Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.

To identify PKA substrates that regulate the excitability changes that are associated with rewarding experiences and to more broadly examine PKA substrates, we developed a phosphoproteomic analysis method that uses affinity beads coated with 14-3-3 proteins to enrich phosphorylated proteins. Using this approach, we comprehensively identified PKA substrates downstream of D1Rs in the striatum to elucidate PKA-mediated signaling pathways. We found more than 100 candidate substrates of PKA, including Rap1 GEF (Rasgrp2). We also found that DA stimulated Rasgrp2 phosphorylation via PKA, thereby activating Rap1, and that Rap1 regulated neuronal excitability and cocaine-induced behavioral reward responses through MAPK (ERK).

Based on pharmacological observations, PKA is thought to regulate not only the excitability of MSNs but also the synaptic plasticity that controls reward-related behaviors (). However, whether and how PKA regulates the excitability of MSNs and reward-related behaviors remain largely unknown. A systematic survey of cAMP-activated phosphoproteins in various brain regions was first carried out by Paul Greengard and his colleagues more than 30 years ago (). Since their achievement, major effort has been made to identify the target substrates of PKA to understand the modes of action of DA, and a few of its substrates, including DARPP-32, GluR1, and NR1, have been reported. DARPP-32 appears to control synaptic plasticity through the inhibition of protein phosphatase-1 (). GluR1 and NR1 phosphorylation appears to enhance surface expression of both AMPA and NMDA receptors, which may be involved in synaptic plasticity (). However, these substrates do not explain how D1Rs and PKA regulate neuronal excitability or how these processes control behavior.

Regulation of phosphorylation of the GluR1 AMPA receptor in the neostriatum by dopamine and psychostimulants in vivo.

It is well known that dopamine (DA) is necessary for motor function, motivation, working memory, and reward (). DA signaling dysfunction has been implicated in various neuropsychological diseases, including Parkinson’s disease, drug addiction, compulsive behavior, attention-deficit/hyperactivity disorder, autism spectrum disorders, and schizophrenia (). The principal target of DA is medium spiny neurons (MSNs), which are a special type of GABAergic inhibitory cell that comprise 95% of the neurons within the striatum, including the nucleus accumbens (NAc). There is a distinct class of spatially intermixed MSNs that express DA type 1 or 2 receptors (D1R-MSNs or D2R-MSNs, respectively). The D1R is coupled to adenylate cyclase through Gand activates protein kinase A (PKA), whereas the D2R inhibits adenylate cyclase through G). DA acts to increase the excitability of D1R-MSNs and their response to glutamatergic synaptic input from the cerebral cortex and thalamus (). Conversely, DA appears to reduce the excitability of D2R-MSNs. When DA levels are relatively low during rest, D1R-MSNs appear to be less excitable than D2R-MSNs (). Thus, the D2R-MSN pathway may predominate under basal conditions. When a substantial increase in DA is induced by reward-related DA release or cocaine administration, the D1R-MSN pathway becomes more dominant than the D2R-MSN pathway. In vivo Caimaging has demonstrated that acute cocaine induces fast activation of D1R-MSNs and progressive deactivation of D2R-MSNs (). The D1R-mediated neuronal pathway has been implicated in reward-related behaviors such as appetitive reward learning and adaptive responses to cocaine ().

G( olf ) and G s in rat basal ganglia: possible involvement of G( olf ) in the coupling of dopamine D 1 receptor with adenylyl cyclase.

Rap1 is known to activate B-raf, resulting in the activation of MAP2K (MEK), followed by MAPK1/3 activation (). Consistently, we found that the activation of Rap1 that was induced by treatment with SKF81297 and forskolin was accompanied by the activation of MAPK1/3 ( Figure 1 and Table 1 ). To determine whether Rap1 mediates the activation of MAPK1/3 by PKA downstream of the D1R, we expressed wild-type MAP2K1 (wtMAP2K1 [MEK1]), constitutively active mutant MAP2K1 (caMAP2K1, the phospho-mimic MAP2K1 [S218D and S222E];), or dominant-negative MAP2K1 (dnMAP2K1, the unphosphorylatable MAP2K1 [S218A and S222A];) in accumbal D1R-MSNs of D1a-Cre transgenic mice ( Figures S5 A–S5C). The number of spikes in caMAP2K1-transfected D1R-MSNs was significantly higher than that in EGFP-transfected control cells, whereas the number of spikes was reduced in dnMAP2K1-transfected D1R-MSNs (p < 0.05; Figure S5 D). Compared to EGFP control transfection, the expression of caMAP2K1 in D1R-MSNs also significantly potentiated cocaine-induced place preference (p < 0.05), whereas the opposite effect was observed in the case of dnMAP2K1 expression (p < 0.05; Figure S5 E). When dnMAP2K1 was cotransfected with caPKA or caRap1 in D1R-MSNs of the NAc, dnMAP2K1 diminished the increased sensitivity to cocaine reward induced by caPKA or caRap1 ( Figures S5 F and S5G). The deficit in cocaine-induced place preference in Rap1 knockout mice was restored by cotransfection with caMAP2K1 (p < 0.05; Figure S5 H). These results indicate that Rap1 mediates the activation of MAPK1/3 by PKA downstream of D1Rs ( Figure 7 D).

Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.

We examined whether endogenous Rap1 in D1R-MSNs of the NAc is required for neuronal firing and cocaine reward. To this end, homozygous loxP-flanked (floxed) Rap1 mice (Rap1) were administered AAV-SP::Cre into the NAc ( Figure 7 A, Figure S3 B). The expression of Cre in D1R-MSNs significantly decreased Rap1 protein expression in the NAc, as revealed by immunoblotting (p < 0.01; Figure 7 A). Localized knockout of Rap1 notably decreased evoked action-potential firing and diminished cocaine-induced place preference compared with the control treatment (p < 0.05 and p < 0.05; Figures 7 B and 7C, respectively). It should be noted that the deficit in cocaine-induced place preference in Rap1 knockout mice was restored by cotransfection with caRap1 (p < 0.05), but not caPKA ( Figure 7 C). Taken together, these results indicate that Rap1 is required for neuronal excitability and cocaine reward ( Figure 7 D).

(D) Working model of D1R-dependent Rap1 signaling. (a) Basal condition. When DA levels are relatively low at rest, D1R-MSNs appear to be less excitable. (b) Hyperdopaminergic condition. The binding of DA to D1Rs activates PKA to phosphorylate Rasgrp2. The phosphorylation of Rasgrp2 leads to Rap1 activation, followed by recruitment of the MAPK pathway, which stimulates the excitability of accumbal D1R-MSNs. The enhancement of D1R-MSN excitability increases spike firing in response to excitatory glutamatergic input from the cortex and/or thalamus. The D1R-MSN pathway is subsequently activated, which eventually results in reward-related behaviors. See also Figures S3–S7

(C) AAV-mediated knockout of Rap1 in D1R-MSNs attenuated cocaine-induced conditioned place preference, and cocaine-induced conditioned place preference was restored by the expression of caRap1. The diagram shows the AAV constructs used in the rescue experiments. The data in (A) and (C) are presented as scatterplots in which each point represents an experimental point. The horizontal bars show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01.

(B) The AAV-mediated knockout of Rap1 decreased the firing rate of D1R-MSNs in the NAc slices. The top panel shows a representative trace of D1R-MSN activity in response to a current pulse at the resting membrane potential. The bottom panel shows pooled data for the firing rates. Data are represented as the mean ± SEM. ∗ p < 0.05 compared with the AAV-SP::EGFP-transfected control group.

(A) Schematic of AAV-mediated conditional Rap1 knockout in accumbal D1R-MSNs. The diagram shows the AAV constructs (top panel) and stereotaxic injection of AAV into the NAc of Rap1 flx/flx mice (middle panel). The bottom panel shows that AAV-mediated knockout of Rap1 in D1R-MSNs decreased the Rap1 levels in the NAc. The bottom left panels show immunoblots for total Rap1. Quantification of the immunoblotting assay is shown in the bottom right panel.

Because Rap1 has been implicated in the control of dendritic spine structural plasticity in cultured cortical and hippocampal pyramidal neurons (), we investigated whether the activation of Rap1 might be involved in the spine morphology of accumbal D1R-MSNs. We coinjected AAV-Flex-wtRap1 or AAV-Flex-caRap1 mutant together with AAV-substance P (SP)::Cre into the NAc of C57BL/6 mice and compared the results to those obtained following injection of the AAV-SP::Cre and AAV-Flex-EGFP control vectors. Dendritic spine analysis revealed that the expression of either wtRap1 or caRap1 in D1R-MSNs had no effect on the number of spines or their morphology ( Figure 6 D), suggesting that the contribution of Rap1 signaling to the morphology of the spines of D1R-MSNs is minimal.

To further study the role of Rap1 in MSNs, wild-type Rap1 (wtRap1) or constitutively active mutant Rap1 (caRap1, the fast-cycling variant of Rap1a [F28L];) was expressed in the D1R-MSNs of the NAc. A GST-RalGDS pull-down assay revealed an increase in the Rap1-GTP level in the NAc of both the AAV-Flex-wtRap1- and AAV-Flex-caRap1-injected Drd1a-Cre transgenic mice, although the level of activated Rap1 in the caRap1-transfected mice was higher than that in the wtRap1-transfected mice (p < 0.05; Figure 6 A). Consistent with our observation for mutant PKA, the number of spikes in caRap1-transfected D1R-MSNs was significantly higher than that in EGFP-transfected control cells (p < 0.05; Figure 6 B), with no differences in the I-V curves ( Figure S4 ). The expression of caRap1 in D1R-MSNs also significantly potentiated cocaine-induced place preference (p < 0.01), whereas the expression of this Rap1 mutant had no effect on conditioned place preference in saline-treated mice ( Figure 6 C). These results indicate that the activation of Rap1 increases neuronal excitability and the rewarding effect of cocaine.

