Figure 1.
Second Messenger Pathways Involved in the Phosphorylation of DARPP-32 on Thr34 and Thr75 in Medium Spiny Projection Neurons
A calcium elevation produced by glutamate leads to calcium binding to CaM (Ca4CaM), which activates both CaMKII and PP2B, the latter dephosphorylating DARPP-32 on Thr34. Stimulation of the dopamine D1 receptor activates the PKA cascade via AC5 and cAMP formation. PKA in its turn increases phosphorylation of DARPP-32 on Thr34, which then inhibits PP1.
Table 1.
Reactions and Rate Constants of Dopamine–PKA Pathway
Table 2.
Reactions and Rate Constants of Ca to CaMKII and PP2B Pathway
Figure 2.
Model Response to Either D1 Activation or Calcium Elevation
Dopamine elevation at time 300 s (A3) leads to a nine times increase in phosphoThr34 within 2 min (A1, solid line), while phosphoThr75 is reduced to 60% of control (A2, solid line). This is quantitatively in accordance with experimental results. The PKA-dependent activation of PP2A is critical for this behavior; if left out of the model, phosphoThr75 increases instead of decreases (blue dashed line). Calcium-dependent activation of PP2A is not critical to simulate the correct response to D1 activation (purple dotted line). Calcium elevation at time 300 s (B3) leads to a reduction in both Thr34 (B1, solid line) and Thr75 phosphorylation (B2, solid line). In contrast to the result in A1 and A2, this response requires calcium-dependent activation of PP2A; if left out of the model (purple dotted line), calcium causes an increase in Thr75 phosphorylation.
Figure 3.
Simulations of Prolonged D1 Receptor Stimulation Paired with Calcium Elevation
(A) Dopamine-dependent increase in phosphoThr34 starting at time 300 s is reduced when, in addition, calcium is elevated at time 1500 s (solid line).
(B) Dopamine-dependent decrease in phosphoThr75 is minimally changed by a subsequent calcium elevation. Reduction of PP2B activity increases the levels of phosphoThr34 and abolishes the calcium-dependent decrease in phosphoThr34 (A, purple dashed line). Furthermore, inhibition of PP2B activity indirectly decreases the levels of phosphoThr75 (B, purple dashed line) because less DARPP-32 is available for phosphorylation on Thr75 since more of it is phosphorylated on Thr34.
(C) Time course of dopamine and calcium elevation.
Figure 4.
Responses to Transient Dopamine and Calcium Elevations
(A1) A brief, high-amplitude (solid line), or slow, low-amplitude (dashed line) dopamine elevation, with approximately equal area under the curve, is used as input to the model network. Dopamine produces an elevation in cAMP (A2), an elevation in PKAc (A3), and an increase in phosphoThr34 (A4). While the cAMP signal follows the dynamics of the dopamine signal, PKAc formation and Thr34 phosphorylation are proportional to total amount of dopamine, as if integrating the cAMP signal because these reactions occur on a slower time scale.
(B1) A fast (solid line) or slow (dashed line) transient calcium signal is used as input to the model. Calcium produces a decrease in cAMP (B2) due to both calcium-dependent activation of PDE1 as well as calcium-dependent inhibition of AC5. PDE1 has a larger effect on the maximal cAMP decrease, whereas AC5 has a larger effect on the later part of the cAMP reduction. Despite a decrease in cAMP, the free PKAc concentration (B3, solid line) is transiently increased following a fast and high calcium input, due to the calcium-dependent activation of PP2A, and causes an increase in phosphoThr34 (B4, solid line). A slower calcium input has minor effects on PKAc (B3, dashed line), but causes an initial decrease in phosphoThr34 (B4, dashed line), mostly due to a prolonged activation of PP2B.
Figure 5.
Effect of Simultaneous, Transient Calcium and Dopamine Inputs
In all panels, the response to dopamine alone is shown with green dashed lines; the response to paired dopamine and calcium is shown with red solid lines.
(A) The cumulative increase in free PKAc following eight high-amplitude, brief dopamine pulses with 20-s intervals, is enhanced when a high-amplitude, brief calcium input is paired with each dopamine pulse.
(B) Phosphorylation of Thr34 is enhanced when calcium and dopamine inputs are paired.
(C) PP1 inhibition is more pronounced when calcium and dopamine inputs are paired due to the increase in phosphoThr34.
(D) The ratio of PKAc to PP1 is enhanced when calcium and dopamine inputs are paired.
(E) Some dephosphorylation on Thr75 occurs following a dopamine input due to the PKA dependent activation of PP2A. When in addition calcium is transiently elevated, calcium-dependent activation of PP2A significantly decreases phosphoThr75.
(F) The total formation of activated PKA (both free and bound to phosphoThr75) is larger with dopamine inputs alone. This is partly due to the calcium-dependent decrease in the cAMP formation, but also the PKAc interaction with phosphoThr75 is important.
Figure 6.
Role of the PKA–PP2A–phosphoThr75 Feedback Loop on PKAc and phosphoThr34
(A) Eight high-amplitude, brief, dopamine pulses, 20 s apart, lead to a larger buildup of free PKAc (A1) or Thr34 (A2) compared with control (black) if the reaction rates are set to zero in the PKA phosphorylation of PP2A only (purple; reaction (i) in (E) opened), in the phosphoThr75 inhibition of PKA only (light blue; reaction (iii) in (E) opened), or in both reactions (orange). Thus, both reactions (i) and (iii) in the PKA–PP2A–phosphoThr75 loop work as a sink for the PKAc signal.
