouped into two classes, with the D1-like receptor class composed of the D1 and D5 receptor subtypes, and the D2-like receptor class composed of the D2, D3, and D4 receptor subtypes ([1]). At the molecular level, most signaling properties are shared among all of the subtypes within a class; similarity of signal transduction pathways is one of the criteria by which subtypes are grouped into classes. In this review of dopamine receptor signaling, we avoid repetition by discussing each class as a whole rather than treating each subtype separately when little is known about subtype-specific properties, making specific reference to a subtype primarily when there are data supporting a unique property of that subtype.

All of the dopamine receptors are G protein-coupled receptors (GPCRs), whose signaling is primarily mediated by interaction with and activation of heterotrimeric GTP-binding proteins (G proteins). Members of this superfamily are also called 7-transmembrane receptors because they traverse the cell membrane seven times, or serpentine receptors because of the manner in which they wind back and forth across the membrane.

D1-Like Receptors and G Proteins

As receptors that stimulate adenylate cyclase, the D1-like receptors were assumed to couple to the adenylate cyclase stimulatory G protein Gα_s. Because Gα_s is ubiquitously expressed, the ability of D1-like receptors to stimulate adenylate cyclase in virtually any cell line ([2]), together with physical and functional coupling of both D1 and D5 receptors to Gα_s ([3], [4]), strongly support the notion that Gα_s mediates the D1-like receptor signaling in some tissues. In the neostriatum, however, the brain region with the densest dopamine innervation and the highest expression of the D1 receptor, expression of Gα_s is very low, whereas Gα_olf is abundantly expressed ([5]). The nucleus accumbens and olfactory tubercle also express abundant Gα_olf and little Gα_s ([6]). Gα_olf is the heterotrimeric G protein involved in olfaction, is very closely related to Gα_s (88% amino acid homology), and also stimulates adenylate cyclase ([7]). Gα_olf null mutant mice exhibit little dopamine-stimulated adenylate cyclase; they also lack other functional and behavioral responses to D1 receptor stimulation, including cocaine or D1 agonist-induced locomotor activation and induction of c-fos expression in the neostriatum or nucleus accumbens, strongly suggesting that Gα_olf mediates D1 receptor signaling to adenylate cyclase in these basal ganglia nuclei ([5], [8]).

Less is known about the βγ subunits that combine with Gα_s or Gα_olf to mediate D1-like receptor activation of adenylate cyclase. In human embryonic kidney (HEK) 293 cells, depletion of endogenous γ₇ subunit reduces D1 receptor stimulation of adenylate cyclase, but not D5 receptor stimulation, indicating that γ₇ contributes to this pathway for the former subtype ([9]). Depletion of γ₇ also decreases the abundance of the β₁ subunit, consistent with the formation of a β₁γ₇ dimer; the presence of Gα_s in HEK293 cells suggests that a G protein heterotrimer that mediates D1 receptor activation of adenylate cyclase is Gα_sβ₁γ₇. Because γ₇ is abundantly expressed in neostriatal medium spiny neurons ([10]), particularly in neurons that also express D1 receptor mRNA ([9]), neostriatal D1 receptors may signal via a G protein heterotrimer that includes both Gα_olf and γ₇. In other brain regions, including dopamine target areas that express D1 and/or D5 receptors such as the cerebral cortex and hippocampus where the expression of Gα_olf is much lower than that of Gα_s ([6]), it seems likely that Gα_s mediates D1 and D5 receptor signaling to adenylate cyclase. Coupling of the D1 receptor to other heterotrimeric G proteins, such as Gα_o and Gα_q, and of the D5 receptor to Gα_z, has also been described ([4], [11]). D1 receptor interactions with Gα_q are particularly interesting in light of the possibility that D1 or D1-like receptors activate phospholipase C, a phenomenon that will be discussed in greater detail below.

D1-like Dopamine Receptor Signaling

D1-Like Receptor Stimulation of Adenylate Cyclase

The first biochemical evidence for a dopamine receptor was the identification in 1972 of dopamine-stimulated adenylate cyclase activity and cyclic AMP accumulation in the retina ([12]) and in rat neostriatum ([13]). Gα_s and, presumably, Gα_olf bind primarily to the C2 cytosolic domain of adenylate cyclase, bringing the C1 and C2 domains together in a way that enhances the catalytic efficiency of the enzyme ([14]). Adenylate cyclase catalyzes the conversion of ATP to cyclic AMP, which binds to the regulatory subunits of the protein kinase A (PKA) holoenzyme to disinhibit the catalytic subunits. The selective concentration of the type 5 subtype of adenylate cyclase in the neostriatum suggested that it mediates dopamine receptor signaling ([15]), a hypothesis confirmed with the creation of adenylate cyclase 5 null mutant mice, in which neostriatal D1 receptor-stimulated adenylate cyclase is markedly diminished ([16], [17]). Thus, in the rodent neostriatum the primary pathway for D1-like receptor signaling is likely to be D1 receptor → Gα_olf/γ₇ → adenylate cyclase 5 → PKA (Figure 1). PKA phosphorylates a number of proteins involved in signal transduction and regulation of gene expression ([2], [18]).

Figure 1. D1-like receptor signaling pathways. Stimulatory effects are indicated with a solid line ending in an arrowhead, and inhibitory effects with a dashed line ending in a bar. Intervening steps (e.g., MAPKKK → MAPKK → MAPK) are frequently omitted from the figure for simplicity and ion channels are indicated generically, but are identified individually in Fig. 2. AC5, adenylate cyclase type 5; CREB, cyclic AMP response element binding protein; DARPP-32, dopamine-related phosphoprotein, 32 kDa; MAPK, mitogen-activated protein kinase; NHE, Na⁺/H⁺ exchanger; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PP1 or PP2A, protein phosphatase 1 or 2A.

D1-Like Receptor Regulation of PKA Substrates

PKA Substrates—DARPP32

DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa) is a neostriatum-enriched bifunctional signaling protein that inhibits protein phosphatase 1 (PP1) when phosphorylated on Thr34 by PKA or several other kinases ([19], [20]) and inhibits PKA when phosphorylated on Thr75 by cyclin-dependent kinase 5 ([21]). Because Thr75 is dephosphorylated by the PKA-stimulated protein phosphatase-2A ([22]), D1-like receptor signaling is amplified by both positive feedback and feedforward loops. D1 receptor stimulation simultaneously activates PKA by stimulating the production of cyclic AMP and disinhibits PKA by phosphorylation-dependent activation of protein phosphatase-2A and Thr75 dephosphorylation of DARPP-32. At the same time, D1-like receptor activation of PKA not only stimulates PKA-catalyzed phosphorylation of numerous substrates including some described below but also prevents PP1-catalyzed dephosphorylation of many of the same phosphoproteins by phosphorylating DARPP-32 on Thr34. Studies with DARPP-32 null mutant mice have shown that DARPP-32 contributes to acute D1 receptor-mediated responses, both at the cellular and behavioral levels ([20]). Consistent with a signal amplification role for DARPP-32, many deficits associated with genetic deletion of the PP1 inhibitor are observed only at low doses of dopamine receptor agonist ([23], [24], [25]).