(D) Expression of wild-type (WT) and caRap1 in D1R-MSNs had no effect on their spine morphology. The left panels show representative confocal images of viral-mediated EGFP expression in the secondary dendrites of D1R-MSNs. The right panels show the quantification of the spine analysis. AAV-SP::Cre and AAV-Flex-Rap1 mutant were injected into the NAc of C57BL/6 mice. Coronal sections (150 μm) were immunostained using an antibody against GFP 3 weeks after the virus injection. Scale bar represents 20 μm. The data in (A), (C), and (D) are presented as scatterplots in which each point represents an experimental point. The horizontal bars show the mean ± SEM.p < 0.05,p < 0.01. See also Figures S4 and S7

(B) AAV-mediated expression of caRap1 enhanced the firing rate of D1R-MSNs in NAc slices. The top panel shows a representative trace of D1R-MSN activity in response to a current pulse at the resting membrane potential. The bottom panel shows the pooled firing-rate data. The data are presented as the mean ± SEM. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the EGFP-transfected control group.

(A) Schematic of AAV-mediated Rap1 expression in accumbal D1R-MSNs. The diagram shows the AAV constructs (top panel) and stereotaxic injection of AAVs into the NAc of Drd1-Cre transgenic mice (middle panel). The bottom panel shows that AAV-mediated expression of caRap1 in D1R-MSNs increased Rap1-GTP levels in the NAc 3 weeks after AAV injection. Bottom left panels show immunoblots for Rap1-GTP that was pulled down using GST-RalGDS and for total Rap1. Quantification of the immunoblotting assay is shown in the bottom right panel.

Next, we performed whole-cell current-clamp recordings of mutant PKA-transfected D1R-MSNs in the NAc to examine their intrinsic membrane excitability, as measured by the number of action potentials generated by a positive current injection. Interestingly, the caPKA-transfected cells showed a significantly higher number of spikes than the EGFP-transfected control cells (p < 0.05; Figure 5 C). No marked differences in the current-voltage (I-V) curves were observed between the three groups of mice ( Figure S4 ). We also confirmed that treatment of the accumbal slices with forskolin increased the number of spikes (data not shown), as described previously (). Moreover, to assess the behaviorally relevant consequences of PKA activity, we used a cocaine-induced conditioned place-preference model. At a conditioning dose of 10 mg/kg, cocaine induced place preference to the drug-paired side in EGFP-transfected mice (p < 0.01), whereas saline treatment had no effect on place preference ( Figure 5 D). As expected, the rewarding effects of cocaine were markedly potentiated in mice that expressed caPKA in D1R-MSNs of the NAc (p < 0.05; Figure 5 D). These results indicate that PKA activation increases the phosphorylation of Rasgrp2 and the excitability of D1R-MSNs in the NAc to enhance the sensitivity of mice to the cocaine reward.

Although PKA has been implicated in reward signals downstream of D1Rs using pharmacological approaches (), there is no direct evidence that PKA in D1R-MSNs regulates neuronal excitability and reward-related behaviors. We next established a system in which wild-type PKA (wtPKA) or constitutively active mutant PKA (caPKA, a form of the PKA catalytic subunit that is not regulated by cAMP [H87Q and W196R];) was expressed in the NAc under the control of the D1R promoter using adeno-associated virus (AAV)-mediated conditional transgenic techniques ( Figure 5 A, Figure S3 A). We injected AAV-Flex-wtPKA or AAV-Flex-caPKA mutant into the NAc of Drd1a-Cre transgenic mice and compared the results to those obtained following injection of the AAV-Flex-EGFP control vector. Immunoblot analysis revealed that the expression of caPKA increased the phosphorylation level of Rasgrp2 at S116/117, S554, and S586 but did not alter the level of total Rasgrp2 compared to the expression of EGFP (p < 0.05; Figure 5 B), demonstrating that PKA activity can be manipulated in accumbal D1R-MSNs and that the activation of PKA promotes Rasgrp2 phosphorylation in vivo.

