Characterization of the inhibition of protein phosphatase-1 by DARPP-32 and inhibitor-2.
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Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, Mr 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 approximately 1 nM) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6-11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1-8 (I2-(9-204)) or 100-204 (I2-(1-99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9-13 (I2-(14-204)) resulted in a large decrease in inhibitory potency (IC50 approximately 800 nM), whereas further COOH-terminal deletion (I2-(1-84)) caused a moderate decrease in inhibitory potency (IC50 approximately 10 nM). Within residues 9-13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6-20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6-38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1-120) but not I2-(85-204). These results indicate that distinct amino acid motifs contained within the NH2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14-84 of inhibitor-2 and residues 6-38 of phospho-DARPP-32 share elements that are important for interaction with PP1. (+info)
Enhancement of (-)-stepholidine on protein phosphorylation of a dopamine- and cAMP-regulated phosphoprotein in denervated striatum of oxidopamine-lesioned rats.
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AIM: To study effects of (-)-stepholidine (SPD) on the phosphorylation of a dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in the striatum of oxidopamine-lesioned rats. METHODS: The amount of dephospho-DARPP-32 was measured by a back-phosphorylation assay. RESULTS: In the striatum of control rats, SPD per se had no effect on the phosphorylation of DARPP-32, but it antagonized the decrease by 28% of dephospho-DARPP-32 induced by the D1 agonist SK&F-38393. In the denervated striatum of oxidopamine-lesioned rats, SPD decreased the amount of dephospho-DARPP-32 by 44%. The effect of SPD was completely counteracted by the concomitant administration of the D1 antagonist Sch-23390. CONCLUSION: SPD exhibits D1 agonistic action on DARPP-32 phosphorylation in the denervated striatum of oxidopamine-lesioned rats, but it acts as a D1 antagonist in normal striatum. (+info)
Expression of the striatal DARPP-32/ARPP-21 phenotype in GABAergic neurons requires neurotrophins in vivo and in vitro.
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The medium spiny neuron (MSN) is the major output neuron of the caudate nucleus and uses GABA as its primary neurotransmitter. A majority of MSNs coexpress DARPP-32 and ARPP-21, two dopamine and cyclic AMP-regulated phosphoproteins, and most of the matrix neurons express calbindin. DARPP-32 is the most commonly used MSN marker, but previous attempts to express this gene in vitro have failed. In this study we found that DARPP-32 is expressed in <12% of E13- or E17-derived striatal neurons when they are grown in defined media at high or low density in serum, dopamine, or Neurobasal/N2 (Life Technologies), and ARPP-21 is expressed in <1%. The percentage increases to 25% for DARPP-32 and 10% for ARPP-21 when the same cells are grown in Neurobasal/B27 (Life Technologies) for 7 d. After growth in Neurobasal/B27 plus brain-derived neurotrophic factor (BDNF) for 7 d, E13-derived MSNs are 53.7% DARPP-32-positive and 29. 0% ARPP-21-positive; E17-derived MSNs are 66.8% DARPP-32-positive and 51.5% ARPP-21-positive. The percentage of calbindin-positive neurons also is increased under these conditions. Finally, ARPP-21 expression is reduced in mice with a targeted deletion of the BDNF gene. We conclude that BDNF is required for the maturation of a large subset of patch and matrix MSNs in vivo and in vitro. In addition, we introduce a culture system in which highly differentiated MSNs may be generated, maintained, and studied. (+info)
Effect of protein kinase A-induced phosphorylation on the gating mechanism of the brain Na+ channel: model fitting to whole-cell current traces.