PKA Substrates—Ion Channels

D1-like receptor activation of PKA increases the phosphorylation of numerous voltage- and ligand-gated ion channels by various combinations of direct PKA-catalyzed phosphorylation of channel subunits and DARPP-32-mediated inhibition of PP1. For example, there are at least five potential PKA phosphorylation sites in the LI-II region of the pore-forming α-subunit of voltage-gated Na⁺ channels ([26]), and activation of DARPP-32 also decreases PP1-catalyzed dephosphorylation at Ser573. Enhanced phosphorylation at Ser573 decreases Na⁺ currents through a decrease in the open probability of the channel ([26], [27], [28], [29], [30]). D1 receptor stimulation of PKA also decreases K⁺ currents through several types of inwardly rectifying channels, increases L-type, and decreases N and P/Q type Ca²⁺ channel activity, increases NMDA receptor activity via phosphorylation of the NR1 subunit, enhances AMPA currents, and modulates GABA currents. Because D1-like receptor regulation of ion channels often involves multiple, incompletely defined signaling pathways in addition to PKA, this topic is discussed in greater detail below.

PKA Substrates—CREB

D1 receptor stimulation induces the expression of a number of transcription factors ([31], [32]), at least some of which are dependent on initial activation of the transcription factor cyclic AMP response element-binding protein (CREB) ([31], [33]). D1 receptor activation of CREB involves PKA-dependent CREB phosphorylation of Ser133, which permits binding of CREB to CREB-binding protein and transcriptional activation of genes with cyclic AMP response elements ([34]). D1 receptor stimulation may also increase Ser133-phosphorylated CREB via activation of extracellular signal-regulated kinase (ERK) ([35]). Phosphorylation of CREB is also regulated by Ca²⁺, so the transcription factor is likely to be an important site of integration for signals from dopamine and N-methyl-D-aspartate (NMDA) glutamate receptors (Figure 1). CREB-mediated changes in gene expression are important in synaptic plasticity and probably contribute to synaptic rearrangements that underlie the persistence of drug addiction ([36]).

D1-Like Receptor Stimulation of Phospholipase C

One finding that is difficult to reconcile with a model of D1 receptor signaling that includes a central role for a cyclic AMP/PKA cascade is that a null mutation of adenylate cyclase 5 enhances D1 agonist-stimulated locomoter activity even though D1 receptor stimulation of adenylate cyclase activity is almost abolished ([16], [17]). Mice with genetic deletion of adenylate cyclase 5 have altered abundance of several proteins involved in dopamine receptor signaling and also disrupted D2 receptor signaling, making interpretation of behavioral effects complicated, but one possible explanation is that a cyclic AMP-independent signaling pathway mediates D1 receptor locomotor activation, and perhaps other behavioral effects of D1 receptor stimulation. One such alternative pathway for D1-like receptor signaling is phospholipase C-mediated mobilization of intracellular calcium ([37]). There are at least two distinct potential mechanisms for D1-like receptor activation of phospholipase C. Bergson and colleagues demonstrated that heterologously expressed D1 and D5 dopamine receptors, when co-expressed with calcyon, stimulate the release of calcium from intracellular stores following priming of the cells with a Gα_q-coupled receptor agonist ([38]). The effect of calcyon is dependent on its binding to the C terminus of the D1 receptor. Endogenous D1-like receptors in neocortical or hippocampal neurons, but not neostriatal neurons, display a similar priming-dependent ability to mobilize calcium ([39]). Whether this involves calcyon-dependent enhancement of coupling to Gα_q and direct activation of phospholipase C is unknown, although the lack of effect of cyclic AMP analogues indicates that it is not mediated by PKA.

A second potential cyclic AMP-independent mechanism for D1-like receptor signaling invokes a novel SCH23390-binding receptor that is linked to phospholipase C via Gα_q ([40], [41]). The regional distribution and pharmacological profile of D1-like receptor-stimulated phospholipase C differ from both D1 and D5 receptors. Some compounds efficacious for stimulating adenylate cyclase are weak agonists or antagonists at the phospholipase C-coupled receptor, whereas the drug SKF83959 has little efficacy for adenylate cyclase but is one of the most potent and efficacious agonists known to stimulate the phospholipase C-coupled receptor ([42], [43]). D1-like receptor-stimulated phospholipase C is most abundant in the amygdala and hippocampus ([40]); it is of interest that the amygdala has a relatively dense dopaminergic innervation and abundant D1 receptors but little or no D1 receptor-stimulated adenylate cyclase ([44]). D1-like receptors are also functionally and physically coupled to Gα_q, with the distribution of Gα_q-coupled receptors roughly paralleling that of D1-like receptor-stimulated phospholipase C ([4], [41], [45]). This putative D1-like Gα_q-coupled receptor does not react with a D1 receptor antibody ([4], [45]) and is not a product of the drd1 gene because D1-like receptor-stimulated phospholipase C is spared in D1 receptor null mutant mice ([46]); indeed, the existence of a genetically distinct phospholipase C-coupled D1-like receptor has been used to explain the conservation of D1 agonist-stimulated behaviors in D1 null mutant mice ([47]), although the mixed genetic background of the mice also affected the observed phenotype ([48]). A major inconsistency in this otherwise intriguing hypothesis is that [³H]SCH23390 binding sites are undetectable in most brain regions of the D1 null mutant mouse ([49]), including in amygdala where stimulation of phospholipase C is most robust, a finding difficult to reconcile with numerous reports that SCH23390 is a potent antagonist ligand of the Gα_q- and phospholipase C-coupled D1-like receptor.

D1-Like Receptor Regulation of Ion Channels

D1-Like Receptor Regulation of K⁺ Channels

Activation of D1-like or D2-like receptors has opposing effects on K⁺ currents in most cells. In general, D1-like receptors attenuate these currents via stimulation of the PKA-DARPP-32 signaling cascade, whereas the D2-like receptors enhance them via inhibition of that pathway as well as by release of Gβγ subunits (Figure 2). We focus here on voltage-gated (I_A/I_Af, I_D/I_As/I_KS, I_K/I_KSS) and inwardly rectifying potassium currents that primarily act to regulate firing rate, stabilize membrane potential, and integrate subthreshold inputs.

Figure 2. Regulation of ion channels by D1-like (top) and D2-like (bottom) dopamine receptors. Stimulatory (solid line with arrowhead) or inhibitory (dashed line with bar) effects of dopamine receptors on GABA receptors (GABA), NMDA glutamate receptors (NMDA), AMPA glutamate receptors (AMPA), L- and N,P,Q-type Ca²⁺ channels, persistent (P) and transient (T) Na⁺ channels, G protein-regulated inwardly rectifying K⁺ channels (GIRK), and voltage-gated K⁺ channels (VGK⁺C) are schematically depicted, as well as whether the effect is thought to be mediated by protein kinase A (PKA), protein kinase C (PKC), G protein βγ subunits (Gβγ), or inhibition of the cyclic AMP/PKA pathway (↓PKA).

The family of outwardly rectifying voltage-gated K⁺ channels in neocortical neurons has been separated into three different components based on their inactivation kinetics (I_Af, I_KS, I_KSS). The terminology differs among studies so that it can sometimes be difficult to understand exactly which component is being measured. Here, we have separated the components according to the criteria developed by Foehring and Surmeier ([50]). I_Af is the rapidly inactivating component of this current and can also be referred to as the fast A-type current (I_A) because it shows fast inactivation kinetics (around 12 msec at −10 mV). I_KS, also referred to as I_D, I_As, or the slow A-type current, is the slowly inactivating component of this voltage-gated K⁺ current and has an inactivation time constant of 293 msec at −10 mV. Finally, I_KSS, also referred to as I_K, is the very slowly inactivating component, with an inactivation time constant between 2 and 20 sec.