(D) AAV-mediated expression of caPKA in D1R-MSNs potentiated cocaine-induced conditioned place preference. The data in (B) and (D) are presented as scatterplots in which each point represents an experimental point. The horizontal bars show the mean ± SEM.p < 0.05,p < 0.01. See also Figures S3, S4, and S7

(C) AAV-mediated expression of caPKA potentiated the firing rate of D1R-MSNs in NAc slices. The left panel shows a representative trace of D1R-MSN activity in response to a current pulse at the resting membrane potential. The right panel shows the pooled data for the firing rates. The data are presented as the mean ± SEM. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the EGFP-transfected control group.

(B) AAV-mediated expression of caPKA in D1R-MSNs stimulates Rasgrp2 phosphorylation in the NAc. The left panels show representative immunoblots. Quantification of the immunoblotting assay is shown in the right panels.

(A) Schematic of AAV-mediated PKA expression in accumbal D1R-MSNs. The diagram shows the AAV constructs (left panel) and stereotaxic injection of AAVs into the NAc of Drd1-Cre transgenic mice (middle panel). Representative coronal brain slices showing the expression of EGFP 3 weeks after AAV injection into the NAc (right panel). The scale bar represents 1 mm.

Rasgrp2 has been shown to be predominantly expressed in the MSNs of the striatum (). Consistent with previous reports, immunohistochemistry using specific Rasgrp2 antibodies revealed that Rasgrp2 was highly expressed in the NAc and striatum compared with other brain regions, including the cerebral cortex ( Figure 4 A). Next, we examined the phosphorylation of Rasgrp2 in the NAc by immunoblotting after cocaine injection. Treatment with a single dose of cocaine significantly and dose-dependently increased the phosphorylation level of Rasgrp2 at S116/S117, S554, and S586 in the NAc 15 min after treatment (p < 0.05; Figure 4 B). To investigate if cocaine stimulates the phosphorylation of Rasgrp2 in D1R-MSNs, we performed immunohistochemical analysis in Drd1-mVenus transgenic mice, in which D1R-MSNs express a variant of yellow fluorescent protein (mVenus). Cocaine-induced phosphorylation of Rasgrp2 was detected in D1R-MSNs in the NAc of Drd1-mVenus transgenic mice ( Figure 4 C). Furthermore, cocaine administration significantly increased the level of activated Rap1 in the NAc 15 min after cocaine injection, as assessed by Rap1-GTP levels, which were measured via a GST-RalGDS pull-down assay (p < 0.05; Figure 4 D). These results demonstrate that cocaine stimulates PKA-Rasgrp2 signaling, thereby activating Rap1 in the NAc of mice.

(D) Cocaine administration increased the level of activated Rap1 in the NAc. The top panels show immunoblots for Rap1-GTP that was pulled down by GST-RalGDS and for total Rap1. Quantification of the immunoblotting assay is shown in the bottom panel. The data for (B) and (D) are presented as scatterplots in which each point represents an experimental point. The horizontal bars show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01.

(C) Phosphorylated Rasgrp2 was detected in D1R-MSNs in the NAc after cocaine injection. Drd1-mVenus transgenic mice were administered saline or cocaine (30 mg/kg, i.p.), and immunohistochemical analysis was performed 15 min after the treatment. Immunofluorescence staining using an anti-Rasgrp2 antibody specific for phosphorylation at S116/S117 (left panels) or an anti-GFP antibody (middle panels) is shown. The scale bar represents 10 μm.

(B) Cocaine administration dose-dependently stimulated the phosphorylation of Rasgrp2 in the NAc 15 min after cocaine injection. Left panels show representative immunoblots. Quantification of the immunoblotting assay is shown in the right panels.

(A) The Rasgrp2 protein was highly expressed in the NAc and striatum. Immunofluorescence staining using an anti-Rasgrp2 antibody is shown. The scale bar represents 1 mm.

To examine whether SKF81297 and forskolin affect the levels of the GTP-bound form of Rap1 in striatal slices, we utilized beads coated with RalGDS, an effector of Rap1, and performed an affinity precipitation assay to specifically pull down the GTP-bound form of Rap1 (Rap1-GTP). Treatment of the slices with SKF81297 and forskolin increased the levels of Rap1-GTP (p < 0.05; Figure 3 C). Taken together, these results suggest that D1R agonists activate Rap1 via PKA-mediated phosphorylation of Rasgrp2.

