Oestradiol increases phosphorylation of a dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32) in female rat brain

Recent studies suggest that oestrogen and progestin receptors may be activated by the neurotransmitter dopamine, as well as by their respective ligands. Because intracerebroventricular infusion of D(1), but not D(2), dopaminergic receptor agonists increases oestrous behaviour in oestradiol-primed rats, we wanted to determine if treatment with oestradiol alters the activity of D(1) receptor-associated processes in steroid receptor-containing areas in female rat brain. One D(1) receptor-associated phosphoprotein that may be influenced by oestradiol is a dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000 (DARPP-32). Because DARPP-32 is phosphorylated in response to dopamine acting via a cAMP-dependent protein kinase, it provides a useful marker to examine where in the brain a particular stimulus might be altering the activity of D(1) receptor-containing neurones. To determine if oestradiol alters the phosphorylation of DARPP-32, we stained immunocytochemically brain sections of female rats treated with behaviourally relevant doses of oestradiol or oil vehicle with an antibody that detects only the threonine 34-phosphorylated form of DARPP-32. Behaviourally effective doses of oestradiol increase the phosphorylation of DARPP-32 within the medial preoptic nucleus, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus and the ventromedial nucleus of the hypothalamus, 48 h after treatment. These data suggest that oestradiol increases the activity of D(1) dopamine receptor-associated processes in oestrogen receptor-containing areas of female rat forebrain.     

Regulation of extracellular signal-regulated kinase phosphorylation in cultured rat striatal neurons

Recent studies demonstrate that activation of Ca²⁺-permeable N-methyl-D-aspartate (NMDA) receptors upregulates phosphorylation of mitogen-activated protein kinases (MAPKs) in heterologous cells and neurons. In cultured rat striatal neurons, the present work systematically evaluated the role of a number of protein kinases in forming a signaling cascade transducing NMDA receptor signals to MAPKs. It was found that a brief NMDA application consistently induced rapid and transient phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2), a best characterized subclass of MAPKs. This ERK1/2 phosphorylation was resistant to the inhibition of protein kinase C, p38 MAPK, cyclin-dependent kinase 5, receptor tyrosine kinase (epidermal growth factor receptors), or non-receptor tyrosine kinases (including Src) by their selective inhibitors. However, the increase in ERK1/2 phosphorylation was partially blocked by a protein kinase A (PKA) inhibitor. The inhibitors for Ca²⁺/calmodulin-dependent protein kinase (CaMK) or phosphatidylinositol 3-kinase (PI3-kinase) completely blocked the NMDA-stimulated ERK1/2 phosphorylation. In an attempt to characterize the sequential role of CaMK and PI3-kinase, we found that NMDA increased PI3-kinase phosphorylation on Tyr⁵⁰⁸, which kinetically corresponded to the ERK1/2 phosphorylation and was blocked by the CaMK inhibitor. These results indicate that the protein kinases are differentially involved in linking NMDA receptors to ERK1/2 in striatal neurons.     

μ- and δ-opioid receptor agonists inhibit DARPP-32 phosphorylation in distinct populations of striatal projection neurons

In the striatum, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) is highly expressed by virtually all projection medium-sized spiny neurons. cAMP-dependent phosphorylation of DARPP-32 is stimulated via activation of dopamine D1 receptors in striatonigral neurons, and via activation of adenosine A2A receptors in striatopallidal neurons. In this study, we have examined the contribution of mu-, delta- and kappa-opioid receptors to the regulation of DARPP-32 phosphorylation, in rat striatal slices. The results show that, at low concentrations (100 pm-1 nm), the mu-opioid agonist, Tyr-D-Ala-Gly-N-Me-Phe-glycinol (DAMGO), inhibits the increase in DARPP-32 phosphorylation induced by activation of D1, but not by activation of A2A receptors. Conversely, the delta-receptor agonist, Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE), inhibits DARPP-32 phosphorylation induced by activation of A2A, but not by activation of D1 receptors. The kappa-receptor agonist, U50488, does not affect DARPP-32 phosphorylation induced by either D1 or A2A agonists. Thus, mu-opioid receptors interact with dopamine D1 receptors on striatonigral neurons, whereas delta-opioid receptors interact with adenosine A2A receptors on striatopallidal neurons. These results suggest that regulation of DARPP-32 phosphorylation is involved in mediating some of the effects exerted by enkephalin on striatal medium-sized spiny neurons.     