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The activity of the voltage-gated Na+ channel is subjected to modulation through covalent modifications. It has been previously shown that brain Na+ currents are reduced following the activation of the protein kinase A (PKA) pathway, but the effect of the phosphorylation on the gating mechanism of the channel has not been demonstrated so far. In this study, we analyze the whole-cell Na+ current recorded in the absence or presence of forskolin, which stimulates the PKA pathway. A minimal molecular model of the gating mechanism of the Na+ channel is defined to fit the experimental data: it consists of three closed states, one open state, and two inactivated states. We experimentally demonstrate that the kinetics of inactivation from the closed states are not affected by phosphorylation. The results obtained by computer fitting indicate that, among all the kinetic parameters describing the transitions between states, only one parameter is significantly modified in the presence of forskolin, and corresponds to the acceleration of the inactivation from the open state. This conclusion is supported by the analysis of current traces obtained from cells in the presence of a phosphatase inhibitor or loaded with the PKA catalytic unit, and is in agreement with previously reported single channel records. (+info)
Inhibitor-1 is not required for the activation of glycogen synthase by insulin in skeletal muscle.
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Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle. (+info)
D(2) dopamine receptors induce mitogen-activated protein kinase and cAMP response element-binding protein phosphorylation in neurons.
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Dopamine, by activating D(1)- and D(2)-class receptors, plays a significant role in regulating gene expression. Although much is known about D(1) receptor-regulated gene expression, there has been far less information on gene regulation mediated by D(2) receptors. In this study, we show that D(2) receptors can activate the mitogen-activated protein kinase (MAPK) and the cAMP response element-binding protein (CREB) in neurons. Treatment of brain slices with the D(2) receptor agonist quinpirole induced rapid phosphorylation of MAPK and CREB. The neuroleptic drug eticlopride, a highly selective D(2) receptor antagonist, blocked the quinpirole-induced phosphorylation of MAPK and CREB. D(2) receptor-induced MAPK phosphorylation depended on intracellular Ca(2+) elevation, protein kinase C activation, and MAPK kinase activation, but not on the protein tyrosine kinase Pyk2, even though quinpirole stimulated Pyk2 phosphorylation. D(2) receptor-induced CREB phosphorylation was mediated by activation of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase, but not MAPK. The dopamine and cAMP-regulated phosphoprotein DARPP-32 also was required for the regulation of MAPK and CREB phosphorylation by D(2) receptors. Our results suggest that MAPK and CREB signaling cascades are involved in the regulation of gene expression and other long-term effects of D(2) receptor activation. (+info)
Dopamine in the developing kidney.
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The adult kidney has a high rate of dopamine (DA) production, metabolism, and signalling. The non-neuronal DA system in the adult kidney is of utmost importance for the regulation of salt metabolism. DA may also act as a transcription factor and may be of importance for tissue differentiation. In the central nervous system, D1 receptors require the dopamine- and cAMP-regulated phosphoprotein with a molecular weight of 32,000 Dalton (DARPP-32) to mediate their actions. The renal D1 mediates DARPP-32 activation via a cascade involving cAMP and PKA, and protein kinase C (PKC) activation via phospholipase C. Active DARPP-32 has a specific inhibitory effect on protein phosphatase 1 (PP1), leaving, e.g. Na+,K+-ATPase in a phosphorylated, inactive, state. Thus, dopamine acts as a natriuretic hormone in the mature kidney. Here, we discuss the age-dependent distribution and some functional aspects of several parts of the renal dopamine system (dopamine, AADC, COMT, D1 receptor, and DARPP-32) during renal morphogenesis. (+info)
Requirement for DARPP-32 in progesterone-facilitated sexual receptivity in female rats and mice.
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DARPP-32, a dopamine- and adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (32 kilodaltons in size), is an obligate intermediate in progesterone (P)-facilitated sexual receptivity in female rats and mice. The facilitative effect of P on sexual receptivity in female rats was blocked by antisense oligonucleotides to DARPP-32. Homozygous mice carrying a null mutation for the DARPP-32 gene exhibited minimal levels of P-facilitated sexual receptivity when compared to their wild-type littermates. P significantly increased hypothalamic cAMP levels and cAMP-dependent protein kinase activity. These increases were not inhibited by a D1 subclass dopamine receptor antagonist. P also enhanced phosphorylation of DARPP-32 on threonine 34 in the hypothalamus of mice. DARPP-32 activation is thus an obligatory step in progestin receptor regulation of sexual receptivity in rats and mice. (+info)