To measure guanine nucleotide exchange activity on Rap1, we utilized beads coated with nucleotide-free Rap1 (Rap1 G15A) and performed an affinity precipitation assay to specifically pull down Rasgrp2 that was activated in intact cells, as described previously (). When COS-7 cells were transfected with EGFP-Rasgrp2, a small amount of EGFP-Rasgrp2 precipitated with Rap1 G15A ( Figure 3 A). The coexpression of the catalytic unit of PKA (myc-PKA) dramatically increased the amount of EGFP-Rasgrp2 that precipitated with Rap1 G15A and stimulated the phosphorylation of Rasgrp2 at S116, S117, S554, and S586 (p < 0.01; Figure 3 A). The coexpression of myc-PKA did not affect the amount of unphosphorylatable mutant Rasgrp2 (Rasgrp2-116A/117A/554A/586A) that precipitated with Rap1 G15A or the phosphorylation of this mutant ( Figure 3 A). When COS-7 cells were transfected with phospho-mimic mutant Rasgrp2 (Rasgrp2-116D/117D/554D/586D and Rasgrp2-116E/117E/554E/586E), a large amount of Rasgrp2 precipitated with Rap1 G15A compared to that precipitated in EGFP-Rasgrp2-transfected control cells (p < 0.05; Figure 3 B). These results indicate that PKA phosphorylates Rasgrp2 and activates its guanine nucleotide exchange activity on Rap1 in COS-7 cells.

(C) cAMP and D1R stimulation increased Rap1 activation in striatal slices. Striatal slices were treated with either forskolin (10 μM) or SKF81297 (1 μM) for 10 min. The left panels show immunoblots for Rap1-GTP that was pulled down by GST-RalGDS and for total Rap1. Quantification of the immunoblotting assay is shown in the right panel. The data for (A)–(C) are presented as scatterplots in which each point represents an experimental point. The horizontal bars show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01.

(B) The phosphomimic mutant Rasgrp2 exhibited increased guanine nucleotide exchange activity in COS-7 cells. The left panels show immunoblots for EGFP-Rasgrp2 that was pulled down by GST-Rap1 G15A and for EGFP-Rasgrp2. Quantification of the immunoblotting assay is shown in the right panel.

(A) PKA increased the guanine nucleotide exchange activity of Rasgrp2 via its phosphorylation in COS-7 cells. The left panels show immunoblots for EGFP-Rasgrp2 that was pulled down by GST-Rap1 G15A and for phosphorylated Rasgrp2, EGFP-Rasgrp2, and myc-PKA. Quantification of the immunoblotting assay is shown in the right panel.

We also performed a pull-down assay using affinity beads coated with 14-3-3 and found that treating the slices with SKF81297 and forskolin increased the amount of Rasgrp2 bound to 14-3-3 (p < 0.01; Figure 2 D). These results suggest that PKA phosphorylates Rasgrp2 and induces its association with 14-3-3. Because 14-3-3 has been shown to bind to phosphorylated proteins and to alter their localization, activity, and stability (), whether the phosphorylation of Rasgrp2 affects its guanine nucleotide exchange activity was an intriguing question.

We next examined whether SKF81297 and forskolin induced the phosphorylation of Rasgrp2 in acute striatal slices using these phospho-specific antibodies. Treatment of the slices with SKF81297 and forskolin stimulated the phosphorylation of Rasgrp2 at S116, S117, S554, and S586 (p < 0.01; Figure 2 C). To investigate whether the phosphorylation of Rasgrp2 is mediated by PKA in the brain, striatal slices were pretreated with the PKA inhibitor Rp-cAMPS before SKF81297 or forskolin treatment as previously described (). SKF81297- and forskolin-stimulated phosphorylation of Rasgrp2 was blocked by the PKA inhibitor Rp-cAMPS (p < 0.05; Figure 2 C). These findings suggest that DA stimulates the phosphorylation of Rasgrp2 by acting through D1R-mediated activation of PKA and, together with the phosphoprotein screening data, reveal a novel PKA-Rasgrp2 signaling pathway.

SKF81297-induced phosphorylation of Rasgrp2 at Ser 116 (S116), Ser 117 (S117), and Ser 554 (S554) was observed in mouse striatal slices ( Table 1 ). To confirm whether PKA directly phosphorylates these sites, GST-Rasgrp2 was subjected to in vitro phosphorylation analysis. Because Ser 586 (S586) is a putative PKA phosphorylation site in addition to S116, S117, and S554, a tetra-substituted Ala mutant (Rasgrp2-116A/117A/554A/586A) was examined. GST-Rasgrp2 was efficiently phosphorylated by PKA in vitro, and the tetra-substituted mutant exhibited an approximately 50% reduction in the phosphorylation level ( Figure 2 A). We further produced phosphorylation state-specific antibodies for S116/S117 (pS116/pS117), S554 (pS554), and S586 (pS586). These antibodies specifically recognized PKA-mediated phosphorylated Rasgrp2 but did not cross-react with nonphosphorylated Rasgrp2 or with the site-specific Ser-to-Ala Rasgrp2 mutant ( Figure 2 B, Figure S2 ). These results indicate that PKA directly phosphorylates Rasgrp2 at S116, S117, S554, and S586 in vitro.