Immunohistochemical localization of DARPP-32 in striatal projection neurons and striatal interneurons: implications for the localization of D1-like dopamine receptors on different types of striatal neurons.

Immunohistochemical double-label techniques were used to study the localization of DARPP-32, a phosphoprotein that is enriched in neurons possessing members of the D1 subfamily of dopamine receptors, in several different types of striatal neurons in the rat basal ganglia. The vast majority (94.1%) of striatonigral projection neurons (the vast majority of which contain substance P), identified by retrograde labeling with fluorogold, were observed to contain DARPP-32. Similarly, the vast majority of striatopallidal projection neurons (87.7%), identified by immunofluorescence labeling for enkephalin (ENK), were found to label for DARPP-32. In contrast, cholinergic and neuropeptide Y-containing striatal interneurons were never observed to contain DARPP-32. These results suggest that essentially all major types of striatal medium spiny projection neurons may possess members of the D1 subfamily of dopamine receptors, but that striatal local circuit neurons do not possess members of the D1 subfamily of receptors.     

Immunohistochemical localization of DARPP-32 in striatal projection neurons and striatal interneurons: implications for the localization of D1-like dopamine receptors on different types of striatal neurons

Immunohistochemical double-label techniques were used to study the localization of DARPP-32, a phosphoprotein that is enriched in neurons possessing members of the D₁ subfamily of dopamine receptors, in several different types of striatal neurons in the rat basal ganglia. The vast majority (94.1%) of striatonigral projection neurons (the vast majority of which contain substance P), identified by retrograde labeling with fluorogold, were observed to contain DARPP-32. Similarly, the vast majority of striatopallidal projection neurons (87.7%), identified by immunofluorescence labeling for enkephalin (ENK), were found to label for DARPP-32. In contrast, cholinergic and neuropeptide Y-containing striatal interneurons were never observed to contain DARPP-32. These results suggest that essentially all major types of striatal medium spiny projection neurons may possess members of the D₁ subfamily of dopamine receptors, but that striatal local circuit neurons do not possess members of the D₁ subfamily of receptors.     

Nuclear protein kinase substrates in AtT-20 cells: translocation of a 14 kDa phosphoprotein

In this work we characterize some acidic nuclear substrates of protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA), using intact anterior pituitary corticotrophic tumor cells (AtT-20/D16-16). It was found that, as in the cytosolic fraction, substrates for both PKC and PKA exist in the nucleus and that changes in the phosphorylation states of a few of these phosphoproteins are mediated by both kinases. One of the phosphoproteins examined, a 14 kDa phosphoprotein (pp14) described previously, exhibited a phorbol-ester induced translocation from nucleus to cytosol in pulse-chase experiments utilizing 35S-methionine labeling. These results suggest that pp14 may be involved in signal transduction in AtT-20 cells. Although its identity remains to be determined, a 14 kDa DNA-binding protein was also seen in nuclear extracts of AtT-20 cells.     

Estrogenic stimulation of neurite growth in midbrain dopaminergic neurons depends on cAMP/protein kinase A signalling