In medium spiny neurons of the striatum and nucleus accumbens, the I_A type current is important for regulation of firing rate. This current has at least two components that are activated depending on the membrane potential of the cell. The fast component (I_Af) is activated at more depolarized membrane potentials and does not appear to be modulated by D1 receptor stimulation. However, at more hyperpolarized membrane potentials, such as during the down state, the slower component of this current (I_KS/I_As/I_D) is available. In the nucleus accumbens, combined activation of D1 and D2 receptors increases neuronal firing rate by inhibition of I_D. The precise signaling mechanism used by each receptor has not been worked out, but this response is dependent on activation of cAMP/PKA and on D2-like receptor-mediated liberation of Gβγ. These pathways converge on the A-type K⁺ current to attenuate the outward flow of K⁺ ions ([51]). These authors speculated that D1-Gα_s/Gα_olf and D2-Gβγ are acting cooperatively to increase adenylate cyclase and PKA activity and produce the observed changes in potassium currents ([52], [53]). This is one of several mechanisms that may contribute to D1/D2 receptor synergism ([54]).

D1-like suppression of K⁺ currents in the prefrontal cortex has important effects on the excitability of both interneurons and pyramidal cells; elucidating these effects is important for understanding how dopamine will modulate these cortical activity states. D1 stimulation increases evoked GABA release from interneurons via an increase in their excitability. This increase in excitability appears to be due to D1-PKA-stimulated decrease of K⁺ currents in one class of interneurons, the so-called fast-spiking parvalbumin positive cells ([55]). Dopamine via D1-like receptors suppresses an inwardly rectifying K⁺ current, a K⁺-dependent leak current, and the slowly inactivating membrane outward rectification in prefrontal cortex fast-spiking interneurons. Collectively, these D1-mediated modulations of K⁺ currents act to depolarize and increase the evoked output of a specific subclass of interneurons. Pyramidal cell excitability is regulated by D1 receptor- and PKA-mediated decreases in K⁺ currents in a similar manner. D1 reduction of the voltage-gated, slowly inactivating K⁺ current (I_D) in pyramidal cells results in a stronger effect of subthreshold inputs because a decrease in I_K will lesson opposition to depolarization and latency to first spike ([56], [57]). These D1 effects on pyramidal cells may be mediated through elevation of PKA and increased phosphorylation of DARPP-32. In contrast, the inwardly rectifying K⁺ channels may be modulated in a slightly different manner from other K⁺ currents. It appears that D1-like receptor stimulation inhibits voltage-gated K⁺ channels as a result of direct binding of cyclic AMP to the channel, thus increasing cell excitability ([58], [59]). Overall, in the prefrontal cortex, dopamine acts via D1-like receptors to increase the excitability of both pyramidal neurons and fast-spiking interneurons through inhibition of several K⁺ currents.

D1-Like Receptor Regulation of Ca²⁺ Channels

Dopamine modulates high-voltage-activated Ca²⁺ currents in several types of vertebrate and invertebrate neurons in vitro ([60], [61], [62], [63]). D1 receptor stimulation increases L-type and decreases N, P/Q-type Ca²⁺ channel conductances in most brain areas (Figure 2). In cortical cells L-type channels are associated with the somatic region of cells and exert a powerful effect on signal integration. N- and P/Q-type channel activation produces Ca²⁺ spikes mainly within more distal dendritic regions of somotosensory ([64]) but not prefrontal cortical neurons ([65]).

In striatum, D1-like receptor-mediated decreases in N- and P/Q-type conductances inhibit spike-induced Ca²⁺ influx, whereas increases in L-type currents lead to depolarization ([62]). This effect on L-type channels becomes even more pronounced in conjunction with the D1-mediated decrease in K⁺ currents discussed above and the increase in NMDA current. D1 receptor-stimulated increases in L-type conductances, which are very slow to inactivate, might help to stabilize the prolonged depolarizations ([63]).

D1-like receptors decrease N- and P/Q-type Ca²⁺ channels via activation of PKA and DARPP-32 ([62]). The α1 subunit of these channels contains phosphorylation sites for PKA, but phosphorylation of these sites by PKA activates the channels ([66]). Surmeier et al. ([62]) speculate that the D1-mediated decrease in N- and P/Q-type currents is therefore due not to a direct phosphorylation of the channel by PKA, but rather to a transient increase in PP1-catalyzed dephosphorylation caused by PKA-dependent phosphorylation of PP1-targeting proteins. This effect would be rapidly reversed by comcomitant PKA phosphorylation of DARPP-32, which attenuates PP1 dephosphorylation of the channel ([67]). According to this hypothesis, however, D1-like receptor inhibition of these channels should be prolonged in DARPP-32 null mutant mice; in contrast, D1 agonist-induced inhibition of the channels is decreased in DARPP-32 null mutant mice ([23]).

D1-like receptor effects on Ca²⁺ channels in the prefrontal cortex are similar to those in the striatum, although the pathways have not been completely worked out, and appear to serve as a gain control mechanism to amplify somatic inputs via increases in L-type Ca²⁺ currents and attenuate dendritic inputs via decreases in N- and P/Q-type currents ([56], [58]). Direct D1-like receptor stimulation induces a complex modulation of Ca²⁺ potentials in prefrontal cortex neurons. D1-like agonists suppress full Ca²⁺ spikes via a Ca²⁺- and PKC-dependent pathway ([56], [68]). In contrast, subthreshold depolarizations produced by L-type Ca²⁺ channels ([65]) are augmented transiently (∼7 min) by D1-like receptor agonists ([68]), an effect that is blocked by PKA inhibitors. In this way, D1-like receptor stimulation activates PKA to potentiate subthreshold L-type Ca²⁺ currents, yet it acts via PKC to suppress large amplitude Ca²⁺ spikes, thereby tuning Ca²⁺ currents to have the greatest activation in the voltage range necessary to produce spikes. Coupled with the D1 receptor-mediated increase in I_Nap (see below) and decrease in K⁺ currents, D1 receptor activation greatly prolongs the output of prefrontal pyramidal neurons.

D1-Like Receptor Regulation of Na⁺ Channels

Voltage-gated Na⁺ channels are important for determining threshold for action potential initiation, the duration and frequency of firing, EPSP amplification, and resonance properties of neurons ([69], [70], [71]). D1-like receptor stimulation exerts a powerful influence over cellular activity through manipulation of the phosphorylation state of these channels. D1 effects may differ depending on the specific type of Na⁺ channel activated and the brain area in which DA is acting.

In the neostriatum and hippocampus, down-regulation of transient Na⁺ currents by a D1-PKA-DARPP-32 pathway that increases phosphorylation of Ser573 of the Na⁺ channel α-subunit decreases excitability of cells and increases rheobase current by increasing the action potential threshold to more depolarized levels ([20], [23], [26], [27], [72], [73], [74]). The functional effect of this modulation is to decrease the output of these neurons, but the modulation may be state dependent. Neostriatal cells have rhythmic oscillations in membrane potential (up- and downstates), and it appears that D1 receptor activation creates more selectivity for depolarizing inputs that will evoke an upstate because it opposes the activation of Na⁺ currents ([27]). As a result, depolarizing inputs must be stronger to evoke a postsynaptic response and thus cells are more selective about which inputs drive upstates. The D1-PKA-DARPP-32 modulation of striatal Na⁺ channels, therefore, tends to counteract the effectiveness of depolarizing inputs that drive these neurons into upstates.

