Dopamine receptor-interacting proteins: the Ca2+ connection in dopamine signaling

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Dopamine receptor-interacting proteins: the Ca21 connection in dopamine signaling Clare Bergson1, Robert Levenson2, Patricia S. Goldman-Rakic3 and Michael S. Lidow4 1

Department Department 3 Department 4 Department 2

of of of of

Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USA Pharmacology, Pennsylvania State College of Medicine, Hershey, PA 17033, USA Neurobiology, Yale School of Medicine, New Haven, CT 06510, USA Biomedical Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA

Abnormal activity of the dopamine system has been implicated in several psychiatric and neurological illnesses; however, lack of knowledge about the precise sites of dopamine dysfunction has compromised our ability to improve the efficacy and safety of dopaminerelated drugs used in treatment modalities. Recent work suggests that dopamine transmission is regulated via the concerted efforts of a cohort of cytoskeletal, adaptor and signaling proteins called dopamine receptorinteracting proteins (DRIPs). The discovery that two DRIPs, calcyon and neuronal Ca21 sensor 1 (NCS-1), are upregulated in schizophrenia highlights the possibility that altered protein interactions and defects in Ca21 homeostasis might contribute to abnormalities in the brain dopamine system in neuropsychiatric diseases. The neurotransmitter dopamine (DA) plays a prominent role in a variety of vital brain functions including motor control, short-term memory, attention and reward [1– 3]. Imbalances in DA transmission have been associated with stress-induced alterations in brain function [4] and drug dependence [5]. Abnormal activity of the DA system has been implicated in psychosocial and neurological illnesses, including Parkinson’s disease, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder (ADHD) and Gilles de la Tourette syndrome [6– 8]. However, lack of knowledge about the precise sites of the DA dysfunction in schizophrenia, bipolar disorder and ADHD has compromised our ability to improve the efficacy and safety of dopamine-related drugs used in treatment modalities. In the CNS, DA modulates neuronal excitability by regulating ligand- and voltage-gated ion channels [9]. The actions of DA are mediated by a family of seventransmembrane G-protein-coupled receptors (GPCRs), D1, D2, D3, D4 and D5 receptors, encoded by five distinct genes. Studies with recombinant receptors indicate that each receptor subtype displays a unique set of properties with respect to affinity for DA, potential for alternative splicing, and specificity of coupling to heterotrimeric GTP-binding G proteins [10]. For example, although both D1-like receptors (D1 and D5 receptor subtypes) stimulate Corresponding author: Clare Bergson ([email protected]).

adenylyl cyclase, the D1 receptor subtype, unlike the D5 receptor subtype, preferentially couples to G-protein heterotrimers that contain g7 subunits [11], and the D5 receptor exhibits tenfold higher affinity for DA than does the D1 receptor [10]. Similarly, although each D2-like receptor (D2, D3 and D4 receptor subtypes) inhibits adenylyl cyclase, the D4 receptor displays a tenfold higher affinity for the atypical antipsychotic clozapine than does either the D2 or the D3 receptor [10]. In addition, the recent identification of a group of DA receptor-interacting proteins (DRIPs) suggests that the intracellular activity of individual DA receptor subtypes is regulated by the concerted actions of a cohort of cytoskeletal, adaptor and signaling proteins (Table 1). DA receptors can also interact with themselves or other receptors to form novel heterodimers with distinct receptor pharmacologies [12– 20]. Dopamine receptor signaling complexes Elucidating the complete array of DRIPs has now taken center stage in efforts to identify the players that are involved in the regulation of dopamine-mediated signaling. Many of the known DRIPs have been identified using the yeast two-hybrid (Y2H) system in which segments of a DA receptor are used to ‘fish out’ interacting proteins from brain cDNA libraries. A striking aspect of these screens is the finding that the D1 and D2 receptor subtypes appear to interact with two different sets of DRIPs (Table 1). A possible explanation for this non-overlap is the low homology (, 40%) between D1 and D2 receptor cytoplasmic segments, indicating a lack of conserved protein interaction domains. This observation suggests that D1 and D2 receptors are components of distinct signaling complexes. Furthermore, functional studies indicate that DRIPs regulate most stages of the life-cycle of a DA receptor, including biosynthesis, trafficking to the plasma membrane, signaling and internalization (Figure 1) [21 –31]. The identification of DRIPs and other previously unknown molecular partners provides both an initial picture of the variety of molecules that interact with DA receptors and clues for fully elucidating the diverse signaling mechanisms that are involved in DA neurotransmission in brain.

