Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine-and cAMP-regulated phosphoprotein of M r 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca 2+ -dependent activation of the Ca 2+ -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca 2+ DARPP-32 (dopamine-and cAMP-regulated phosphoprotein of M r 32 kDa) is a cytosolic protein that is selectively enriched in medium spiny neurons in the neostriatum . When DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on Thr34, it is converted into a potent inhibitor of protein phosphatase-1 (PP-1) (Hemmings et al. 1984b). DARPP-32 Thr34 phosphorylation leads to an increase in the state of phosphorylation of downstream PP-1 substrates, including various neurotransmitter receptors and voltage-gated ion channels . We have recently reported that DARPP-32 is phosphorylated at Thr75 by cyclin-dependent kinase 5 (Cdk5) in vitro and in neostriatal neurons (Bibb et al. 1999). DARPP-32 phosphorylated at Thr75 inhibits PKA activity and thereby reduces the efficacy of dopamine signaling. Thus, DARPP-32 is a bifunctional signal transduction molecule that controls activities of PP-1 and PKA through the phosphorylation of Thr34 and Thr75, respectively. Mice lacking DARPP-32 exhibit profound deficits in their molecular, electrophysiological and behavioral responses to dopamine, drugs of abuse and antipsychotic medication, demonstrating the importance of DARPP-32 in most of the actions of dopamine (Fienberg et al. 1998;Fienberg and Greengard 2000). Received September 11, 2002; revised manuscript received February 6, 2002; accepted February 7, 2002. Address correspondence and reprint requests to Dr Akinori Nishi, Department of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. E-mail: nishia@med.kurume-u.ac.jp Abbreviations used: AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionate; cAMP, cyclic AMP; Cdk5, cyclin-dependent kinase 5; DARPP-32, dopamine-and cAMP-regulated phosphoprotein of M r 32 kDa; PAGE, polyacrylamide gel electrophoresis; PKA, cAMPdependent protein kinase; PMSF, phenylmethylsulfonyl fluoride; PP-1, protein phosphatase-1; PP-2A, protein phosphatase-2A; SDS, sodium dodecyl sulfate.

D opamine-and cAMP-regulated phosphoprotein of M r 32,000 (DARPP-32) is a signal transduction molecule that is selectively enriched in medium-sized spiny neurons in neostriatum, and plays an obligatory role in dopaminergic signaling. Mice lacking DARPP-32 exhibit profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse and antipsychotic medication, demonstrating the importance of DARPP-32 in most of the actions of dopamine (1).When DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on Thr-34, it is converted into a potent inhibitor of protein phosphatase-1, and thereby controls the phosphorylation state and activity of many downstream physiological effectors, including various neurotransmitter receptors and voltage-gated ion channels (for review, see ref.2). Phosphorylation and dephosphorylation of DARPP-32 at Thr-34 are regulated by dopamine, adenosine, glutamate, serotonin, and other neurotransmitters. For example, dopamine, by activating dopamine D1-type receptors (3), and adenosine, by activating adenosine A 2A receptors (4), stimulate the phosphorylation of DARPP-32 at Thr-34.Metabotropic glutamate (mGlu) receptors are subdivided into three groups on the basis of agonist pharmacology, sequence homology, and G-protein effector coupling: group I (mGlu1 and mGlu5 receptors), group II (mGlu2 and mGlu3 receptors), and group III (mGlu4, mGlu6, mGlu7, and mGlu8 receptors; ref. 5). Group I mGlu receptors are coupled to the phospholipase C (PLC) pathway (6), the extracellular signal-regulated kinase (ERK) pathway (7), and the p38 pathway (8); group II and III mGlu receptors are negatively coupled to adenylyl cyclase (9). Individual subtypes of mGlu receptors are assumed to mediate distinct facilitatory (group I) or inhibitory (group II and III) actions on neuronal transmission (10). Recently, mGlu receptors were shown to be involved in cocaine self-administration (11) and methamphetamine-induced neurotoxicity (12). However, the molecular mechanisms underlying the actions of mGlu receptors are not clearly understood. In this study, we investigated the regulation of DARPP-32 phosphorylation at Thr-34 by mGlu receptors by using neostriatal slices. The results obtained indicate that the actions of mGlu5 receptors are linked to A 2A adenosine receptors by means of a mechanism involving the ERK cascade.

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