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Articles

Bidirectional Regulation of DARPP-32 Phosphorylation by Dopamine

Akinori Nishi, Gretchen L. Snyder and Paul Greengard
Journal of Neuroscience 1 November 1997, 17 (21) 8147-8155; DOI: https://doi.org/10.1523/JNEUROSCI.17-21-08147.1997
Akinori Nishi
1Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021
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Gretchen L. Snyder
1Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021
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Paul Greengard
1Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021
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  • Fig. 1.
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    Fig. 1.

    Effect of dopamine on the level of phospho-DARPP-32 in neostriatum. Slices were incubated with dopamine (100 μm) in the presence of the dopamine uptake inhibitor nomifensine (10 μm) for the indicated times.A, Phospho-DARPP-32 was detected at a molecular mass of ∼32 kDa using mAb-23 against thr34-phospho-DARPP-32. Note that the phospho-DARPP-32 mAb also detected a cross-reactive protein band at a molecular mass of ∼75 kDa, the levels of which were not affected by dopamine. B, Total DARPP-32 was detected in the same membrane as shown in A using mAb C24–5a against DARPP-32. C, The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with untreated tissue. Data represent mean ± SEM for the number of experiments shown in parentheses. * p < 0.05, ** p < 0.01 compared with 0 min; †p < 0.05 compared with 2 min.

  • Fig. 2.
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    Fig. 2.

    Effects of D1 agonist (SKF82526) and D2 agonist (quinpirole) on the basal level of phospho-DARPP-32 in neostriatum. Slices were incubated with (A) SKF82526 (1 μm) or (B) quinpirole (1 μm) for the indicated times. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with untreated tissue. Data represent mean ± SEM for 4–12 experiments. * p < 0.05, ** p < 0.01 compared with 0 min; †p < 0.05 compared with 5 min.

  • Fig. 3.
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    Fig. 3.

    Opposing effects of D1 agonist (SKF82526) and D2 agonist (quinpirole) on the level of phospho-DARPP-32 in neostriatum. Slices were preincubated with the indicated concentrations of quinpirole (1 nm to 1 μm) for 5 min and then incubated with quinpirole plus SKF82526 (1 μm) for an additional 5 min. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. Data represent mean ± SEM for four to five experiments. * p < 0.01 compared with SKF82526 alone.

  • Fig. 4.
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    Fig. 4.

    Effect of the antipsychotic drug raclopride on the level of phospho-DARPP-32 in (A) neostriatum and (B) nucleus accumbens. Slices were incubated for a total of 20 min. Raclopride (1 μm) was added at 0 min, quinpirole (100 nm) at 10 min, and SKF82526 (1 μm) at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. A, Data represent mean ± SEM for six to nine experiments. *p < 0.01 compared with no addition; †p < 0.01 compared with SKF82526 alone; §p < 0.05 compared with quinpirole alone; ¶p < 0.01 compared with SKF82526 plus quinpirole.B, Data represent mean ± SEM for five to seven experiments. * p < 0.05 compared with no addition; † p < 0.01 compared with SKF82526 alone; §p < 0.01 compared with quinpirole alone; ¶p < 0.02 compared with SKF82526 plus quinpirole.

  • Fig. 8.
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    Fig. 8.

    Postulated pathways by which dopamine may regulate DARPP-32 phosphorylation. Activation of D1 receptors increases cAMP, leading to the activation of PKA and the phosphorylation of DARPP-32 on thr34, converting it into a potent inhibitor of protein phosphatase-1. Activation of D2 receptors decreases DARPP-32 phosphorylation by two mechanisms (which might occur in the same or in different groups of neurons): one involves an inhibition of adenylyl cyclase, a decrease in cAMP, a decrease in activity of PKA and a decreased phosphorylation of DARPP-32; the other involves an increase in intracellular Ca2+, an activation of calcineurin, and an increased dephosphorylation of thr34-phospho-DARPP-32. This scheme is supported by evidence showing that NMDA receptor activation (Halpain et al., 1990) and thapsigargin (present study), both of which raise intracellular Ca2+, cause the dephosphorylation of thr34-phospho-DARPP-32.

  • Fig. 5.
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    Fig. 5.

    Effect of D2 agonist (quinpirole) on stimulated levels of phospho-DARPP-32 in neostriatum. Slices were preincubated with quinpirole (1 μm) for 5 min and then incubated with quinpirole plus either (A) SKF82526 (1 μm), (B) forskolin (10 μm), or (C) 8-bromo-cAMP (1 mm) for an additional 5 min. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526, forskolin, or 8-bromo-cAMP alone. Data represent mean ± SEM for three to four experiments. *p < 0.01 compared with SKF82526, forskolin, or 8-bromo-cAMP alone.

  • Fig. 6.
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    Fig. 6.

    Effect of Ca2+-free/EGTA medium on the level of phospho-DARPP-32 in neostriatum. Slices were incubated in control or Ca2+-free/EGTA medium for 20 min.A, Phospho-DARPP-32 was detected using a phosphorylation state-specific mAb (mAb-23). B, The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with control. Data represent mean ± SEM for four experiments. * p < 0.01 compared with control.

  • Fig. 7.
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    Fig. 7.

