Metabotropic glutamate receptors and dopamine receptors cooperate to enhance extracellular signal-regulated kinase phosphorylation in striatal neurons

Abstract

Striatal medium spiny neurons are an important site of convergence for signaling mediated by the neurotransmitters dopamine and glutamate. We report that in striatal neurons in primary culture, signaling through group I metabotropic glutamate receptors (mGluRs) 1/5 and the D1 class of dopamine receptors (DRs) 1/5 converges to increase phosphorylation of the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2). Induction of mitogen-activated protein kinase kinase-dependent signaling cascades by either mGluR1/5 or DR1/5 gave rise to increases in phosphorylation of ERK2. Coactivation of mGluR1/5 and DR1/5 with (S)-3,5-dihydroxyphenylglycine and (+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride enhanced the phosphorylation of ERK2. This interaction between mGluR1/5 and DR1/5 required protein kinase C (PKC), because the PKC inhibitors calphostin C, bisindolylmaleimide I, and Gö6976 blocked DR1/5-enhanced phosphorylation of ERK2. Use of the phosphatase inhibitors calyculin and okadaic acid indicated that inhibition of protein phosphatases 1 and 2A dramatically enhanced ERK2 phosphorylation by mGluR1/5. Coactivation of mGluR1/5 and DR1/5 also enhanced cAMP-response element binding protein (CREB) phosphorylation (compared with each receptor agonist alone) but did not enhance CREB-mediated transcriptional activity. Thus, signal transduction pathways activated by DR1/5 and mGluR5 interact to modify downstream events in striatal neurons while retaining numerous regulatory checkpoints.

Figure 1.

Metabotropic glutamate receptor agonists DHPG and CHPG evoke a cytoplasmic calcium rise in E18 striatal neurons. Agonists were applied for 20 s (black bar on x-axis), and antagonists were applied 3 min before stimulation with agonist. A, Traces show DHPG-evoked intracellular calcium signals. Trace 1, 10 μm; trace 2, 1 μm; trace 3, 4 μm DHPG in the presence of 20 μm MPEP. B, Concentration-response curves for DHPG (open squares; n = 45) and CHPG (open diamonds; n = 100). C₁, Effect of the mGluR antagonists AIDA (10 μm; n = 286), LY367385 (10 μm; n = 322), and MPEP (20 μm; n = 312) on the response to 4 μm DHPG. C₂, Removal of extracellular calcium blocks calcium rise by 4 μm DHPG (n = 302). C₃, Blocking calcium channels with cadmium inhibits calcium rise by DHPG (n = 132). Data are presented as a percentage of the DHPG response remaining in the presence of the antagonist. ^*p < 0.001. D, An example of a calcium response in a single striatal neuron before application of DHPG and during the response to the agonist (4 μm).

Figure 2.

Group I metabotropic glutamate receptors stimulate ERK2 phosphorylation in E18 striatal neurons. A, Western blot analysis of mGluR1 and mGluR5 expression in cultured striatal neurons as a function of DIV. Protein content per lane for these blots was identical. Film exposure times were also identical to demonstrate the magnitude of the difference in protein levels of mGluR1 versus mGluR5. B, Immunofluorescence using subtype-specific antibodies shows that both mGluR1 and mGluR5 are localized in somata and processes, with mGluR5 more abundant than mGluR1. C, Time course of ERK2 phosphorylation by DHPG. D, Concentration-response of ERK2 phosphorylation by DHPG. E, Pharmacological characterization indicates that mGluR5 is the predominant group I mGluR mediating ERK2 phosphorylation in striatal neurons. mGluR agonist DHPG (10 μm) or CHPG (750 μm) was applied for 2 min. Receptor antagonist LY367385, 4CPG, or MPEP (see concentrations in Results) was applied 30 min before stimulation with DHPG. F, Cells were stimulated with DHPG and double labeled using anti-phosphoERK with anti-NeuN to mark neurons. White arrows indicate cells positive for NeuN; blue arrows indicate cells negative for NeuN. DAPI staining indicates the location of all cells.

Figure 4.