(D) Forskolin and SKF81297 stimulated the association of Rasgrp2 with 14-3-3 in striatal slices. Striatal slices were treated with either forskolin (10 μM) or SKF81297 (1 μM) for 10 min. The left panels show an immunoblot for Rasgrp2 that was pulled down by GST-14-3-3 and for total Rasgrp2. Quantification of the immunoblotting assay is shown in the right panels. The data in (C) and (D) are presented as scatterplots in which each point represents an experimental point. The horizontal bars show the mean ± SEM.p < 0.05,p < 0.01. See also Figure S2

(C) Forskolin and SKF81279 stimulated the phosphorylation of Rasgrp2 in striatal slices. Striatal slices were treated with either forskolin (1 μM) or SKF81297 (1 μM) for 10 min after they were pretreated with Rp-cAMPS (1 mM) 60 min before. The top panels show representative immunoblots. Quantification of the immunoblotting assay is shown in the bottom panels.

(B) The specificity of the antibodies against phosphorylated Rasgrp2 mediated by PKA. A total of 500 fmol of GST-Rasgrp2-1-374 aa (for the pS116/pS117 antibody) or GST-Rasgrp2-495-609 aa (for the pS554 and pS586 antibodies) containing the indicated amount of phosphorylated or nonphosphorylated protein was subjected to SDS-PAGE followed by immunoblot analysis using phospho-specific antibodies.

(A) pEF-BOS-GST-Rasgrp2-WT or the pEF-BOS-GST-Rasgrp2-4A mutant was transfected into COS-7 cells. GST-fusion proteins were precipitated using glutathione Sepharose beads and incubated with [γ- 32 P] ATP in the presence or absence of GST-PKACα. The proteins were visualized via autoradiography (left panel) and silver staining (right panel).

Among the putative PKA substrates in the Rap1 pathway, Rasgrp2 (CalDAG-GEF1) and Rap1gap are positive and negative regulators of Rap1, respectively (). Rap1 is a small GTPase that has been implicated in synaptic plasticity in hippocampal neurons and in memory formation (). Thus, we speculated that the Rap1 pathway is involved in the regulation of neuronal functions via D1R signaling, and we subsequently focused on the Rap1 pathway, especially Rasgrp2, because guanine nucleotide exchange factors (GEFs) are primarily responsible for the activation of small GTPases.

The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13.

Because we were interested in the direct target substrates of PKA located downstream of the D1R, we identified the putative PKA substrates that were phosphorylated upon stimulation with SKF81297 and entered these substrates into a curated pathway database, Reactome ( http://www.reactome.org ), to identify the signaling pathways related to the obtained PKA substrates. We identified Rap1 signaling, potassium channels, and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels as major PKA-related pathways ( Table S2 ).

We then developed a phosphoprotein screening method to more comprehensively identify the PKA substrates downstream of the D1R, as depicted in Figure 1 B. Striatal slices were treated with forskolin to induce PKA-mediated phosphorylation. Extracts from these striatal slices were then applied to affinity beads coated with 14-3-3 protein to enrich for Thr- or Ser-phosphorylated proteins. The bound proteins were digested using trypsin and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the phosphorylated proteins and their phosphorylation sites. Treatment with forskolin stimulated the phosphorylation of more than 200 proteins. The phosphorylation sites that exhibited stimulus-induced activation that was at least 5-fold greater than the control level are summarized in Table 1 . Detailed information about the phosphorylation sites is summarized in Table S1 , available online (the information will be available in the Kinase-Associated Neural Phospho-Signaling Database; https://srpbsg01.unit.oist.jp/index.php?ml_lang=en ). Motif analysis of the phosphopeptides that were identified using this method revealed that approximately 60% of the phosphorylation sites contained basic residues at the −3 and −2 positions, suggesting that PKA or a related kinase was responsible for these phosphorylation events. Approximately 20% of these sites contained Pro at the +1 position, suggesting that they are phosphorylation sites for MAPK1/3 or other Pro-oriented kinases. We also found that the addition of SKF81297 increased the phosphorylation of more than 100 proteins ( Table 1 ). In total, 47 of these phosphoproteins were also identified following forskolin stimulation; again, these sites were predominantly categorized as putative PKA and MAPK1/3 target sites ( Figure S1 Table 1 ). Of note, most of these proteins and phosphorylation sites have not yet been reported as part of the DA-D1R signaling pathway.