In neocortical neurons, one type of voltage-gated Na⁺ channel carries two Na⁺ currents by virtue of its ability to switch between gating modes ([75], [76], [77]). The fast-gating mode is similar to the transient Na⁺ current discussed above, whereas the other current is a slowly inactivating or persistent Na⁺ current (I_NaP) ([78], [79]). Individual Na⁺ channels can exhibit this persistent gating mode, but only a small fraction of the channels are in this gating mode at any time.

As in neostriatal neurons, D1-like receptor stimulation of PKA decreases transient Na⁺ currents in neocortical pyramidal cells ([80]). On the other hand, D1-like receptors may enhance I_NaP ([56], [81]), decrease it ([82]), or have no effect ([80]). The D1-mediated modulation reported by Gorelova and Yang ([81]) occurs at subthreshold membrane potentials by shifting I_NaP activation to more negative membrane potentials and slowing its inactivation ([56], [81]). Rather than being mediated by PKA, the D1 receptor effect on I_NaP in intact prefrontal cortical neurons from brain slices is mediated by PKC ([81]). Maurice et al. ([80]), who observed no effect of D1-like receptor activation on I_NaP in acutely dissociated prefrontal cortical neurons, proposed that the Nav1.6 α-subunit, which lacks the phosphorylated residue Ser573, contributes disproportionately to the persistent current, whereas the transient current is attributable to the PKA-sensitive Nav1.1/1.2 channel, again supporting the notion that modulation of the persistent current is not through PKA (Figure 2).

Figure 3. D2-like receptor signaling pathways. Stimulatory effects are indicated with a solid line ending in an arrowhead, and inhibitory effects with a dashed line ending with a bar. Intervening steps (e.g., MAPKKK → MAPKK → MAPK, or the multiple possible steps between Gβγ and RTK) are frequently omitted from the figure for simplicity. See Fig. 2 for more specific description of ion channels. AA, arachidonic acid; AC2 or AC5, adenylate cyclase type 2 or 5; CREB, cyclic AMP response element-binding protein; DARPP-32, dopamine- and cyclic AMP-regulated phosphoprotein, 32 kDa; MAPK, mitogen-activated protein kinase; NHE, Na⁺/H⁺ exchanger; PA, phosphatidic acid; PC, phosphatidylcholine; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; PP1 or PP2A, protein phosphatase 1 or 2A; RTK, receptor tyrosine kinase.

D1-Like Receptor Regulation of Glutamate Receptors

D1-like receptors interact with glutamate receptors at multiple levels, including signal integration by CREB and PKA-dependent phosphorylation of the glutamate receptors. D1-like receptors increase NMDA receptor-mediated responses in neostriatal ([83], [84], [85], [86]), hippocampal ([87]), and cortical neurons ([88], [89], [90], [91]) and enhance phosphorylation of the NMDA-NR1 subunit. DARPP-32 is required for D1 receptor-stimulated phosphorylation of NR1 and for the increased NMDA-evoked responses ([20], [86], [92]). A contribution of D1-like receptor activation of L-type Ca²⁺ channels and protein kinase C has also been described ([93], [94]), whereas other studies have focused on the role of PKA ([95]). PKA/DARPP-32 may enhance NMDA receptor function by a complex combination of mechanisms including direct phosphorylation of NR1 by PKA, DARPP-32-mediated inhibition of dephosphorylation of NR1, and depolarization via DARPP-32-mediated activation of L-type Ca²⁺ channels ([20], [91]).

An additional consideration is that D1 effects may be state dependent. If the target cell is in a depolarized state, then D1 receptor stimulation increases NMDA currents, but if it is in a hyperpolarized state when dopamine is applied, then D1-like receptor activation decreases NMDA receptor currents ([27], [96], [97]). A positive feedback situation may exist in that NMDA receptor activation in depolarized cells may benefit from the simultaneous D1 receptor-mediated modulation of I_Nap, Ca²⁺ channels, and NMDA receptors; greater depolarization is produced, these voltage-dependent currents become larger, thus evoking more depolarization, thereby enhancing the subtle actions of dopamine.

Dopamine appears to exert opposing effects on AMPA currents via activation of D1 and D2 receptors. Both receptor subtypes can activate kinases that target specific residues on AMPA receptors, but as for NMDA receptors, experiments to elucidate signaling pathways regulating these voltage-dependent receptors are complicated by the necessity to exclude dopamine receptor effects on other ion currents.

D1 receptor activation may have variable effects on AMPA currents in the striatum, with a small effect to enhance the currents, and a larger effect to stabilize them by delaying AMPA receptor current “rundown” ([98], [99], [100]). The stabilization may be mediated by both PKA-catalyzed phosphorylation of the GluR1 subunit at Ser845 and DARPP-32 inhibition of PP1-catalyzed dephosphorylation of the residue ([100], [101], [102], [103]), whereas enhanced amplitude may reflect D1-like receptor activation of L-type Ca²⁺ channels ([62], [93], [99]), also mediated by PKA/DARPP-32. Thus, D1 receptor-mediated enhancement of AMPA currents would depend on the co-presence of the Ca²⁺ channels, perhaps accounting for some variability in results. To complicate matters further, there is some debate about whether the effects of D1 stimulation occur pre- or postsynaptically. In prefrontal cortex, D1 receptor stimulation attenuates AMPA currents by decreasing the probability of glutamate release from presynaptic cells. This implies that rather than a direct effect of D1 receptor activation on AMPA receptors, decreased synaptic glutamate levels manifest themselves as decreases in AMPA currents ([89]). Later work supported this idea by removing the presynaptic component of this response. When only the postsynaptic effect is examined, then D1-like receptor stimulation slightly increases AMPA currents via a Ca²⁺-dependent mechanism ([104]).

D1-like receptor stimulation also enhances the trafficking of NMDA receptors and the GluR1 subunit of AMPA receptors to the membrane in neurons ([105], [106]). Enhanced trafficking of GluR1 is mimicked by forskolin-induced activation of adenylate cyclase ([106]), whereas that of NMDA receptors is independent of DARPP-32 but requires the Src-family protein tyrosine kinase Fyn ([107]). Finally, a direct protein-protein interaction between the C termini of the D1 receptor and the NR1-1a and NR2A NMDA receptor subunits mediates reciprocal regulation of receptor function and trafficking ([108], [109], [110]). In particular, this physical interaction permits D1 receptor inhibition of NMDA receptor currents ([108]).

D1-Like Receptor Regulation of GABA Receptors

Dopamine exerts varied effects on GABA_A currents depending on the brain area and cell type being examined. One reason for these various effects could be the subunit composition of the GABA receptors located in these different areas, because different isoforms of α-, β-, and γ-subunits are differentially affected by protein kinases. D1-like receptor stimulation decreases GABA receptor activation in medium spiny neurons from neostriatum and nucleus accumbens ([24], [111]), an effect associated with phosphorylation of β1/β3 subunits and activation of PKA/DARPP-32 ([24]). In large cholinergic interneurons of the neostriatum, D1-like agonists have been reported to have no effect on GABA receptor IPSPs ([112]) or to enhance the activity of a subpopulation of zinc-sensitive GABA_A receptors via the D5 receptor, PKA, and PP1 ([113]).