Previous work from this laboratory indicates that the differentiation of mouse midbrain dopaminergic neurons is influenced by estrogen. These effects may be transmitted either through classical nuclear receptors or via "nongenomic" mechanisms, including the interaction with hypothetical membrane receptors coupled to distinct intracellular signalling pathways. The latter mechanism seems to be of particular interest for the observed interactions of estrogen with developing dopaminergic neurons, insofar as estrogen has been shown to increase intracellular calcium levels within seconds. This study focuses on signal transduction cascades that might be activated by estrogen during differentiation of dopaminergic cells. Treatment with 17beta-estradiol or a membrane-impermeable estrogen-BSA construct (E-BSA) increased neurite growth and arborization of dopaminergic neurons. This effect was inhibited by antagonists of cAMP/ protein kinase A (PKA) and calcium signalling pathways but not by the estrogen receptor antagonist ICI. In addition, estrogen exposure stimulated the phosphorylation of CREB in midbrain dopaminergic cells as studied by quantitative double-labelling immunocytochemistry and gel shift assay. Again, this effect was antagonized only by the simultaneous treatment with inhibitors of the cAMP/PKA or calcium pathways and not by ICI pretreatment. These data together with our previous findings demonstrate that estrogen can interact with membrane binding sites on dopaminergic neurons, thereby stimulating the cAMP/PKA/phosphorylated cAMP-responsive element binding protein (CREB) signalling cascade, most likely through the activation of calcium-dependent kinases. In conclusion, rapid "nongenomic" estrogen signalling represents another mechanism, in addition to the activation of classical nuclear estrogen receptors, that is capable of influencing neuronal differentiation in the mammalian brain.     

Dopamine-independent action of cocaine on striatal and accumbal neurons

Increasing evidence suggests that dopamine (DA) mechanisms alone cannot fully explain the psychoemotional and behavioural effects of cocaine, including its ability to induce drug-taking behaviour. Although it is known that cocaine, after intravenous administration or smoking, may reach brain levels high enough to inhibit Na+ transport, the role of this action remains unclear. To examine the contribution of local anaesthetic and DA mechanisms to changes in striatal and accumbal neuronal activity induced by cocaine, single-unit recording was combined with iontophoresis in awake, unrestrained rats. Most spontaneously active and glutamate-stimulated neurons were highly sensitive to brief cocaine applications (0-40 nA); cocaine-induced inhibitions occurred at small ejection currents (0-5 nA), were dose-dependent, highly stable during repeated applications and strongly dependent on basal activity rates. These neuronal responses remained almost unchanged after systemic administration of either a selective D1 antagonist (SCH-23390, 0.2 mg/kg) or a combination of SCH-23390 (1 mg/kg) and eticlopride (1 mg/kg), a D2 antagonist. Whereas SCH-23390 alone had a weak attenuating effect, no effect and even a slight enhancement of responses to cocaine occurred in fast-firing glutamate (GLU)-stimulated units after the combined blockade of D1 and D2 receptors. Responses to cocaine were mimicked by iontophoretic procaine (0-40 nA), a short-acting local anaesthetic with minimal effect on DA uptake. Procaine-induced inhibitions occurred at the same low currents, had a similar time-course, and were also strongly dependent on basal discharge rate. Our data support the existence of a DA-independent mechanism for the action of cocaine involving a direct interaction with Na+ channels. Although further studies are required to clarify this mechanism and its interaction with other pharmacological and behavioural variables, a direct interaction with Na+ channels may contribute to changes in neuronal activity induced by self-injected cocaine, thereby playing a role in mediating the psychoemotional and behavioural effects of this drug.     

Anti-apoptotic actions of vasopressin in H32 neurons involve MAP kinase transactivation and Bad phosphorylation

Vasopressin (VP) secreted within the brain modulates neuronal function acting as a neurotransmitter. Based on the observation that VP prevented serum deprivation-induced cell death in the neuronal cell line, H32, which expresses endogenous V1 receptors, we tested the hypothesis that VP has anti-apoptotic properties. Flow cytometry experiments showed that 10 nM VP prevented serum deprivation-induced cell death and annexin V binding. Serum deprivation increased caspase-3 activity in a time and serum concentration dependent manner, and VP prevented these effects through interaction with receptors of V1 subtype. The signaling pathways mediating the anti-apoptotic effect of VP involve mitogen activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK), Ca²⁺/calmodulin dependent kinase (CaMK) and protein kinase C (PKC). Western blot analyses revealed time-dependent decreases of Bad phosphorylation and increases in cytosolic levels of cytochrome c following serum deprivation, effects which were prevented by 10 nM VP. These data demonstrate that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochrome c and caspase-3 activation. The data suggest that VP has anti-apoptotic activity in neurons and that VP may act as a neuroprotective agent in the brain.     

