Reevaluation of the dopamine D₂ receptor in the treatment of schizophrenia: Novel intracellular mechanisms as predictors of antipsychotic efficacy
Since the advent of antipsychotic medications, there have been many speculations about their precise mechanisms of therapeutic action. Although it is apparent that blockade of dopamine D₂ receptors (D₂R) is crucial to the efficacy of all current antipsychotic medications, it is not clear which signaling events downstream of the D₂R must be blocked for the therapeutic actions of antipsychotics and which events, when blocked, lead instead to side effects.

The best characterized D₂R-mediated signaling pathways involve coupling of the receptor to pertussis toxin-sensitive G proteins of the G_i and G_o subfamilies (Sidhu and Niznik, 2000), through which D₂R activation results in a decrease in cyclic AMP (cAMP). D₂R activation can also have a number of other effects, including enhancement of specific potassium currents, inhibition of L-type calcium currents, mediation of extracellular signal-regulated kinase 1 (ERK1) and potentiation of arachidonic acid release (Beom et al., 2004; Missale et al., 1998; Perez et al., 2006; Hernández-López et al., 2000). There is growing evidence that D₂Rs can interact with a number of membrane-bound or intracellular proteins, which may further modulate signaling specificity (reviewed in Terrillon and Bouvier, 2004; Ferré et al., 2007a). In particular, D₂R heteromerization may result in a switch from G_i/_o coupling to G_s (i.e., through D₂R and cannabinoid 1 receptor interaction) (Kearn et al., 2005) or to coupling with G_q (as suggested in D₂R and D₁R interactions) (Rashid et al., 2007). Moreover, heteromerization between D₂R and other receptors such as the adenosine A_2A receptor may allow for reciprocal modulation of D₂R function (Ferré et al., 2007a; Ferré et al., 2007b). It also has been suggested that calcium signaling mechanisms may modulate D₂R’s signaling efficacy; interaction between D₂R and calcium-binding protein S100B results in enhanced D₂R intracellular signaling (Liu et al., 2008; Stanwood, 2008).

The interaction between D₂R and arrestin has received increasing attention. Following D₂R activation, D₂R signaling is attenuated by recruitment of arrestin 3 to the cell surface where it binds to the receptor (Klewe et al., 2008; Lan et al., 2008a ; Lan et al., 2008b), leading to inactivation and internalization of the D₂R. Arrestin 3 also binds Akt—a serine/threonine kinase involved in multiple cellular functions and implicated clinically in schizophrenia (Arguello and Gogos; 2008; Beaulieu et al., 2005; Brazil and Hemmings, 2001; Brazil et al., 2004; Emamian et al., 2004; Kalkman, 2006). Following D₂R activation by dopamine, the signaling scaffold formed by arrestin 3, while facilitating receptor desensitization and internalization, also recruits Akt into a complex with the phosphatase PP2A, which dephosphorylates and consequently inactivates Akt (Beaulieu et al., 2007a ). Thus, D₂R activation inhibits Akt activity through an arrestin-dependent but G protein-independent pathway (Beaulieu et al., 2007a ; Beaulieu et al., 2007b). Curiously, the mood stabilizer, lithium, has been shown to disrupt the arrestin 3-Akt-PP2A complex, thereby preventing dopamine-induced dephosphorylation of Akt and blocking amphetamine-induced locomotion (Beaulieu et al., 2008). Moreover, amphetamine-induced locomotion is greatly diminished in arrestin 3 knockout mice, suggesting that this pathway is critical to at least some psychostimulant effects (Beaulieu et al., 2005).

Using newly developed BRET (bioluminescent resonance energy transfer) biosensors in assays that measure direct protein-protein interactions within the living cell, recent studies have demonstrated that antipsychotic medications prevent arrestin 3 recruitment by blocking D₂R activation (Klewe et al., 2008; Masri et al., 2008). Masri et al. (2008) hypothesized that antipsychotic drugs achieve their therapeutic effect through a common mechanism involving blockade of arrestin-mediated signaling (Masri et al., 2008). Masri et al. (2008) also demonstrated that nearly all antipsychotics tested (including haloperidol, clozapine, olanzapine, desmethylclozapine, chlorpromazine, quetiapine, risperidone and ziprasidone) behave as inverse agonists to decrease constitutive G protein signaling as well as to prevent the agonist quinpirole from inhibiting cAMP synthesis (via D₂R-mediated G_i/_o signaling). The lone exception, aripiprazole, behaved as a partial agonist instead of as an inverse agonist of the G protein mediated effects. The latter finding is consistent with previous studies highlighting aripiprazole’s ability to differentially modulate various G protein-mediated effector pathways, a property termed “functional selectivity” (Mailman, 2007; Urban et al., 2007). Using the BRET assay, Klewe et al. (2008) and more recently Masri et al. (2008) demonstrated that all antipsychotics, including aripiprazole, block arrestin 3 recruitment. This finding has led Masri et al. (2008) to suggest that blockade of arrestin 3 recruitment to the D₂R, and not modulation of G-protein-mediated pathways, is a common and specific property of all current antipsychotics and may be used to predict the antipsychotic efficacy of drugs in development (Masri et al., 2008). This hypothesis remains to be tested and at present appears to lean heavily on the evidence for aripiprazole’s atypical effects on constitutive (non-agonist-dependent) D₂R-mediated G-protein signaling. Indeed, the fact that lithium acts to prevent arrestin-mediated signaling in response to amphetamine but is not an effective antipsychotic in monotherapy suggests that antipsychotic action may be more complex than simple blockade of D₂R-mediated arrestin signaling. In addition, the ability of antipsychotics, including aripiprazole, to block agonist binding to the D₂R and thus activation of the receptor, makes it likely that agonist-induced activity in multiple signaling pathways will also be blocked by these drugs.