As for the D1 and NMDA receptors, mutually inhibitory modulation between D5 and GABA_A receptors results from a protein-protein interaction between the D5 receptor and the γ₂ subunit of the GABA_A receptor ([114]). These direct interactions between D1-like and ion channel-coupled receptors may represent G protein-independent mechanisms of signaling, although it is also possible that G protein activation is required for formation of the multiprotein signaling complex.

D1-Like Receptor Regulation of Other Signaling Pathways

Inhibition of Na⁺-,K⁺-ATPase

D1-like receptors inhibit Na⁺-,K⁺-ATPase in many peripheral and neural tissues ([115], [116]). Multiple mechanisms mediate this effect. Activation of DARPP-32 plays a key role in both renal tubule cells ([117]), where inhibition of Na⁺-, K⁺-ATPase contributes to D1 receptor-stimulated natriuresis and diuresis, and neostriatal neurons ([23]). Phospholipase C-dependent mechanisms have also been described ([118], [119]), and D1-like receptor inhibition of Na⁺/H⁺ exchange would also be expected to inhibit Na⁺-,K⁺-ATPase activity by decreasing the intracellular concentration of Na⁺ (for an extensive review, see 115).

Activation of Mitogen-Activated Protein Kinases

Several reports describe D1-like receptor activation of mitogen-activated protein (MAP) kinases, including ERK ([35], [120]), p38 MAP kinase, and c-jun amino-terminal kinase ([121]). Regulation of the latter two MAP kinase pathways is mediated by PKA, whereas D1-like receptor activation of ERK may be partially independent of PKA or cyclic AMP. One possible PKA-independent but cyclic AMP-dependent mechanism for activation of ERK by the D1 receptor is activation of the Rap GTPase ([122]) by the cyclic AMP-activated guanine nucleotide-exchange factor Epac ([123]).

D2-Like Receptors and G Proteins

D2-like receptor signaling is mediated primarily by activation of the heterotrimeric G proteins Gα_i/o, a class of G proteins inactivated by pertussis toxin-catalyzed ADP-ribosylation ([124], [125]). For the D2 receptor, the possibility that the alternatively spliced insert in the third cytoplasmic loop of D2_L might influence G protein interactions and result in differential G protein selection by D2_S and D2_L has meant that analyses of G protein selection often focus on comparisons between the two isoforms. There is considerable disagreement in the literature concerning which G proteins interact with D2_S and D2_L ([126], [127]). It seems likely that both receptor isoforms are inherently able to activate multiple Gα_i/o subtypes, including Gα_i2, Gα_i3, and Gα_o ([128], [129]), but that interactions with particular G proteins are restricted in a cell-type dependent manner due to compartmentalization or the availability of appropriate effectors and scaffolding proteins. D2_S and D2_L can also activate the pertussis toxin-insensitive G protein Gα_z ([130], [131]). This interaction could explain some reports of pertussis toxin-insensitive signaling by the D2 receptor ([132], [133], [134]). Nevertheless, accumulating evidence from a variety of approaches has identified Gα_o as the Gα_i/o subtype that is most robustly activated by D2_L ([135], [136], [137]) and by D2_S ([138], [139], [140]) and, furthermore, the G protein subtype that is predominantly coupled to D2-like receptors in mouse brain ([141]).

The human D4 receptor is similar to D2 in that it activates multiple pertussis toxin-sensitive G proteins, including Gα_i2, Gα_i3, and Gα_o ([142], [143]). The rat D4 receptor has been reported to couple preferentially to Gα_z ([131]) and to the pertussis toxin-sensitive transducin subtype, Gα_t2 ([144]). Work by several groups has identified Gα_o as being activated by the D3 receptor and mediating D3 signaling, with some evidence for D3 receptor signaling via Gα_z and Gα_q/11 ([131], [142], [145], [146]). An example of the importance of cell-type specific factors in the selective activation of G proteins by GPCRs is that the D3 receptor couples more efficiently to Gα_o in SH-SY5Y cells than in HEK293 cells, despite the abundance of that G protein subtype in both cell lines ([146]). Zaworski et al. ([146]) suggest that the additional presence in SH-SY5Y cells of effectors regulated by the D3 receptor contributes to the efficient activation of Gα_o by the D3 receptor in those cells, a hypothesis consistent with other work showing that receptors form complexes with effectors and that G proteins participate in complex formation ([147]).

An unusual feature of the D3 receptor is that it binds agonists with a high affinity that is relatively insensitive to GTP ([126]). The affinity of GPCRs for agonists is typically reduced by GTP, reflecting GTP-induced destabilization of the agonist-receptor-G protein ternary complex. The insensitivity of the D3 receptor to GTP could reflect GTP-resistant coupling to G proteins or a receptor structure that is constrained in a comformation with high affinity for agonists regardless of interactions with G proteins; interesting work by Leysen and colleagues expressing the D3 receptor in E. coli, and thus in the absence of endogenous G proteins with which the receptor can interact, indicates that the latter explanation is more likely ([148]).

D2-like Dopamine Receptor Signaling

D2-Like Receptor Signaling via Gα_i/o—Inhibition of Adenylate Cyclase

The first signaling pathway identified for D2-like receptors was inhibition of cyclic AMP accumulation ([149], [150]). As for D1 receptor stimulation of adenylate cyclase, genetic deletion of adenylate cyclase 5 abolishes D2 receptor-mediated inhibition of adenylate cyclase in the mouse neostriatum. That this null mutation also eliminates the locomotor inhibitory effects of D2 receptor-blocking antipsychotic drugs shows the behavioral significance of this signaling pathway ([16]). The lack of responsiveness to antipsychotic drugs is a phenotype also seen in D2 receptor ([151]) and DARPP-32 ([20]) null mutant mice, further indicating that this signaling pathway contributes to D2 receptor-stimulated locomotor activity. D2 and D4 receptors inhibit adenylate cyclase activity in a variety of tissues and cell lines ([2], [152]). Inhibition of adenylate cyclase by the D3 receptor is weaker and often undetectable although it is of interest that the D3 receptor robustly inhibits adenylate cyclase type 5 ([153], [154]), in contrast to several other adenylate cyclase subtypes including the closely related type 6. On the other hand, co-expression of D2 and D3 receptors in COS-7 cells with adenylate cyclase 6 substantially increases the potency of D2-like receptor agonists for inhibition of cyclic AMP accumulation, suggesting that the D2/D3 heteromer has increased potency for agonists and/or couples more efficiently to adenylate cyclase 6 ([154], [155]).

D2-like receptor inhibition of adenylate cyclase is presumed to be mediated by Gα_i/o, because adenylate cyclase 5 is directly inhibited by Gα_i and is insensitive to Gβγ ([156]). Gα_i binds primarily to the C1 cytosolic domain of Gα_i-inhibited forms of adenylate cyclase and reduces C1/C2 domain interaction ([157]). One inconsistency is that adenylate cyclase 5 expressed in insect Sf9 cells is insensitive to purified Gα_o ([156]), whereas work cited above supports a prominent D2 → Gα_o → adenylate cyclase 5 signaling path.