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Table 1. Dopamine receptor-interacting proteins (DRIPs)a,b Receptor subtype interactionc DRIP Receptors and channels Dopamine D2 receptor Dopamine D3 receptor NMDA receptor NR1-1a subunit NMDA receptor NR2A subunit Somatostatin sst5 receptor Adenosine A1 receptor Adenosine A2A receptor GABAA receptor Kir3 Kþ channel Cytoskeletal proteins Neurofilament M 4.1N 4.1B 4.1G Filamin A Gamma COP Spinophilin Signaling proteins NCS-1 Calcyon Kinases GRK2 Adaptors or chaperones DRIP78 Nck Grb2

D1

D2

D3

D4

D5

– – Yes [15,16] Yes [15] – Yes [17] – – –

Yes [12] Yes [13] – – Yes [12] – Yes [18] – Yes [20]

Yes [13] Yes [14] – – – – – – –

– – – – – – – – –

– – – – – – – Yes [19] –

Yes [25] – – – – Yes [22] –

– Yes Yes Yes Yes – Yes

– Yes Yes Yes Yes – –

– – – – – – –

– – – – – – –

– Yes [30]

Yes [29] –

– –

– –

– –

–

Yes [29]

–

–

–

Yes [21] – –

– – –

– – Yes [28]

– Yes [27] Yes [27]

– – –

[26] [26] [26] [23,24] [31]

[26] [26] [26] [23,24]

a

This list of dopamine receptor-interacting proteins was compiled by searching the PubMed database (http://www.ncbi.nlm.nih.gov) and the Database of Interacting Proteins (http://dip.doe-mbi.ucla.edu/). The proteins are grouped into five classes based on function. Abbreviations: gamma COP, gamma subunit of the coatomer protein complex; GRK2, G-protein-coupled receptor kinase 2; NCS-1, neuronal Ca2þ sensor 1. c Those receptors that interact with listed proteins are indicated with associated references. b

DRIPs: the Ca21 connection An intriguing idea to emerge from the functional characterization of DRIPs is the possibility that intracellular Ca2þ plays an important role in D1 and D2 receptor signaling. This notion is somewhat surprising in the face of more than a decade of research implicating cAMP as the principle effector in DA-mediated signaling [10]. Specifically, the D1-like receptor DRIP calcyon has been found to potentiate D1 and D5 receptor stimulation of the release of intracellular Ca2þ without compromising the ability of the receptors to activate adenylyl cyclase [30]. Also, the D2 receptor DRIP neuronal Ca2þ sensor 1 (NCS-1) has been shown to potentiate D2 receptor signaling by inhibiting D2 receptor desensitization in a Ca2þ-dependent fashion [29]. Calcyon and NCS-1 therefore appear to link Ca2þ directly to the function of both D1 receptors and D2 receptors in the brain. In light of the crucial role that Ca2þ plays in neuronal plasticity [32], DA receptor interactions with NCS-1 and calcyon provide a new conceptual framework for understanding the dopamine-mediated modulation of cognitive, motor and reward behaviors. Below, we explore various mechanisms by which calcyon and NCS-1 might regulate DA neurotransmission, and examine a potential connection of these Ca2þ signaling DRIPs to schizophrenia and other psychiatric disorders. Calcyon and D1-like DA receptor Ca21 signaling Calcyon is a single-pass type III transmembrane protein oriented such that the N- terminus of the protein is http://tips.trends.com

intravesicular (or extracellular) and the C-terminus is cytoplasmic [30]. The sequence of calcyon is closely related to two integral membrane proteins P19 (also called NEEP21) and P21 [33 –35]. NEEP21 was recently found to localize to early endosomes and to influence glutamate receptor recycling in neurons [35]. Initially isolated in a Y2H screen, subsequent pull-down and co-immunoprecipitation assays indicate that C-terminal residues in calcyon bind to residues 421– 435 in the D1 receptor cytoplasmic tail [30]. The expression of calcyon in heterologous cells enables recombinant D1 and D5 receptors to stimulate the release of intracellular Ca2þ with kinetics and a magnitude similar to that produced following the activation of conventionally Gq/11-linked GPCRs [30]. A direct interaction between D5 receptors and calcyon has not been shown; however, D5 receptor residues 453– 448 display , 80% sequence similarity with the D1 receptor calcyon-binding motif. It is also possible that the D5 receptor might interact with calcyon with residues outside of this domain. Although functional evidence from the brain [36] and kidney [37,38] had provided evidence for a Gq/11-coupled D1-type DA receptor, the novel mechanism by which calcyon confers on the known D1 and D5 receptors the ability to elevate intracellular levels of Ca2þ was not expected. Indeed, calcyon enables D1-like receptor Ca2þ signaling via an activity-dependent mechanism that involves the initial priming of cells with a heterologous Gq/11-linked GPCR agonist before D1 or D5 receptor

