Dopamine-glutamate interactions in the neostriatum determine psychostimulant action, but the underlying molecular mechanisms remain elusive. Here we found that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between dopamine D2 receptors (D2R) and NMDA receptor NR2B subunits in the neostriatum in vivo. The D2R-NR2B interaction is direct and occurs in the confined postsynaptic density microdomain of excitatory synapses. The enhanced D2R-NR2B interaction disrupts the association of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site (Ser1303), and inhibits NMDA receptor-mediated currents in medium-sized striatal neurons. Furthermore, the regulated D2R-NR2B interaction is critical for constructing behavioral responsiveness to cocaine. Our findings here uncover a direct and dynamic D2R-NR2B interaction in striatal neurons in vivo. This type of dopamine-glutamate integration at the receptor level may be responsible for synergistically inhibiting the D2R-mediated circuits in the basal ganglia and fulfilling the stimulative effect of psychostimulants.

Summary Ca2+/calmodulin-dependent protein kinase II (CaMKII) is central to synaptic transmission. Here we show that synaptic CaMKIIα binds to the third intracellular loop of the limbic dopamine D3 receptor (D3R). This binding is Ca2+-sensitive and is sustained by autophosphorylation of CaMKII, providing an unrecognized route for the Ca2+-mediated regulation of D3Rs. The interaction of CaMKIIα with D3Rs transforms D3Rs into a biochemical substrate of the kinase and promotes the kinase to phosphorylate D3Rs at a selective serine site (S229). In accumbal neurons in vivo, CaMKIIα is recruited to D3Rs by rising Ca2+ to increase the CaMKIIα-mediated phosphorylation of D3Rs, thereby transiently inhibiting D3R efficacy. Notably, the D3R inhibition is critical for integrating dopamine signaling to control behavioral sensitivity to the psychostimulant cocaine. Our data identify CaMKIIα as a recruitable regulator of dopamine receptor function. By binding and phosphorylating limbic D3Rs, CaMKIIα modulates dopamine signaling and psychomotor function in an activity-dependent manner.

Plastic changes in glutamatergic synapses that lead to enduring drug craving and addiction are poorly understood. By focusing on the turnover and trafficking of NMDA receptors, we found that chronic exposure to the psychostimulant amphetamine induces selective downregulation of NMDA receptor NR2B subunits in the confined surface membrane pool of rat striatal neurons at synaptic sites. Remarkably, this downregulation is a long-lived event and results from the destabilization of surface-expressed NR2B due to accelerated ubiquitination and degradation of crucial NR2B-anchoring proteins by the ubiquitin-proteasome system. The biochemical loss of synaptic NR2B further translates to the significant modulation of synaptic plasticity in the form of long-term depression at cortico-accumbal glutamatergic synapses. Behaviorally, genetic disruption of NR2B induces, whereas restoration of NR2B loss prevents, behavioral sensitization to amphetamine. Our data identify NR2B as a key regulator in the remodeling of excitatory synapses and persistent psychomotor plasticity in response to amphetamine.

The glutamate receptor adaptor protein Homer is concentrated in the postsynaptic density of excitatory synapses and is critical for normal operation of synaptic transmission. In this study, we investigated the responsiveness of Homer family proteins to dopamine stimulation with the psychostimulant cocaine in rat striatal neurons both in vivo and in vitro. We found that a single dose of cocaine specifically induced a rapid and transient increase in protein levels of the Homer1a, but not Homer1b/c and Homer2a/b, isoforms in the striatum. This selective Homer1a induction was mediated primarily through activation of dopamine D1, but not D2, receptors. Both protein kinase A and Ca 2ϩ /calmodulin-dependent protein kinases are important for mediating the cocaine stimulation of Homer1a expression. At the transcriptional level, cAMP response element-binding protein serves as a prime transcription factor transmitting the signals derived from D1 receptors and associative pathways to the CaCRE sites within the Homer1a promoter. From a functional perspective, non-cross-linking Homer1a, once induced, competed with the cross-linking isoforms of Homer proteins (Homer1b/c and Homer2a/b) to uncouple the connection of group I metabotropic glutamate receptors (mGluRs) with inositol-1,4,5-triphosphate receptors. These results indicate that cocaine possesses the ability to stimulate Homer1a expression in striatal neurons through a specific synapse-to-nucleus pathway. Moreover, inducible Homer1a expression may represent a transcription-dependent mechanism underlying the dynamic regulation of submembranous macromolecular complex formation between group I mGluRs and their anchoring proteins.Synaptic Homer proteins are important for synaptic construction and function Sheng and Kim, 2002). Long-form Homer proteins (Homer1b/c, Homer2a/b, and Homer3) contain the N-terminal EVH1 (Enabled/ VASP homology 1) domain, which binds the C terminus of group I metabotropic glutamate receptors (mGluRs), whereas the C-terminal coiled-coil structure and leucine zipper motifs render a capability for self-assembly (Brakeman et al., 1997;Xiao et al., 1998;Xiao et al., 2000). Thus, as a prominent scaffolding molecule concentrated in the postsynaptic density of excitatory synapses, Homer crosslinks group I mGluRs to other targets in a specific subcellular microdomain to regulate a specific signaling activity. Emerging evidence indicates that Homer proteins play an essential role in the membrane trafficking of mGluR1␣/5 (Ango et al., 2000); the coupling of mGluR1/5 to inositol-1,4,5-triphosphate (IP 3 ) receptors and the cation (Ca 2ϩ or K ϩ ) channel (Yuan et al., 2003); the development of spines, axons, and synapses (Shiraishi et al., 2003); and drug addiction (Swanson et al., 2001;Szumlinski et al., 2004Szumlinski et al., , 2005; also see below).One distinctive member of Homer family is Homer1a (Brakeman et al., 1997;Xiao et al., 1998Xiao et al., , 2000. Unlike the long-form of Homer proteins, this short-form of Homer lacks the C-terminal coiled-co...

The metabotropic glutamate receptor 1 (mGluR1) is a Gαq protein-coupled receptor and is distributed in broad regions of the mammalian brain. As a key element in excitatory synaptic transmission, the receptor regulates a wide range of cellular and synaptic activities. In addition to regulating its targets, the receptor itself is believed to be actively regulated by intracellular signals, although underlying mechanisms are largely unknown. Here we found that a synapse- enriched protein kinase, Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα), directly binds to the intracellular C terminus (CT) of mGluR1a. This binding is augmented by Ca2+ in vitro. The direct interaction promotes CaMKIIα to phosphorylate mGluR1a at a specific threonine site (T871). In rat striatal neurons, the mGluR1 agonist triggers the receptor- associated phosphoinositide signaling pathway to induce Ca2+-dependent recruitment of CaMKIIα to mGluR1a-CT. This enables the kinase to inhibit the response of the receptor to subsequent agonist exposure. Our data identify an agonist-induced and Ca2+-dependent protein-protein interaction between a synaptic kinase and mGluR1, which constitutes a feedback loop facilitating desensitization of mGluR1a.