Striatal slices were treated with forskolin to induce PKA-mediated phosphorylation. The extracts of these striatal slices were then applied to affinity beads coated with 14-3-3 protein to enrich the phosphorylated proteins. The bound proteins were digested using trypsin and subjected to LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites. Treatment with forskolin stimulated the phosphorylation of more than 200 proteins. The reproducible phosphorylation sites, which were stimulated by more than 5-fold compared to the control at least twice in more than three independent experiments, are summarized. See also Figure S1 and Tables S1 and S2

To explore novel phosphorylation events downstream of the D1R, we utilized acute slices of the mouse striatum. Because D1R agonists have been shown to activate PKA to phosphorylate DARPP-32 at Thr 34 (T34), GluR1 at Ser 845 (S845), and NR1 at Ser 897 (S897) (), we first confirmed the phosphorylation of these proteins in striatal slices after stimulation with SKF81297 (a D1R agonist) and forskolin, which specifically produces cyclic AMP and subsequent activation of PKA. The addition of SKF81297 and forskolin induced the phosphorylation of not only DARPP-32 at T34 (p < 0.01), GluR1 at S845 (p < 0.01), and NR1 at S897 (p < 0.05) but also MAPK1/3 (ERK1/2) at Thr 202/Tyr 204 (T202/Y204, p < 0.01; Figure 1 A). In the striatum, the phosphorylation and activation of MAPK1/3 downstream of the D1R appear to be mediated by PKA ().

(B) Scheme and chart for the phosphoproteomic analysis used to identify PKA substrates downstream of the D1R. Striatal slices were treated with SKF81297 or forskolin to induce PKA-mediated phosphorylation. Extracts of these striatal slices were then applied to affinity beads coated with 14-3-3 to enrich phosphorylated proteins. The bound proteins were digested using trypsin and subjected to LC-MS/MS to identify the phosphorylated proteins and their phosphorylation sites.

(A) SKF81279 and forskolin stimulated the phosphorylation of DARPP-32, GluR1, NR1, and MAPK1/3. Striatal slices were treated with either forskolin (1 μM) or SKF81297 (1 μM) for 10 min. The left panels show immunoblots and the corresponding total proteins. Quantification of the immunoblot assay is shown in the right panels. The data are presented as scatterplots in which each point represents an experimental point. The horizontal bars show the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01.

Regulation of phosphorylation of the GluR1 AMPA receptor in the neostriatum by dopamine and psychostimulants in vivo.

Discussion

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Keller-McGandy C.E.

Hernandez L.F.

Kett L.R.

Young A.B.

Standaert D.G.

Graybiel A.M. Dysregulation of CalDAG-GEFI and CalDAG-GEFII predicts the severity of motor side-effects induced by anti-parkinsonian therapy. Crittenden et al., 2010 Crittenden J.R.

Dunn D.E.

Merali F.I.

Woodman B.

Yim M.

Borkowska A.E.

Frosch M.P.

Bates G.P.

Housman D.E.

Lo D.C.

Graybiel A.M. CalDAG-GEFI down-regulation in the striatum as a neuroprotective change in Huntington’s disease. Crittenden and Graybiel, 2011 Crittenden J.R.

Graybiel A.M. Basal Ganglia disorders associated with imbalances in the striatal striosome and matrix compartments. Rasgrp1–4 are members of a family of genes that is characterized by the presence of a Ras superfamily GEF domain (). In the brain, Rasgrp1 (CalDAG-GEFII) and Rasgrp2 are enriched in striatal neurons, whereas Rasgrp3 is enriched in oligodendrocytes of the cerebellum and cerebral cortex (). Although Rasgrp1 and Rasgrp2 have been implicated in basal ganglia disorders, including L-DOPA-induced dyskinesias and Huntington’s disease (), it remains unclear how DA controls the GEF activity of Rasgrp2. The present study provides the first demonstration of a mechanistic link with the D1R. We found that PKA phosphorylates Rasgrp2 at S116, S117, S554, and S586, promoting its binding to 14-3-3 and activating its guanine nucleotide exchange activity on Rap1. Multiple-alignment analysis of mouse Rasgrp1–4 revealed that the PKA phosphorylation sites identified in Rasgrp2 were not conserved in other Rasgrp proteins ( Figure S6 ), suggesting that the regulatory mechanism of Rasgrp2 by PKA is not conserved among Rasgrp family proteins. We also found that treatment of striatal slices with a D1R agonist and peritoneal injection of cocaine induced Rasgrp2 phosphorylation and Rap1 activation. These results suggest that Rap1 is activated via PKA-mediated phosphorylation of Rasgrp2 downstream of D1Rs. Because most GEFs that act on the Ras family of GTPases are maintained in an inactive form, it is conceivable that the binding of Rasgrp2 to 14-3-3 changes the conformation of Rasgrp2 from an inactive state to an active state.

Caron, 2003 Caron E. Cellular functions of the Rap1 GTP-binding protein: a pattern emerges. Stork, 2003 Stork P.J. Does Rap1 deserve a bad Rap?. Woolfrey et al., 2009 Woolfrey K.M.

Srivastava D.P.

Photowala H.

Yamashita M.

Barbolina M.V.

Cahill M.E.

Xie Z.

Jones K.A.

Quilliam L.A.

Prakriya M.

Penzes P. Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines. Xie et al., 2005 Xie Z.