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Effector coupling, signaling • Calcyon • Spinophilin

Trafficking, synaptic localization • Filamin A (APB280) • Neurofilament M • Protein 4.1

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Desensitization • NCS-1 • GRK2 Presynaptic terminal

Internalization • Grb2 • Nck

Synaptic vesicles

VDCC

Biosynthesis, post-translational processing • DRIP78 • Gamma-COP

stimulation. The function of priming is not fully understood but when followed by a D1-like receptor agonist, priming appears to stabilize or enhance the formation of the calcyon – D1 receptor protein complex possibly as a result of phosphorylation of calcyon [30]. Ligand binding studies show that in the presence of calcyon, the majority of D1 receptors are found in a high-affinity agonist binding conformation after priming [39]. Evidence from studies of other GPCRs indicates that the high-affinity agonist binding sites might correspond to D1 receptors preferentially coupled to Gq/11, and the low-affinity agonist binding sites might correspond to D1 receptors preferentially linked to Gs [40]. This kind of scenario is consistent with the permissive effect of priming agents on D1 receptor Ca2þ signaling in the presence of calcyon. Calcyon localizes to dendrites and spines of D1 receptorexpressing neurons in the neocortex and hippocampus. A markedly different situation is found in the striatum where the density of D1 receptors is the highest in the brain. Whereas D1 receptors are abundant in the spines of striatal medium spiny neurons [41], calcyon is predominantly detected within the cell bodies of D1 receptorpositive neurons in the striatum [30]. This regional difference in the subcellular localization of calcyon and D1 receptors might explain the inability to detect D1-like receptor agonist-evoked Ca2þ responses in striatal cultures [42]. Similar to recombinant receptors coexpressed with calcyon, endogenous D1-like receptors stimulate http://tips.trends.com

NMDA AMPA receptor receptor Ca DAG Ins(1,4,5)P3 Ca2+

Gq Calcyon

2+

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Fig. 1. Dopamine receptor-interacting protein (DRIP) discovery indicates that protein –protein interactions probably regulate all aspects of dopamine receptor biology from biosynthesis to desensitization. For example, DRIP78, a resident endoplasmic reticulum (ER) protein, functions as a chaperone by assuring the proper folding and post-translational modification of the dopamine D1 receptor [21], whereas gamma-COP (gamma subunit of the coatomer protein complex) is important for D1 receptor export from the ER [22]. Various DRIPs linked to the cytoskeleton, including filamin A [23,24], neurofilament M [25] and protein 4.1 [26], regulate D1 and D2 receptor surface expression, and might anchor the receptors at sites of synaptic activity. Subtype-specific association of D1 and D2 receptors with adaptors such as Grb2 and Nck [27,28], kinases such as G-proteincoupled receptor kinase 2 (GRK2) [29], and signaling proteins such as neuronal Ca2þ sensor 1 (NCS-1) [29] appear to regulate dopamine receptor activity via mechanisms involving desensitization and internalization. By contrast, calcyon potentiates D1 receptor Ca2þ signaling via a novel effector coupling mechanism [30] (see main text for more details). The functional significance of D2 receptor interaction with spinophilin [31] has not been elucidated, but spinophilin might perform a scaffolding function through its interaction with actin and protein phosphatase I.