D2 receptor signaling via inhibition of adenylate cyclase would be expected to act in opposition to agents that stimulate adenylate cyclase, decreasing the phosphorylation of PKA substrates (Figure 3). For example, stimulation of D2-like receptors decreases PKA-stimulated phosphorylation of DARPP-32 at Thr34 and increases phosphorylation at Thr75 ([22], [158]). Both of these effects may be at least partially mediated by Gα_i-dependent inhibition of cyclic AMP, although for Thr34, the lack of effect of quinpirole in Ca²⁺-free medium is difficult to reconcile with a major role for adenylate cyclase inhibition; calcium-dependent stimulation of calcineurin (protein phosphatase 2B) also contributes to D2 receptor dephosphoryation of Thr34 ([158]). It is of interest that the opposing effects of D1 and D2 receptors on DARPP-32 both lead to inhibition of the Na⁺,K⁺-ATPase in neostriatal neurons ([159]). Another response potentially mediated by D2-like receptor inhibition of adenylate cyclase is autoreceptor suppression of tyrosine hydroxylase activity. Stimulation of D2-like synthesis-inhibiting autoreceptors reverses PKA-dependent phosphorylation of tyrosine hydroxylase at Ser40 and activation of the enzyme ([160]). Although it is reasonable to speculate that inhibition of adenylate cyclase contributes to this response, a role for other mechanisms, such as calcium-stimulated protein phosphatases, has not been excluded. Inhibition of adenylate cyclase is also likely to be the mechanism of D4 receptor-induced inhibition of GABA currents in rat globus pallidus, perhaps by decreasing PKA-dependent phosphorylation of the β₃ subunit of the GABA_A receptor ([161]), and the proximal mechanism of D4 receptor-induced inhibition of NMDA receptor currents in rat prefrontal cortex, for which the complete pathway is apparently disinhibition of PP1, dephosphorylation of autophosphorylated calcium-, calmodulin-dependent kinase II thus reducing its calcium-independent kinase activity, and decreased phosphorylation of the NR1 subunit of the NMDA receptor which in turn decreases NMDA receptor expression in the plasma membrane ([162]). On the other hand, inhibition of NMDA receptor transmission by the D4 receptor in rat hippocampus apparently involves platelet-derived growth factor receptor-dependent mobilization of calcium rather than inhibition of PKA-dependent phosphorylation ([163]).

D2-Like Receptor Signaling Mediated by G Protein βγ Subunits

As is typical of Gα_i/o-coupled receptors, D2-like receptors modulate many signaling pathways in addition to adenylate cyclase, including phospholipases, ion channels, MAP kinases, and the Na⁺/H⁺ exchanger ([2]). Many of these pathways are regulated by G protein βγ subunits that are released by receptor activation of Gα_i/o proteins (Figure 3).

Gβγ-Stimulated Adenylate Cyclase

Gβγ has a permissive effect on adenylate cyclase 2 and 4, so that the stimulatory effect of other activators, including Gα_s and protein kinase C, is enhanced in the presence of free Gβγ ([52], [53], [156]). Gβγ binds to residues in several cytosolic domains of adenylate cyclase 2, with stimulation primarily due to binding to the less conserved region of cytosolic loop 1, C1b ([164], [165]). This Gβγ-mediated stimulatory effect is primarily observed for Gα_i/o-coupled receptors, perhaps because activation of only this relatively abundant class of G proteins liberates a sufficient concentration of Gβγ subunits. D2 and D4 receptors markedly increase the activity of adenylate cyclase 2, whereas the D3 receptor has little or no effect ([53], [153]). D2-like receptor activation of Gβγ-stimulated adenylate cyclase response has only been observed by using heterologously expressed receptors and adenylate cyclase. It is not known if it contributes to D2-like receptor signaling in neurons, although it has been speculated that Gβγ-stimulated adenylate cyclase contributes to synergistic activation of spike firing in nucleus accumbens neurons by D1-like and D2-like receptors ([51]).

Gβγ-Stimulated K⁺ Channels

D2 stimulation exerts a powerful influence over K⁺ currents likely via dissociation of Gβγ subunits rather than by Gα_i-dependent inhibition of adenylate cyclase activity. Unlike the generally excitatory effect of D1 receptor stimulation, D2 stimulation decreases cell excitability by increasing K⁺ currents in most brain areas (Figure 2).

All of the D2-like receptors activate a G protein-regulated inwardly rectifying potassium channel (GIRK or Kir3), a channel that carries one of several potassium currents modulated by dopamine in midbrain dopamine neurons ([166], [167]), and neostriatal D2 receptor-expressing neurons ([168]), with activation presumably via Gβγ ([169], [170], [171]). The D3 receptor is approximately as efficient as the D2_L receptor at coupling to homomeric GIRK2 ([172]), the GIRK subtype predominantly expressed by dopamine neurons in the rat ventral mesencephalon ([173], [174]), and regulation of GIRK channels contributes to inhibition of secretion by the D3 receptor heterologously expressed in AtT-20 mouse pituitary cells ([175]). D2 and D4 receptors both co-precipitate with GIRK channels in a heterologous expression system, and the rat neostriatal D2 receptor co-precipitates with GIRK2, suggesting the existence of a stable complex that forms during receptor/channel biosynthesis ([147]). The formation of large multiprotein complexes that include GPCRs and their effectors may be a general characteristic of GPCR signaling ([176]). Evidence that dopamine release-regulating autoreceptors are coupled to potassium channels ([177]) rather than to inhibition of adenylate cyclase ([178]), together with the robust regulation of GIRK currents by D2 receptors in substantia nigra dopamine neurons ([179]), suggests that D2 receptor activation of GIRK currents contributes to D2 autoreceptor inhibition of dopamine release and dopamine neuronal activity. The hyperactivity and facilitation of D1 receptor signaling observed in GIRK2 null mutant mice ([180]) is also consistent with a loss of inhibitory autoreceptor function.

The effect of D2 stimulation on voltage-gated outward potassium currents appears to depend on the brain area being examined. Medium spiny neurons in the striatum show an increase in the amplitude of the slowly inactivated K⁺ current (I_D) in the presence of D2 agonists ([181]), but pyramidal neurons of the cortex show no effect of D2 stimulation on this same current ([57]). More work is needed to understand the mechanism by which D2 receptors exert their effects on voltage-gated K⁺ channels.

Gβγ-Mediated Regulation of Ca²⁺ Channels

All D2-like receptors decrease the activity L, N, and P/Q-type channels via pertussis toxin-sensitive G proteins ([128], [182], [183], [184], [185]). D2 receptors in neostriatal large aspiny (cholinergic) interneurons inhibit N-type Ca²⁺ channels by a membrane-delimited pathway that probably involves Gβγ subunits ([186], [187]). Gβγ subunits target the I-II linker region and/or the COOH terminus of the α-subunit of these Ca²⁺ channels, decreasing the amount of current carried ([188], [189], [190]). D2 receptors in striatal medium spiny neurons decrease L-type Ca²⁺ currents ([191]). This response is also mediated by Gβγ subunits, although rather than being membrane delimited the pathway involves Gβγ stimulation of cytosolic phospholipase C, mobilization of Ca²⁺, and activation of calcineurin (Figure 3).

The functional effects of this modulation are possibly to regulate neurotransmitter release when the N-type Ca²⁺ channel is targeted; thus, D2 receptor activation of neostriatal interneurons inhibits the release of acetylcholine ([192]), and D2-like receptor stimulation decreases glutamate release in striatum, presumably via effects on N-type Ca²⁺ channels ([193], [194]). Inhibition of glutamate release may implicate tonic D2 receptor activity in the prevention of excessive glutamate release; together with D2 receptor inhibition of L-type channels in neostriatal medium spiny neurons, this would be expected to promote the downstate in neostriatal projection neurons. Facilitory effects of D1-like receptor stimulation would take over when synchronized inputs arrive drive the cell into the upstate. In this way, D2 receptor activation may serve as a gatekeeper over striatal upstates ([85]). Voltage-dependent Ca²⁺ channels are inhibited by D2 receptors in the anterior pituitary ([128]) and by the D3 receptor heterologously expressed in AtT-20 cells ([184]); inhibition of Ca²⁺ channels in these cells would be expected to decrease secretion of pituitary hormones.