Huganir R.L.

Penzes P. Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6. Zhu et al., 2002 Zhu J.J.

Qin Y.

Zhao M.

Van Aelst L.

Malinow R. Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Sweatt, 2001 Sweatt J.D. The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. A current by MAPK ( Stacey et al., 2012 Stacey D.

Bilbao A.

Maroteaux M.

Jia T.

Easton A.C.

Longueville S.

Nymberg C.

Banaschewski T.

Barker G.J.

Büchel C.

et al. IMAGEN Consortium

RASGRF2 regulates alcohol-induced reinforcement by influencing mesolimbic dopamine neuron activity and dopamine release. Fasano et al., 2009 Fasano S.

D’Antoni A.

Orban P.C.

Valjent E.

Putignano E.

Vara H.

Pizzorusso T.

Giustetto M.

Yoon B.

Soloway P.

et al. Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) controls activation of extracellular signal-regulated kinase (ERK) signaling in the striatum and long-term behavioral responses to cocaine. We found that Rap1 is involved in the neuronal excitability of D1R-MSNs. Rap1 has various effectors, including AF-6/afadin, B-raf, RalGDS, and Riam (). Rap1 and AF-6 have been implicated in the control of spine-neck elongation in cultured cortical and hippocampal pyramidal neurons (). However, the contribution of Rap1 signaling to the spine morphology of D1R-MSNs appears to be minimal because the expression of caRap1 in D1R-MSNs had no effect on their spine morphology. Rap1 is known to activate B-raf, which results in the activation of MAP2K1/2, followed by MAPK1/3 activation (). We found that the activation of Rap1 that was induced by treatment with SKF81297 and forskolin was accompanied by the activation of MAPK1/3. Inhibition of MAPK1/3 attenuated the effects of caPKA and caRap1 on the rewarding effect of cocaine, whereas activation of MAPK1/3 restored the deficit in cocaine-induced place preference in Rap1 knockout mice, suggesting that Rap1 mediates the activation of MAPK1/3 by PKA downstream of D1Rs. MAPK1/3 appears to phosphorylate several potassium and HCN channels ( Table 1 ), which are potentially involved in neuronal excitability. Rasgrf2 (a specific Ras GEF) has been shown to play a role in controlling the excitability of DA neurons through Icurrent by MAPK (). Moreover, Rasgrf1 knockout mice show reduced DA-stimulated MAPK1/3 phosphorylation in striatal neurons and organotypic slices as well as deficient behavioral responses to cocaine (). Thus, our findings can be explained by a similar recruitment of MAPK1/3 and provide a novel connection between D1R, PKA, Rap1, and MAPK1/3 pathways in MSNs. Further intensive studies are required to understand how Rap1 regulates neuronal excitability via the MAPK pathway.

Rittinger et al., 1999 Rittinger K.

Budman J.

Xu J.

Volinia S.

Cantley L.C.

Smerdon S.J.

Gamblin S.J.

Yaffe M.B. Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Yaffe et al., 1997 Yaffe M.B.

Rittinger K.

Volinia S.

Caron P.R.

Aitken A.

Leffers H.

Gamblin S.J.

Smerdon S.J.

Cantley L.C. The structural basis for 14-3-3:phosphopeptide binding specificity. Morrison, 2009 Morrison D.K. The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development. Obsil and Obsilova, 2011 Obsil T.

Obsilova V. Structural basis of 14-3-3 protein functions. In the present study, we succeeded in identifying more than 100 candidate PKA substrates through phosphoproteins screening, and most of these substrates have not yet been reported as part of the DA-D1R signaling pathway. It was not possible to implement this method without enriching the phosphorylated proteins using 14-3-3-coated affinity beads. In fact, many housekeeping proteins, such as actin and tubulin, were obtained as candidate PKA substrates rather than Rap1 regulators if the 14-3-3-coated affinity beads were not used. 14-3-3 binds to phospho-Ser/Thr-containing motifs in target proteins via RSXpSXP (mode 1) and RXXXpSXP (mode 2), where pS represents phosphoserine (). Additionally, 14-3-3 regulates the function, localization, and stability of target proteins depending on their phosphorylation state (). Accordingly, the phosphorylated substrates detected in the present study may play functional roles in D1R signaling in MSNs. Because a 14-3-3 binding motif must be present within the substrate molecule for it to be identified, our proteomic screening failed to detect SKF81297- and forskolin-induced phosphorylation of DARPP-32 at T34, GluR1 at S845, NR1 at S897, and MAPK1/3 at Thr 202/Tyr 204 ( Figure 1 A and Table 1 ). This potential limitation leaves open the possibility for other phosphorylated substrates. We hope that novel and well-known signaling pathways will be found via the application of protein-coated beads that recognize other phospho-binding domains.