D1 receptor PSD

mGlu5 receptor

Priming

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Ins(1,4,5)P3 receptor

M1 receptor

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Fig. 2. Model of the mechanism involved in dopamine (DA) D1-like receptor-stimulated release of intracellular Ca2þ in neurons. The schematic diagram of a dendritic spine and excitatory amino acid axon terminal includes G-protein-coupled receptors, ion channels and accessory proteins localized on or near the postsynaptic density (PSD) of neocortical dendritic spines. Following Gq-coupled activation of muscarinic (M1) acetylcholine receptors or group I metabotropic glutamate (mGlu1) receptors, stimulation of D1-like receptors by an agonist evokes the release of intracellular Ca2þ in neurons, a response that is not detected following exposure of neurons to DA receptor agonists alone. The essential signal(s) provided by the muscarinic receptors and mGlu receptors during the priming step have not yet been determined but probably include Ca2þ, inositol(1,4,5)-trisphosphate [Ins(1,4,5)P3] and/or diacylglycerol (DAG). Studies in heterologous cells indicate that D1 receptor activation significantly increases Ins(1,4,5)P3 production over that produced by priming alone (C. Bergson, unpublished). Because calcyon is a single-transmembrane vesicular protein localized in dendritic spines [30], calcyon might enhance D1 receptor signaling through Ins(1,4,5)P3 by establishing a direct link between the Gq/11-coupled D1 receptor and intracellular Ca2þ stores. Abbreviation: VDCC, voltage-dependent Ca2þ channel.

the release of intracellular Ca2þ in neocortical and hippocampal neurons in culture in a priming-dependent, cAMP-independent fashion [42]. Agonists of a variety of Gq/11-coupled GPCRs, including adrenoceptors and glutamate, 5-HT and muscarinic acetylcholine receptors, in addition to high extracellular Kþ are effective priming agents in neurons. Dendritic spines of pyramidal neurons are sites of excitatory amino acid input, and electron microscopy analysis has localized both calcyon [30] and the D1 receptor [41] to spines. In spines, calcyon is associated with intracellular vesicles, where it might physically link D1 receptors to intracellular Ca2þ stores, thereby enhancing the ability of the D1 receptor to stimulate the release of intracellular Ca2þ. Given the presence of NMDA and AMPA glutamate receptors in spines, it is tempting to speculate that the calcyon – D1 receptor interaction enables DA to modulate glutamate transmission via mechanisms that involve elevated intracellular Ca2þ (Figure 2). Interaction with calcyon might therefore

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allow D1 receptors to influence multiple events in the CNS. For example, release of Ca2þ from intracellular stores has been linked to long-term potentiation (LTP) [43], longterm depression (LTD) [44], activity-dependent protein synthesis [45], changes in dendritic spine shape [46] and synaptic pruning during development [47]. Furthermore, elevated intracellular levels of Ca2þ launch a spectrum of biochemical events that have been shown to influence dendritic growth, including activation of Ca2þ – calmodulin-sensitive kinase II and IV (CaMKII and CaMKIV) and Ca2þ – phospholipid-dependent protein kinase (PKC) [48 – 50]. Therefore, D1-like receptor-evoked Ca2þ signaling provides a means by which DA could modulate the formation, maintenance and remodeling of synaptic circuits in the neocortex and hippocampus. NCS-1 and D2-like DA receptor function NCS-1 was identified as a DRIP in a screen of a brain cDNA library using the carboxyl-terminal tail of the D2 receptor as ‘bait’ [29]. NCS-1 is the mammalian ortholog of Drosophila frequenin, an EF-hand Ca2þ-binding protein [51] implicated in mediating several aspects of neurotransmission including ion channel regulation [52], neurotransmitter release [53] and intracellular protein trafficking [54]. NCS-1 is neuron specific [55] and exhibits an affinity for Ca2þ of 300 nM [56], which is within the physiological range of localized Ca2þ fluxes in neurons [57]. Overexpression of frequenin in flies and frogs causes a chronic facilitation of transmitter release at the neuromuscular junction by unknown mechanisms [58,59]. When expressed in mammalian cells, NCS-1 attenuates DA-induced D2 receptor internalization by a mechanism that involves a reduction in D2 receptor phosphorylation [29]. NCS-1 exerts its effect on D2 receptor signaling through an interaction with G-protein-coupled receptor kinase 2 (GRK2), a kinase also associated with the D2 receptor and NCS-1 in striatal neurons. NCS-1 inhibits GRK2-dependent desensitization of D2 receptors in a Ca2þ-dependent manner as evidenced by the inability to detect GRK2-mediated D2 receptor internalization in the presence of a dominant-negative NCS-1 Ca2þ-binding mutant [29]. NCS-1 was found to bind to GRK2 in a region that partially overlaps with the calmodulin-binding domain [60]. The NCS-1– GRK2 interaction was abolished by the Ca2þ chelator EGTA and by the expression of an NCS-1 Ca2þ-binding mutant, suggesting that the NCS-1– GRK2 interaction is Ca2þ dependent [60]. Because Ca2þ-dependent activation of NCS-1 potentiates signaling through the D2 receptor, pathways converging through internal Ca2þ stores (such as the ER) or Ca2þ channels that alter the Ca2þ activation state of NCS-1 could effectively modulate D2 receptor signaling (Figure 3). Both D2 receptors and NCS-1 have now been localized to presynaptic and postsynaptic terminals. Colocalization of presynaptic D2 receptors with NCS-1 occurs at asymmetric (presumably excitatory) synapses, which suggests that DA might play a prominent role in regulating neurotransmitter release from glutamatergictype terminals. Postsynaptically, D2 receptor– NCS-1 complexes have also been localized in close proximity to intracellular Ca2þ stores [29] providing physical evidence http://tips.trends.com