A 35 kDa Fos-related antigen is co-localized with substance P and dynorphin in striatal neurons

The rat striatum after dopamine denervation followed by repeated apomorphine treatment was examined for the co-expression of c-fos and Fos-related antigens with dynorphin, substance P and [Met5]enkephalin using Western blot and immunohistochemical techniques. Administration of apomorphine, a dopamine agonist, elevated the level of 35 kDa Fos-related antigen which co-localized with dynorphin and substance P, but not enkephalin, in striatal neurons.     

Formation of dendritic spines in cultured striatal neurons depends on excitatory afferent activity

The role of afferent innervation in the formation of dendritic spines was studied in cultured rat striatum. The striatum is a unique structure in that it contains highly spiny GABAergic projection neurons, with no known local excitation. Grown alone in culture, striatal neurons did not express spontaneous network activity and were virtually devoid of dendritic spines. Adding GFP-expressing mouse cortical neurons to the striatal culture caused the appearance of spontaneous and evoked excitatory synaptic currents in the striatal neurons and a 10-fold increase in the density of spines on their dendrites. This effect was blocked by a continuous presence of TTX in the growth medium, while removal of the drug caused a rapid appearance of spines. Exposure to glutamate, or the presence of cortex-conditioned medium did not mimic the effect of cortical neurons on formation of spines in the striatal neurons. Also, the cortical innervation did not cause a selective enhancement of survival of specific subtypes of spiny striatal neurons. These experiments demonstrate that excitatory afferents are necessary for the formation of dendritic spines in striatal neurons.     

Colocalization of excitatory and inhibitory neurotransmitter markers in striatal projection neurons in the rat

The principle neuronal output of the neostriatum comes from medium spiny neurons that project from the caudate/putamen to the globus pallidus and substantia nigra. Although current evidence generally indicates that γ-aminobutyric acid (GABA) is the principal neurotransmitter in this pathway, this cannot account for the excitatory synaptic activity present among cultures of striatal neurons or the short latency excitatory postsynaptic potentials which often proceed or obscure inhibitory activity evoked by striatal stimulation. In this study, retrograde transport of [³H]D -aspartate has been used to demonstrate striatopallidal and striato-nigral neurons that possess a high-affinity uptake system for glutamate and aspartate and are therefore putatively glutamatergic. Injections of [³H]D -aspartate into the globus pallidus or substantia nigra, pars reticularis of the rat retrogradely labeled mediumsized neurons throughout the rostral-caudal extent of the neostriatum. To characterize this population further, adjacent sections were immunoreacted with antibodies to either GABA, glutamic acid decarboxylase (GAD), calbindin, or parvalbumin prior to autoradiographic processing. Under these conditions, autoradiographically labeled neurons displayed positive immunoreactivity for GABA, GAD, or calbindin. Autoradiographic label did not colocalize with parvalbumin immunoreactivity. The colocalization of anatomical markers of GABAergic and glutamatergic neurotransmission raises the possibility that both neurotransmitters are functionally expressed within single striatal projection neurons. © 1994 Wiley-Liss, Inc.     

Modulation of the voltage-gated sodium current in rat striatal neurons by DARPP-32, an inhibitor of protein phosphatase

DARPP-32 is a cyclic adenosine monophosphate-regulated inhibitor of protein phosphatase 1, highly enriched in striatonigral neurons. Stimulation of dopamine D₁ receptors increases phosphorylation of DARPP-32, whereas glutamate acting on N-methyl-d -aspartate receptors induces its dephosphorylation. Yet, to date, there is little direct evidence for the function of DARPP-32 in striatal neurons. Using a whole cell patch-clamp technique, we have studied the role of DARPP-32 in the regulation of voltage-gated sodium channels in rat striatal neurons maintained in primary culture. Injection of phospho-DARPP-32, but not of the unphosphorylated form, reduced the sodium current amplitude. This effect was similar to those induced by okadaic acid, with which there was no additivity and by tautomycin. Our results indicate that, in striatal neurons, sodium channels are under dynamic control by phosphorylation/dephosphorylation, and that phospho-DARPP-32 reduces sodium current by stabilizing a phosphorylated state of the channel or an associated regulatory protein. We propose that the DARPP-32-mediated modulation of sodium channels, via inhibition of phosphatase 1, contributes to the regulation of these channels by D₁ receptors and other neurotransmitters which influence the state of phosphorylation of DARPP-32.     