Despite the paucity of direct evidence for D₂R-arrestin coupling as the mechanism underlying the antipsychotic effects of drugs, the hypothesis remains quite intriguing Given that Akt and its downstream target GSK-3 (glycogen synthase kinase-3) have been implicated in schizophrenia in a number of genetic and postmortem studies, and the Akt/GSK-3 pathway might represent an opening into alternative therapeutics of schizophrenia. Akt is a serine/threonine kinase that may have significant roles in synaptic physiology and neurodegeneration (Brazil et al., 2004). Recruited to the cell surface by binding to phosphatidylinositol 3,4,5 trisphosphate, Akt is activated via phosphorylation of 3-phosphoinoitide-dependent protein kinase 1 (PDK1) and the rictor-mTOR complex (Brazil and Hemmings, 2001; Sarbassov et al., 2005). Once active, Akt phosphorylates GSK-3, thereby inactivating it. Since D₂R activation leads to inactivation of Akt, this also results in increased GSK-3 activity (Beaulieu et al., 2004; Lovestone et al., 2007). GSK-3 activity also plays an important role in modulating the dopaminergic response to amphetamine. Amphetamine’s stimulation of DAT-mediated dopamine efflux and subsequent D₂R stimulation likely results in Akt inactivation and increased GSK-3 activity. Rats treated with the specific GSK-3 inhibitor, AR-A014418, failed to display amphetamine-induced hyperactivity (Gould et al., 2004). Similarly, heterozygous GSK-3β knockout mice (expressing approximately half of wildtype levels of GSK-3β) displayed significantly reduced levels of locomotor activity following amphetamine treatment (Beaulieu et al., 2004). Additionally, treatment of dopamine transporter (DAT) knockout mice with multiple GSK-3 inhibitory drugs inhibited the ordinarily hyperactive behavior of the non-treated DAT knockout mice (Beaulieu et al., 2004).

In a mouse model, acute and chronic haloperidol treatment was shown to increase levels of active, phosphorylated Akt isoform Akt1 and increased phosphorylation and inactivation of GSK-3β (Emamian et al., 2004). Thus, it was suggested that haloperidol treatment may compensate for the decreased levels of endogenous Akt1 in the frontal cortex of people with schizophrenia (Emamian et al., 2004). Atypical antipsychotics also impact on the regulation of Akt and GSK-3β activities. For example, treatment with clozapine results in increased levels of phosphorylated GSK-3β (Kang et al., 2004; Sutton et al., 2007). Interestingly, however, differences between haloperidol and atypical antipsychotics have emerged in the kinetics of Akt/GSK-3 phosphorylation, the levels of proteins expressed following drug exposure, and the signaling pathways that are preferentially activated (Roh et al., 2007).

The abilities of antipsychotic drugs to activate distinct signaling pathways to mediate their ostensible differential pharmacologic effects would suggest clinical variation in their therapeutic effects. However, meaningful differences in the clinical effects of these compounds have not been clearly or consistently evident. The initial reports of superior efficacy of the so-called second generation or atypical antipsychotics on measures of psychosis (Kane et al., 1988), negative symptoms (Tollefson et al., 1997), cognitive deficits (Keefe et al., 1999), relapse prevention (Csernansky et al., 2002), adherence (Wahlbeck et al., 2001) and illness progression (Lieberman et al., 2005a), have not been borne out by more recent studies (Geddes et al., 2000; Lieberman et al., 2005b; Jones et al., 2006; Leucht et al., 2008). Indeed, the differences between antipsychotic drugs are most evident in the types, frequency and severity of side effects rather than in their therapeutic actions (Leucht et al., 1999; Allison et al., 1999; Henderson et al., 2005). In this regard the emerging pattern of variation in the molecular mechanisms of antipsychotic drugs in the face of their common clinical profiles resembles what was previously observed with the variability in neuroreceptor binding profiles (Kinon and Lieberman, 1996). The marked differences in the affinities and selectivity of the various antipsychotics for the receptors of different neurotransmitters were thought to underlie a rich pattern of clinical variation in the therapeutic actions of this group of drugs (Miyamoto et al., 2005). However, this hypothesis has not been supported by clinical studies (Lieberman, 2006; Lewis and Lieberman, 2008).

Nevertheless, there is reason to be hopeful that through functional selectivity, or other potential actions, the abilities of drugs to engage different signaling pathways will confer novel therapeutic effects that will improve the efficacy of treatments. In this context, the studies of Masri et al. (2008) and Klewe et al. (2008) highlight the plausibility that D₂R/arrestin 3 modulation of Akt and GSK-3 activity is an important mechanism underlying psychosis and a potential target for future antipsychotic drugs. Further study of this pathway, including studies designed to reverse the effects of D₂R antagonists or partial agonists (antipsychotic drugs) with systematic differential manipulation of the signaling pathways induced by D₂R activation, is likely to be a fruitful path toward the development of novel treatments for schizophrenia-related disorders.

Acknowledgements: The authors would like to acknowledge the generous support of the Lieber Center for Schizophrenia Research at Columbia University

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