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Presynaptic terminal

Ca2+ stores Ca2+

Ca2+

GRK2 NCS-1 D2 receptor

Ca2+ channels Neurotransmitter

AMPA receptor

NMDA receptor PSD

D2 receptor

Ca2+ NCS-1 Ca

2+

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Fig. 3. Model depicting the role of neuronal Ca2þ sensor 1 (NCS-1) in mediating dopamine D2 receptor signaling. The interaction between the D2 receptor and NCS-1 attenuates D2 receptor desensitization in a Ca2þ-sensitive manner. Interaction of NCS-1 and G-protein-coupled receptor kinase 2 (GRK2) also is Ca2þ dependent, and blocks GRK2-mediated phosphorylation of the D2 receptor. Ca2þ activation of NCS-1 can be regulated via an increase in Ca2þ release from intracellular stores and/or through Ca2þ channels. In postsynaptic terminals, activation of AMPA and NMDA receptors in the postsynaptic density (PSD) might also lead to an increase in cytosolic Ca2þ levels.

for the influence of Ca2þ on D2 receptor-mediated DA signaling. Dopamine receptors, DRIPS, Ca21 and psychiatric disorders There are indications that abnormalities in calcyon and NCS-1 expression are associated with several CNS disorders. In particular, significant increases in the levels of both calcyon (by , 100%) and NCS-1 (by ,50%) have been found in the dorsolateral prefrontal cortex (DLPFC) of schizophrenic patients [61,62]. The increases were unrelated to race, age, gender, alcohol or illicit drug abuse, postmortem period and brain pH. In addition, comparison of these two DRIPs in antipsychotic-treated and untreated clinical cases, in addition to in drug-naive and drug-exposed rhesus monkeys, suggests that the observed increases in calcyon and NCS-1 are unlikely to be the result of antipsychotic medications [61,62]. These findings therefore focus attention on the possibility that DRIPs might contribute to abnormalities in the brain DA system such as predicted by the ‘DA hypothesis’ of schizophrenia [63].

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That abnormal levels of calcyon and NCS-1 were observed in the DLPFC is of particular interest because dysfunction of this area is largely responsible for working memory-dependent executive functions, one of the most consistent and debilitating deficits observed in schizophrenic patients [64]. It is possible to envisage how elevated levels of DRIPS might impact on the neurons and neuronal interactions that carry out the working memory functions of the brain. For example, our studies in cell lines have shown that D1-like receptor-mediated Ca2þ signaling is potentiated in the presence of calcyon [30]. Excessive stimulation of D1-like receptors has a potent depressive effect on excitatory transmission both in cortical slices [65,66] and in vivo in monkeys performing working memory tasks [67] (Figure 4). Such findings point to a specific and potent effect of D1-like receptor signaling that is potentially functionally equivalent to NMDA receptor blockade, a condition that induces and exacerbates psychosis [67]. Because abnormalities of D1-like receptor binding [68,69] have been reported in the DLPFC of schizophrenia patients, it is tempting to speculate that some of the psychotic symptoms observed in these patients might be due to overactivation of D1-like receptors. If increased calcyon expression is among the primary molecular alterations in schizophrenia, a key attribute of the efficacy of antipsychotic medications might actually be their demonstrated ability to block inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-dependent Ca2þ release [70]. Interestingly, a recent genetic study reported that the presence of an allele of the gene encoding the D1 receptor predicted a positive clinical and metabolic response of drug-resistant schizophrenic patients to clozapine [71]. It is a strong possibility that alterations in D1 receptor signaling, whether primary or compensatory, modify the synaptic circuitry that is thought to be essential for working memory, the process now considered to represent a core phenotype in schizophrenia. As mentioned earlier, NCS-1 inhibits D2 receptor desensitization thereby enhancing the influence of D2 P–P