Gβγ-Stimulated MAP Kinases

MAP kinases are components of parallel protein kinase cascades that transmit signals from a variety of extracellular stimuli to the cell nucleus, thus participating in cell proliferation, differentiation, and survival ([195]). Like many other GPCRs, including those coupled to Gα_i/o ([195], [196]), activation of the D2 receptor stimulates MAP kinases, including the two isozymes of extracellular signal-regulated kinase (ERK) ([197], [198], [199], [200], [201], [202], [203], [204]) and stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK) ([199]). D3 ([205]) and D4 ([203], [206]) dopamine receptors also activate ERK. D2-like receptors activate ERK in brain slices ([35], [207]) and in rat brain after administration of agonist in vivo ([208]).

Although the pathway from D2-like receptors to ERK has not been thoroughly elucidated and may differ depending on cell type and receptor subtype, D2-like receptor activation of ERK is frequently mediated by pertussis toxin-sensitive G proteins ([200], [201], [205], [209]), Gβγ ([197], [201], [202]), phosphatidylinositol 3-kinase ([200], [205]), Ras ([199], [206]), and the MAP kinase kinase MEK ([199], [201], [207], [208]). D2-like receptor activation of ERK is in at least some cases mediated by transactivation of a receptor tyrosine kinase (RTK), thus recruiting the RTK-signaling cascade in response to dopamine. Although the epidermal growth factor receptor has frequently been identified as an RTK that is transactivated by GPCRs ([210], [211]), transactivation of the platelet-derived growth factor receptor can be a necessary intermediate step in the activation of ERK by recombinant and endogenous D2 and D4 receptors ([163], [203], [204]). We have observed that the identity of the RTK that is transactivated by D2-like receptors depends on cell type, with D2 receptor stimulation of ERK mediated by the platelet-derived growth factor receptor in nonneuronal cells, but by the epidermal growth factor receptor in neuroblastoma cells and primary neuronal cultures from embryonic rat neostriatum ([209]). It is not clear which of the several identified mechanisms for RTK transactivation ([212]) are used by D2-like receptors, although in some cells D2 receptor stimulation enhances a direct interaction between the D2 receptor and the platelet-derived growth factor receptor ([203]) or the epidermal growth factor receptor ([209], [213]).

D2 receptor activation of ERK stimulates DNA synthesis and mitogenesis in many different cell types ([199], [202], [214], [215]). In post-mitotic neurons, activation of MAP kinases is involved not only in cell survival and in synaptic plasticity ([216], [217], [218]) but also in acute behavioral responses to dopamine receptor stimulation ([208]). D2 receptor signaling to ERK in pituitary lactotrophs may be more complicated; in both primary lactotrophs and a prolactin-secreting cell line, the D2 receptor is reported to inhibit ERK, leading to suppression of prolactin promoter function ([139]). A conflicting report using a different prolactin-secreting cell line describes D2 receptor stimulation of ERK leading to inhibition of cell proliferation ([219]).

Epidermal growth factor receptors signal not only through a Ras-MEK-ERK pathway but also through a phosphoinositide 3-kinase → protein kinase B (Akt) pathway. D2 and D4 receptors activate Akt in several cell types with neuronal characteristics and in immortalized dopaminergic neurons ([206], [213]). In PC12 cells expressing a recombinant D2 receptor, D2 receptor activation of Akt is mediated by Src-dependent transactivation of the epidermal growth factor receptor. This pathway mediates D2 receptor neuroprotection against oxidative stress ([213]).

Other Signaling Pathways Regulated by D2-Like Receptors

D2-Like Receptor Regulation of Phospholipases

D2 receptors in neostriatal medium spiny neurons activate a cytosolic, Gβγ-stimulated form of phospholipase C, PLCβ1, causing inositol trisphosphate-induced calcium mobilization that activates calcium-dependent proteins such as the protein phosphatase calcineurin, ultimately reducing L-type Ca²⁺ currents ([220]). This pathway may contribute to activation of both ERK and CREB by D2-like receptors in neostriatal neurons ([207]) and also to the PKA-independent regulation of DARPP-32 phosphorylation by D2-like receptors described above ([158]). This pathway and the D4 receptor-stimulated transactivation of the platelet-derived growth factor receptor leading to depression of NMDA receptor signaling in hippocampal neurons have interesting similarities, such as dependence on Gβγ, phospholipase C, and calcium mobilization, which are suggestive of a more general role for RTK transactivation in D2-like receptor signaling.

The D2 receptor potentiates arachadonic acid release induced by calcium-mobilizing receptors in heterologous expression systems ([220], [221], [222]), a response that is mediated by cytosolic phospholipase A₂ ([223]). The D4 receptor also activates this pathway ([223]), whereas the D3 receptor has no effect or is inhibitory ([224]). Although the response is inhibited by pertussis toxin, the lack of effect of cyclic AMP and the sensitivity of cytosolic phospholipase A₂ to Gβγ ([225], [226]) suggest that this is a Gβ-mediated response. D2 receptor-stimulated arachidonate release in the absence of a calcium-mobilizing agent has also been observed ([227]). Arachidonic acid and its bioactive lipooxygenase and cyclooxygense metabolites (e.g., prostaglandin E₂) have numerous effects on cellular function and may feed back to regulate D2-like receptor signaling and uptake ([228], [229], [230], [231]).

Many GPCRs, including the D2 receptor, stimulate phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to form choline and phosphatidic acid ([232], [233]). The low molecular weight G protein RhoA is required for this D2 receptor-mediated response ([234]), and work with other phospholipase D-activating GPCRs has identified a direct interaction between the receptors and two monomeric G proteins RhoA and ARF ([232]). D2 receptor stimulation of phospholipase D is not mediated by Gα_i/o, because stimulation is insensitive to pertussis toxin but does require activation of protein kinase Cε ([233]). Activation of Gα₁₃, which in turn stimulates the Rho guanine nucleotide exchange activator (p115RhoGEF), is a mechanism by which some GPCRs activate phospholipase D ([235], [236], [237]).

D2-Like Receptor Regulation of Na⁺ Channels

D2-like receptor activation in the neostriatum exerts variable effects on Na⁺ channels possibly via several intracellular signaling pathways. In the prefrontal cortex, on the other hand, D2-like receptor stimulation does not appear to exert a consistent or strong effect over Na⁺ channels in some studies (81, but see 238), perhaps because D1-like receptors are somehow in a better position to control these channels or simply more abundant. The strong effect of prefrontal cortical D2-like receptors on other ion channels may reflect differences in scaffolding with these channels.