Modulation of perch connexin35 hemi-channels by cyclic AMP requires a protein kinase A phosphorylation site

Retinal neurons are coupled via gap junctions, which function as electrical synapses that are gated by ambient light conditions. Gap junctions connecting either horizontal cells or AII amacrine cells are inhibited by the neurotransmitter dopamine, via the activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway. Fish connexin35 (Cx35) and its mouse ortholog, Cx36, are good candidates to undergo dopaminergic modulation, because they have been detected in the inner plexiform layer of the retina, where Type II amacrine cells establish synaptic contacts. We have taken advantage of the ability of certain connexins to form functional connexons (hemi-channels), when expressed in Xenopus oocytes, to investigate whether pharmacological elevation of cAMP modulates voltage-activated hemi-channel currents in single oocytes. Injection of perch Cx35 RNA into Xenopus oocytes induced outward voltage-dependent currents that were recorded at positive membrane potentials. Incubation of oocytes with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), a membrane permeable cAMP analog, resulted in a dose-dependent and reversible inhibition of hemi-channel currents at the more positive voltage steps. In contrast, treatment with 8-Br-cAMP did not have any effect on hemi-channel currents induced by skate Cx35. Amino acid sequence comparison of the two fish connexins revealed, in the middle cytoplasmic loop of perch Cx35, the presence of a PKA consensus sequence that was absent in the skate connexin. The results obtained with two constructs in which the putative PKA phosphorylation site was either suppressed (perch Cx35R108Q) or introduced (skate Cx35Q108R) indicate that it is responsible for the inhibition of hemi-channel currents. These studies demonstrate that perch Cx35 is a target of the cAMP/PKA signaling pathway and identify a consensus PKA phosphorylation site that is required for channel gating.     

去除蛋白激酶A磷酸化位点的线粒体融合素2基因与血管平滑肌细胞增殖

背景: 线粒体融合素2基因作用于血管平滑肌细胞Ras蛋白,通过胞外信号调节蛋白激酶1/2通路抑制细胞增殖。线粒体融合素2基因氨基酸序列第442位丝氨酸为蛋白激酶A磷酸化位点,与其磷酸化状态密切相关,可能参与其功能调控。 目的：观察大鼠线粒体融合素2基因在去除蛋白激酶A磷酸化位点后对大鼠血管平滑肌细胞增殖的影响及其相关信号通路。 方法：利用已构建的携带绿色荧光蛋白基因、线粒体融合素2基因和去除蛋白激酶A磷酸化位点的线粒体融合素2基因的3种重组腺病毒,感染大鼠主动脉血管平滑肌细胞,将其传代培养3~10代后以抽签法随机分为4组：①不加干预的对照组。②感染携带绿色荧光蛋白的对照组(Adv-GFP组)。③感染携带线粒体融合素2基因的实验组(Adv-Mfn2组)。④感染携带去除蛋白激酶A磷酸化位点的线粒体融合素2基因的实验组(Adv- Mfn2-PKA(△)组)。激光共聚焦显微镜观察完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在细胞中的定位。Western blot检测p-ERK1/2表达水平及完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中的表达。MTT法绘制细胞生长曲线。 结果与结论：完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中均表达蛋白特异性条带。两种基因表达产物都主要分布于线粒体外膜。与对照组和Adv-GFP组相比,Adv-Mfn2组吸光度值在第3,4,5,6天都显著降低(P < 0.01),Adv-Mfn2-PKA(△)组吸光度值无明显变化。与对照组和Adv-GFP组相比,Adv-Mfn2组p-ERK1/2表达水平显著降低(P < 0.01),Adv-Mfn2-PKA(△)组无明显变化。提示去除蛋白激酶A磷酸化位点的线粒体融合素2基因定位于线粒体外膜,对血管平滑肌细胞的增殖无拮抗作用,对胞外信号调节蛋白激酶1/2通路无抑制作用。     