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Fig. 4. Diagram of the ferret brain showing the approximate location from which a cortical slice is taken. Under infrared microscopy, it is possible to visualize neurons and record from selected pairs of cells, and, as described in the text, examine the effect of dopamine on excitatory transmission between pairs of pyramidal neurons (upper right) known to use glutamate as their neurotransmitter. Evidence from electron microscopy has revealed that terminals of excitatory axons bear dopamine D1 receptors. Pharmacological studies in the slice indicate that the amplitude of postsynaptic excitatory potentials (EPSPs) shown to the lower right in the figure are depressed by D1 receptors [65,66]. http://tips.trends.com

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receptors on cell activity [29]. Upregulation of NCS-1 in the DLPFC of schizophrenic patients would also be expected to result in decreased activation of this cortical area. This effect of increased NCS-1 expression on DLPFC activity could occur without any detectable change in the levels of D2 receptors. If so, this inference would closely match the observed decrease in PFC activity in schizophrenic patients [72] with no detectable changes in the levels of D2-like receptor binding sites [73]. Pertinent to treatment strategies for schizophrenia, increased NCS-1 expression would be predicted to result in overactive D2 receptor signaling, and might help explain the efficacy of D2 receptor antagonists in treating the positive symptoms of schizophrenia. In spite of the well-established relationship between D2 receptor affinity and the clinical efficacy of antipsychotic medications, the precise mechanisms and neural sites of dysfunction within the cortical DA system remain obscure. If D2 receptors are overstimulated in schizophrenia, requiring constant dampening with antipsychotic medications, we speculate that overactivity of the circuit elements involved could disrupt specialized neuronal interactions in the PFC different from those affected by D1 receptors, but with equally serious consequences for cognitive functions. Moreover, the elevated levels of NCS-1 in schizophrenic PFC suggest that the deficit might not reside in the D2 receptors themselves but in the D2 receptor signaling pathways. Insight into the functions of NCS-1 and other members of the D2 receptor signaling complex should enhance our ability to improve the efficacy and safety of dopamine-related drugs for the treatment of schizophrenia. Increased NCS-1 expression was observed not only in schizophrenic but also in bipolar patients [62]. This finding was not unexpected because both diseases respond to treatment with antipsychotic medications [74], and there is considerable overlap in the symptom clusters constituting a diagnosis of schizophrenia and bipolar disorder [75]. Additionally, a potential genetic link between calcyon and ADHD has been reported [76]. In a genome-wide scan, the strength of the association of the gene encoding calcyon with ADHD significantly exceeded that of other DA markers, such as DA transporters and receptors. Because, as in schizophrenia, information-processing and stimulusfiltering deficits constitute a core component of ADHD [77], this finding highlights the potential role of calcyon in regulating cognitive performance. Furthermore, this genetic link might explain a suggested increased risk of children diagnosed with ADHD developing schizophrenia later in life [78]. The future The identification of the DRIPs shown in Table 1 represents significant progress in our understanding of the protein interactions that regulate the intracellular activity of DA receptors in the CNS. Despite successes with Y2H approaches, the application of proteomics offers a much greater possibility of identifying the full array of proteins that are directly and indirectly associated with DA receptor signaling complexes in the brain. Recent advances in mass spectrometry permit hundreds of

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proteins to be identified using proteomics [79]. Proteomics offers the potential to elucidate the protein networks involved in regulating DA neurotransmission and expand the number of possible targets available for therapeutic drug development. Functional characterization of DRIPs has implicated novel components of the DA system in certain CNS disorders; for example, the potential connection of calcyon and NCS-1 in schizophrenia, bipolar disorder and ADHD raises the possibility that defects in Ca2þ homeostasis might well be at the core of the involvement of DA in these illnesses. It will be important to complement the postmortem studies with allelic association studies using genetic markers for DRIPs. In this context, a recent search for association and haplotype sharing supported the existence of a risk gene for schizophrenia or bipolar disorder at 10q26, the chromosomal position of the gene encoding calcyon [80]. The generation of NCS-1 and calcyon null and overexpressing mice might lead to the development of a tractable animal model for schizophrenia with which to achieve a better understanding of the etiology of schizophrenia and to develop improved treatments of the underlying pathophysiology. Acknowledgements This article is dedicated to the memory of Patricia S. Goldman-Rakic, who died on 31 July 2003. Center Grant P50 MH44866 (P.S.G-R., Principle Investigator); RO1 MH63271 (C. B.); National Alliance for Research on Schizophrenia and Depression Distinguished Investigator Award (R.L.).

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