Surmeier et al. ([27], [181]) discovered that D2-like receptor stimulation can either increase or decrease Na⁺ currents in neostriatal neurons, perhaps depending on the subtype of D2-like receptors expressed by a given cell. Enhancement of Na⁺ currents requires a soluble second messenger and may represent a process reciprocal to attenuation of voltage-gated Na⁺ currents by D1 receptor-stimulated activation of PKA and DARPP-32 ([23], [116]). That is, the D2-like receptor stimulated increase in Na⁺ current observed in a minority of neostriatal neurons may be due to inhibition of adenylate cyclase and PKA, preventing phosphorylation of the channel. In most D2 agonist-responsive neurons, D2-like receptor stimulation decreases Na⁺ currents in a membrane-delimited manner ([181]) that may involve binding of Gβγ subunits to the Na⁺ channels. The Na⁺ current attenuation involved a shift in inactivation potential to more negative values, with no decrease in the peak amplitude of the current. These results mimic the effects of inhibition of pertussis toxin-sensitive G-proteins (presumably G_i/o) on recombinant Na⁺ channels in CHO cells that are similar to the type expressed in the striatum ([239]).

D2-Like Receptor Regulation of Na⁺/H⁺ Exchange

The Na⁺/H⁺ exchangers are a family of integral membrane proteins that regulate intracellular pH and transcellular Na⁺ absorption ([240]). Heterologously expressed D2 ([133]), D3 ([241], [242]), and D4 receptors ([222]) activate the widely expressed Na⁺/H⁺ exchanger NHE1. Like GPCR activation of phospholipase D, D2 receptor activation of Na⁺/H⁺ exchange is insensitive to pertussis toxin in some cell lines ([133]), and the G protein Gα₁₃ can activate the exchanger via Rho ([243]). It is of interest that several GPCR subtypes, including endogenous D2-like receptors in primary lactotrophs, mediate pertussis toxin-insensitive and cyclic AMP-independent inhibition of Na⁺/H⁺ exchanger activity ([132], [244]). Lin et al. ([244]) have proposed that a potential mechanism for this inhibition is Gα₁₂ competition for the interaction between Gα₁₃ and p115RhoGEF. Na⁺/H⁺ exchange is also inhibited by D1-like receptors by both cyclic AMP-dependent and -independent mechanisms ([115], [245]).

D2-Like Receptor Regulation of Receptor-Linked Ion Channels

Glutamatergic neurotransmission is enhanced in D2 receptor null mutant mice, which probably reflects the loss of both presynaptic control of glutamate release and postsynaptic inhibition of glutamatergic responses by the D2 receptor ([246]). D2-like receptor stimulation decreases NMDA currents, but the mechanism for this effect is still debated. One idea is that D2 stimulation results in changes in K⁺ and Na⁺ permeabilities that hyperpolarize cells and prevent the removal of the Mg²⁺ blockade over these channels, resulting in a decrease in the open probability of the channel ([98]). Another idea is that D2 stimulation causes dephosphorylation of the NR1 subunit by antagonizing D1-like receptor stimulation of the DARPP-32 signaling cascade ([92]). In prefrontal cortex, the D4 receptor decreases NMDA currents by a similar mechanism involving disinhibition of PP1 ([162]). Another means by which D2 activation attenuates NMDA currents in hippocampus is via transactivation of the platelet-derived growth factor receptor ([163]).

A consistent role for the D2 receptor has not been described for GABA transmission, perhaps because both D1-like and D2-like receptors modulate GABAergic transmission by altering GABA release ([247]). In addition to decreasing the probability of GABA release in the prefrontal cortex, D2 receptor agonists reduce postsynaptic responsiveness to a GABA_A agonist by a mechanism that includes transactivation of the platelet-derived growth factor receptor ([247], [248]). This mechanism is consistent with work showing that activation of the platelet-derived growth factor receptor decreases GABA_A currents via activation of phospholipase C and subsequent mobilization of intracellular Ca²⁺ to alter the phosphorylation state of the receptor ([249]).

Novel Protein–Protein Interactions Involved in D2-Like Receptor Signaling

D2-Like Receptor Signaling via Receptor Heteromerization

We have discussed some functional consequences of dopamine receptor heteromerization with ion channels (D1 and NMDA, D5 and GABA_A), receptor tyrosine kinases (D2/D4 and the epidermal growth factor and platelet-derived growth factor receptors), and other dopamine receptor subtypes (D2 and D3) in other contexts in this review. The D2 receptor can also participate in heteromers with other GPCRs including the somatostatin receptor subtype SSTR5 ([251]), and the adenosine A2_A receptor ([251], [252]). Intramembrane interactions of the D2 receptor with neurotensin and metabotropic glutamate receptors are also potentially mediated by direct receptor:receptor interactions ([253]), although competition for a limiting pool of another receptor-interacting protein is another possible mechanism. The formation of heteromers can be constitutive ([252]) or stimulated by the binding of ligands, particularly agonists ([250]), and may underlie mutually inhibitory ([253]) or stimulatory ([250]) interactions. There is considerable behavioral and functional evidence for interactions between brain dopamine and adenosine systems and between dopamine and somatostatin, and receptor heteromerization is likely to be a molecular mechanism of these interactions ([254]).

Other Dopamine Receptor-Interacting Proteins That Modulate Signaling

A number of interactions between the third cytoplasmic loop of D2-like receptors and other proteins are likely to influence D2-like receptor signaling. D2 and D3 receptors, but not D1 or D4 receptors, bind the actin-binding protein filamin A, or ABP-280. Zhou and colleagues report that binding is to a segment in the carboxyl terminus of the third cytoplasmic loop, where both D2 and D3 receptors have a potential site of phosphorylation by PKC, and that D2 and D3 receptors expressed in cells that lack ABP-280 have diminished ability to inhibit adenylate cyclase ([255], [256]). Furthermore, PKC-catalyzed phosphorylation of the D2 receptor on Ser358 may inhibit binding of ABP-280, thus attenuating D2 receptor signaling ([255]). The third cytoplasmic loop of the D2 receptor includes a binding site for spinophilin, a scaffolding protein that also binds and targets PP1 to dendritic spines ([257]). PP1 anchoring by spinophilin is critical for modulation of glutamate receptor activity by dopamine ([100]). Calmodulin binds in a calcium-dependent manner to the amino terminal end of the D2 receptor third cytoplasmic loop and inhibits D2 receptor activation of, but not binding to, Gα_i ([258]). Proteins, such as Nck, Grb2, and c-Src which contain Src homology 3 (SH3) domains, a modular protein-protein interaction domain that is essential for the formation of functional signaling complexes, bind to the third cytoplasmic loop of the D4 receptor, which has multiple copies of the proline-rich SH3 binding motif ([259]). Several SH3 domain-containing proteins also bind to the D3 receptor, although the site of binding has not been identified ([259], [260]). The functional role of SH3 protein binding to D2-like receptors is unknown, although mutation of the SH3 binding motifs in the D4 receptor causes constitutive internalization of the receptor ([260]), and binding of the protein tyrosine kinase c-Src to other GPCRs has important consequences for receptor signaling and desensitization ([261]).

Conclusion

Dopamine receptor signal transduction pathways are complex, so that just one dopamine receptor subtype can activate multiple effectors by distinct or partially overlapping pathways that have many points of intersection. One of the goals of this review was to show how these effectors work in concert to alter cellular function in response to dopamine receptor activation, an area in which considerable progress has been made in recent years. On the other hand, much less is known about how these cellular processes contribute to the function of neuronal networks and, ultimately, to behavior. Another emerging area that is only touched on in this review, despite its probable importance for GPCR function, is the role of protein-protein interactions and signaling complexes in dopamine receptor signaling. Elucidating the mechanisms and significance of receptor oligomerization, of interactions with signal-switching proteins, such as calcyon, and of interactions with scaffolding proteins that support the formation of the signaling complex and target the complex to particular subcellular domains, will be crucial to understanding dopamine receptor function at the molecular level.