(B) High-amplitude, fast calcium inputs lead to a decreased level of free PKAc (B1) and Thr34 (B2) if reactions (i) and (iii) are prevented. Here the PKA–PP2A–phosphoThr75 loop behaves as a positive feedback loop on the PKAc concentration.
(C) Paired fast calcium and dopamine elevations enhance PKAc (C1) and phosphoThr34 levels (C2) when the PKA–PP2A–phosphoThr75 loop is present (black lines), but decrease PKAc and phosphoThr34 when reactions (i) and (iii) are eliminated (orange lines).
(D) Activation of PP2Ac by binding to calcium is required for the stimulatory effect of calcium on PKAc and phosphoThr34. Binding of calcium to PP2A shifts equilibrium of reaction (i) to the substrates, thus PKAc dissociates from PP2A, and shifts the equilibrium of reaction (iii) to the substrates, thus PKAc dissociates from phosphoThr75. If calcium activation of PP2A is prevented, dopamine alone (green lines) results in more PKAc (D1) and phosphoThr34 (D2) than paired calcium and dopamine (red lines). Also, calcium alone (blue lines) produces no significant response in PKAc and even causes a decrease in phosphoThr34.
Figure 7.
Consequences of Pairing Slower Calcium Signals with Dopamine Inputs
(A) The cumulative increase of free PKAc following eight high-amplitude, brief dopamine inputs (green line) is a bit larger than if a slow, low-amplitude calcium signal is paired with the brief dopamine inputs (red line). Also the slower, low-amplitude calcium input alone (blue line) does not give any significant increase in PKAc.
(B) Pairing slow, low-amplitude calcium inputs with dopamine inputs (red line) reduces the cumulative increase in phosphoThr34 compared with a dopamine input alone (green line). A decrease of phosphoThr34 is seen following a calcium input alone (blue line).
(C) The decrease in phosphoThr34 due to the slow, low-amplitude calcium input is due to a prolonged activation of PP2B (dashed line) compared with when a brief, high-amplitude calcium input is given (solid line).
(D) Furthermore, the calcium-activated form of PP2A (PP2Ac) is significantly less activated following a small but slower calcium input (dashed line) than if a high-amplitude calcium input is given (solid line).
Figure 8.
Effects of Dissociation Rate on Calcium Enhancement of Dopamine-Induced PKAc Elevation and PP1 Suppression
In all panels, the response is the maximal ratio of PKAc to PP1. All panels show the effect of dissociation rate on the response to dopamine alone (green dashed line, stars), calcium alone (blue dashed line, circles), and paired dopamine and calcium (red dashed line, squares).
(A) Response is independent of rate of calcium-dependent AC5 inhibition.
(B) Response is independent of rate of Ca4CaM binding to PDE1.
(C) Response is slightly sensitive to the rate of calcium binding to PP2A: for very fast (*100) calcium dissociation rate, the enhancing role of calcium decreases because the activation of PP2A is too brief to dephosphorylate significant amounts of phosphoThr75 following brief calcium inputs.
Figure 9.
Role of Altered Enzyme Quantities on the Integration Properties of DARPP-32
(A) Total concentrations of PKA, PP2B, PP2A, and cdk5 all are varied together between 0.1–10× the control level.
(A1) Independent of this variation, pairing of calcium and dopamine (red, squares) is more effective than either input alone (green dots or blue circles).
(A2) The basal levels of PKAc (black dashed line, triangles) as well as phosphoThr34 (black dotted line, triangles) vary with enzyme quantity. A decrease in enzyme quantity produces a decrease in the change of PKAc during stimulation (dashed line) and an increase in the phosphoThr34 levels (dotted line). Nonetheless, in all cases a simultaneous calcium and dopamine input is most effective in elevating both PKAc and phosphoThr34.
(B) The role of DARPP-32 in signal integration is independent of PKAc. Enzyme quantities were lowered to 10% of control and all the reaction rates in the PKA reactions were increased 100× to make the PKAc formation follow the cAMP signal more effectively.
(B1) The PKAc increase is slightly larger with dopamine alone and decreases almost to basal level in between transient dopamine pulses.
(B2) Thr34 phosphorylation increases with successive paired stimuli, and is higher for paired calcium and dopamine inputs.
Figure 10.
Schematic Drawing of the Reactions Involved in the PKA–PP2A–phosphoThr75 Loop
When dopamine stimulates PKA, a substantial amount of the free catalytic subunit gets bound to PP2A and phosphoThr75, thus dampening the stimulatory effect of dopamine on PKA (A). Calcium stimulation and the formation of the calcium-activated PP2A affects this loop in two ways: 1) by increasing dephosphorylation of phosphoThr75, thus reducing inhibition of PKA; and 2) less PP2A is available for PKA binding, thus shifting this equilibrium to increase the amount of active PKA (B).
Table 3.
Reactions and Rate Constants of Phosphor/Dephosphorylation of DARPP-32
Table 4.
Molecule Quantities