Memory formation in the chick depends on membrane-bound protein kinase C

The role of protein kinase C (PKC) in the formation of memory for a one-trial passive avoidance task in 1-day-old chicks has been studied, following earlier observations that training on this task results in transient and lateralised changes in the phosphorylation state of presynaptic B-50 protein, a PKC substrate. In accord with hypotheses that the activity of PKC is regulated by translocation from cytosol to membrane, a significant increase was found in the fraction of the alpha/beta forms of the enzyme, assayed immunologically, present in a synaptic-membrane-bound, Triton-extractable form in the left intermediate medial hyperstriatum ventrale (IMHV) of chicks 30 min after training on the passive avoidance task. Two inhibitors of PKC, melittin (10 microliters, 120 microM) and H7 (10 microliters, 10 mM), if injected intracerebrally 10 min prior to or 10 min after training, were without effect on the general behaviour of the chicks or their training. However, these injections of the inhibitors produced amnesia in birds tested 3 h later. This effect was lateralised; only left hemisphere injections of the inhibitors produced amnesia. A possible state-dependency interpretation of these results was ruled out. The results are discussed in the context of hypotheses as to the regulatory role of PKC in neural plasticity and memory formation.     

Cortical regulation of striatal projection neurons and interneurons in a Parkinson's disease rat model

Striatal neurons can be either projection neurons or interneurons, with each type exhibiting distinct susceptibility to various types of brain damage. In this study, 6-hydroxydopamine was injected into the right medial forebrain bundle to induce dopamine depletion, and/or ibotenic acid was injected into the M1 cortex to induce motor cortex lesions. Immunohistochemistry and western blot assay showed that dopaminergic depletion results in significant loss of striatal projection neurons marked by dopamine- and cyclic adenosine monophosphate-regulated phosphoprotein, molecular weight 32 k Da, calbindin, and μ-opioid receptor, while cortical lesions reversed these pathological changes. After dopaminergic deletion, the number of neuropeptide Y-positive striatal interneurons markedly increased, which was also inhibited by cortical lesioning. No noticeable change in the number of parvalbumin-positive interneurons was found in 6-hydroxydopamine-treated rats. Striatal projection neurons and interneurons show different susceptibility to dopaminergic depletion. Further, cortical lesions inhibit striatal dysfunction and damage induced by 6-hydroxydopamine, which provides a new possibility for clinical treatment of Parkinson's disease.     

The role of Ca2+ channels in the repetitive firing of striatal projection neurons

Blockade of L-type Ca2+ channels results in a decrease in firing frequency of neostriatal neurons. In contrast, N- and P/Q-types of Ca2+ channel cooperate to tune firing pattern, since both of these channel types have to be blocked to enhance firing frequency. Parameters of the intensity-frequency plot were differentially modified by Ca2+ channel antagonists: while L-type Ca2+ channel block reduced the dynamic range by about 80%, block of N- and P/Q-types of Ca2+ channel generated a steeper intensity-frequency plot. These effects are explained in terms of the sustained depolarization and the afterhyperpolarizing potential known to be dependent upon L- and N-, P/Q-types of Ca2+ channels, respectively.     

DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. II. Purification and characterization of the phosphoprotein from bovine caudate nucleus

DARPP-32 is a neuronal phosphoprotein of Mr = 32,000, originally identified in rat brain (Walaas, S.I., D.W. Aswad, and P. Greengard (1983) Nature 301: 69-72). This protein has now been identified in bovine caudate nucleus cytosol and purified 435-fold to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification procedure involved acid extraction at pH 2, CM-cellulose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, and gel filtration on Ultrogel AcA 44. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 0.96 mol of phosphate/mol of purified DARPP-32. Phosphorylation occurred exclusively on threonine. The isoelectric point of dephospho-DARPP-32 was 4.7, and that of phospho-DARPP-32 was 4.6. The amino acid composition showed a high content of glutamate/glutamine and proline, and a low content of hydrophobic amino acids. DARPP-32 was found to have a Stokes radius of 34 A and a sedimentation coefficient of 2.05 S, indicating that it exists as an elongated monomer.