Pharmacology of DR1/5- and mGluR1/5-mediated ERK2 phosphorylation. A, ERK2 phosphorylation by DHPG (D) is attenuated by the mGluR5 antagonist MPEP (M) (20 μm; 30 min; n = 6). B, The D₁ receptor antagonist SCH23390 (SCH) (20 μm; 30 min; n = 5) diminishes ERK2 phosphorylation by SKF38393 (SKF). C, The PKA inhibitor H89 (H) (5 μm; 60 min; n = 6) blocks DR1/5 phosphorylation of ERK2 without suppressing ERK2 phosphorylation by DHPG. D, Inhibition of MEK with 1 μm U0126 (60 min) suppresses basal as well as DHPG- and SKF38393 (S)-mediated ERK2 phosphorylation. The asterisk indicates a significant increase over control (controls are 0 for treatment with agonist alone or inhibitor for treatment with agonist in the presence of inhibitor).

Figure 3.

Coapplication of DR1/5 and mGluR1/5 agonists enhances phosphorylation of ERK2. A, The DR1/5 agonist SKF38393 (SKF) (10 μm; 2 min) significantly augments DHPG-mediated (10 μm; 2 min) phosphorylation of ERK2 (n = 16). The asterisk indicates a significant increase over control; # indicates significant difference from DHPG (D) or SKF38393 (S) treatment alone. B, DR1/5-mGluR1/5 interaction is cell autonomous, as demonstrated by the absence of an inhibitory effect in the presence of TTX. C, Double-label immunofluorescence reveals a high degree of coexpression of DR1/5 and mGluR5.

Figure 5.

cAMP-mediated increases in ERK2 phosphorylation in striatal neurons are MEK dependent. A, Forskolin (Forsk) elicits a robust increase in ERK2 phosphorylation that is resistant to U0126 concentrations <1 μm (n = 3 for all conditions). B, ERK2 phosphorylation induced by SKF38393 (SKF) is radically reduced and driven to subbasal levels with 0.1 μm U0126 (n = 3 for all conditions). Cells were treated with U0126 for 60 min before agonist application. Asterisks indicate a significant increase over control (0); @ indicates a significant decrease compared with control.

Figure 6.

Protein kinase C mediates mGluR5-D₁ receptor pathway interactions. The protein kinase C inhibitors GF109203X (GF) (A) (500 nm; n = 7) and calphostin C (CalC) (B) (100 nm; n = 5) block the DR1/5-enhanced activation of ERK2 by mGluR without affecting DHPG (D)-mediated ERK2 phosphorylation. Each inhibitor was applied 60 min before treatment with agonist. Graphical summary of results is shown below each panel. Asterisks indicate a significant increase over control (controls are 0 for treatment with agonist alone or inhibitor for treatment with agonist in the presence of inhibitor); # indicates significant difference from DHPG or SKF38393 (SKF; S) treatment alone; @ indicates significant difference from DHPG plus SKF38393 coapplication.

Figure 7.

Inhibition of protein phosphatases PP1 and PP2A before stimulation with DHPG enhances mGluR5-mediated ERK2 phosphorylation. Okadaic acid (A) or calyculin A (CalA) (B) pretreatment enhances ERK2 phosphorylation by DHPG (D) (10 μm; n = 5). Asterisks indicate a significant increase over control; # indicates a significant difference from DHPG treatment alone.

Figure 8.

CREB phosphorylation, but not CREB-mediated transcriptional activity, demonstrates mGluR5/DR1/5 enhancement. A, mGluR5 and DR1/5 agonists each increase CREB phosphorylation; coactivation of both receptors significantly increases phosphoCREB levels beyond that for each agonist alone [10 μm DHPG (D); 10 μm SKF38393 (S); n = 11]. B, CREB-mediated transcriptional activity induced with DHPG, SKF38393, or forskolin was measured using the GAL-4/luciferase reporter system. Cells transfected with pFC2-dbd [dbd(-)CREB, no fusion with CREB] and pFR-Luc served as negative controls. All others were transfected with pFA2-CREB and pFR2-Luc and were either unstimulated [dbd(-) CREB and 0] or stimulated as indicated (n = 4). Asterisks indicates a significant increase over control (0); # indicates a significant difference from DHPG or SKF38393 treatment alone.

Figure 9.

Hypothetical model of elements involved in ERK2 phosphorylation by DR1/5 and mGluR5 activation in striatal neurons. Our data are consistent with the idea that G-protein-coupled phospholipase C activity increases intracellular calcium and elevates ERK2 phosphorylation by mGluR5. The increase in calcium triggers the Ras pathway and increases PKC activity required for DR1/5-mGluR5 pathway interactions. In parallel, DR1/5 activation may increase ERK2 phosphorylation via PKA-mediated increases in Rap 1 and B-Raf activity, also leading to MEK activation. The phosphatases PP1 and PP2A target MAPK for dephosphorylation.