    Absence of effect of D2 agonist (quinpirole) on the level of phospho-DARPP-32 in Ca2+-free/EGTA medium. Neostriatal slices were incubated in Ca2+-free/EGTA medium for a total of 20 min. Buffer was replaced by Ca2+-free/EGTA medium at 0 min, quinpirole (1 μm) was added at 10 min, and (A) SKF82526 (1 mm), (B) forskolin (10 μm), or (C) 8-bromo-cAMP (1 mm) was added at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526, forskolin, or 8-bromo-cAMP alone. Data represent mean ± SEM for three to four experiments.

Tables

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    Table 1.

    Effect of cyclosporin A on the level of phospho-DARPP-32 in neostriatum

    Cyclosporin A (−)Cyclosporin A (+)
    A. D1 agonist SKF82526
     0 min1.0016.0  ± 1.6*
     5 min7.14 ± 0.97*31.6  ± 2.81-160
     30 min3.07 ± 0.66*,1-16528.0  ± 3.11-160
    B. D2 agonist quinpirole
     0 min1.0013.50  ± 1.74*
     2 min0.525 ± 0.051*5.98  ± 1.281-160
     10 min0.580 ± 0.058*5.60  ± 1.051-160
    C. D1 + D2 agonist
     Control1.0011.0  ± 2.5*
     SKF8252610.61 ± 2.28*21.1  ± 2.31-160
     SKF82526 + quinpirole4.78 ± 1.20*,1-16520.3  ± 2.51-160
    • Slices were preincubated in the absence or presence of cyclosporin A (CyA) (5 μm) for 60 min, followed by the addition of SKF82526 and/or quinpirole. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with control. A, Slices were incubated with SKF82526 (1 μm) for the indicated times. Data represent means ± SEM for five to nine experiments.

    • ↵*  p < 0.01 compared with 0 min/CyA (−);

       p < 0.01 compared with 0 min/CyA (−);

       p < 0.01 compared with control/CyA (−);

    • ↵F1-160  p < 0.01 compared with 0 min/CyA (+);

       p < 0.01 compared with 0 min/CyA (+). C, Slices were preincubated with quinpirole (1 μm) for 5 min and then incubated with quinpirole plus SKF82526 (1 μm) for an additional 5 min. Data represent means ± SEM for six to nine experiments.

       p < 0.01 compared with control/CyA (+);

    • ↵F1-165  p < 0.01 compared with 5 min/CyA (−). B, Slices were incubated with quinpirole (1 μm) for the indicated times. Data represent means ± SEM for five to seven experiments.

       p < 0.05 compared with SKF82526/CyA (−).

    • View popup
    Table 2.

    Effect of D2 agonist on the level of phospho-DARPP-32 is not mediated through the NMDA receptor

    MK801 (−)MK801 (+)
    Control0.103 ± 0.021*0.247  ± 0.0442-160
    SKF825261.0001.115  ± 0.078
    SKF82526 + NMDA0.042 ± 0.009*1.042  ± 0.085
    SKF82526 + quinpirole0.598 ± 0.137*0.616  ± 0.0842-165
    • Neostriatal slices were incubated for a total of 20 min in the absence or presence of the NMDA receptor antagonist MK801 (100 μm). MK801 was added at 0 min, NMDA (100 μm) or quinpirole (1 μm) at 10 min, and SKF82526 (1 μm) at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. Data represent means ± SEM for four to seven experiments.

    • ↵*  p< 0.01 compared with SKF82526/MK801 (−);

    • ↵F2-160  p < 0.05 compared with control/MK801 (−);

    • ↵F2-165  p < 0.01 compared with SKF82526/MK801 (+).

    • View popup
    Table 3.

    Effect of D2 agonist on the level of phospho-DARPP-32 is not reduced by tetrodotoxin (TTX)

    TTX (−)TTX (+)
    Control0.686  ± 0.1091.0003-150
    SKF825263.282  ± 0.5753-1605.492  ± 0.7803-165,3-168
    SKF82526 + quinpirole1.716  ± 0.4093-150,3-1651.968  ± 0.5593-168,3-150
    • Neostriatal slices were incubated for a total of 20 min in the absence or presence of TTX (1 μm). TTX was added at 0 min, quinpirole (1 μm) at 10 min, and SKF82526 (1 μm) at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with TTX alone. Data represent means ± SEM for six to seven experiments.

    • ↵F3-160  p < 0.01,

    • ↵F3-150  p < 0.05 compared with control/TTX (−);

    • ↵F3-165  p < 0.05 compared with SKF82526/TTX (−);

    • ↵F3-168  p < 0.01 compared with control/TTX (+);

      * p < 0.01 compared with SKF82526/TTX (+).

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The Journal of Neuroscience: 17 (21)
Journal of Neuroscience
Vol. 17, Issue 21
1 Nov 1997
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Bidirectional Regulation of DARPP-32 Phosphorylation by Dopamine
Akinori Nishi, Gretchen L. Snyder, Paul Greengard
Journal of Neuroscience 1 November 1997, 17 (21) 8147-8155; DOI: 10.1523/JNEUROSCI.17-21-08147.1997

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Bidirectional Regulation of DARPP-32 Phosphorylation by Dopamine
Akinori Nishi, Gretchen L. Snyder, Paul Greengard
Journal of Neuroscience 1 November 1997, 17 (21) 8147-8155; DOI: 10.1523/JNEUROSCI.17-21-08147.1997
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Keywords

  • DARPP-32
  • dopamine
  • D2 receptor
  • phosphorylation
  • neostriatum
  • calcineurin
  • raclopride

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