Regulation Of DARPP-32 Phosphorylation Research Articles

Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels.

Regulation of psychomotor functions by dopamine: integration of various approaches

(1)The basal ganglia circuitry mediates a wide rage of brain functions such as motor control, behavioral planning, and reward prediction. Dopamine (DA) transmission plays an essential role in the regulation of these brain functions. DA action not only regulates the firing activity of target neurons but also is involved in the pattern formation of their firing. The striatopallidal neurons containing dopamine D(2) receptor plays a dual role in motor coordination dependent on DA transmission. (2)Activation of presynaptic D(2)-like receptors on GABAergic terminals onto striatal cholinergic interneurons selectively blocks N-type Ca(2+) channels, thereby inhibiting GABA release. In addition, contribution of N-type channels and D(2)-like receptor-mediated presynaptic inhibition decreases in parallel with development, implying some relationship between basal ganglia-related function or dysfunction and age. (3)As an approach to determine dopamine neuronal activity, we monitored neuronal activities by measuring cytosolic Ca(2+) concentration in VTA dopamine neurons. The present study indicates that VTA dopamine neurons are the direct targets of orexin-A and psychostimulants, and the [Ca(2+)](i) signaling is thought to play a significant role in the regulation of dopamine neuronal activity. (4)The excitability of neostriatal neurons is regulated by a balance of glutamatergic and dopaminergic inputs. Glutamate has been shown to modulate dopaminergic signaling. Studies on the regulation of DARPP-32 phosphorylation by glutamate provide a molecular basis for both the synergistic and antagonistic effects of glutamate on dopaminergic signaling. (5) Impairment of function of stem/progenitor cells may be implicated in the pathogenesis of schizophrenia. To test this hypothesis, several experiments are currently ongoing in our laboratory, and the preliminary results obtained are described here.

Co-localization of the D 1 dopamine receptor in a subset of DARPP-32-containing neurons in rat caudate–putamen

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, apparent molecular weight of 32,000) is part of the D 1 dopamine receptor signal transduction cascade. Both the D 1 receptor and DARPP-32 are found in the caudate–putamen, but it is not known if they co-localize in the medium-sized spiny neurons. In the present study, double-labelling immunocytochemistry was used to simultaneously localize the D 1 receptor and DARPP-32 in the rat caudate–putamen. The neuropil was heavily and uniformly immunoreactive for both the D 1 receptor and DARPP-32. All cell bodies immunopositive for the D 1 receptor were immunopositive for DARPP-32. The D 1 receptor was not detectable, however, in nearly half of the DARPP-32-containing cell bodies. DARPP-32 is present in striatopallidal and striatonigral projections. The D 1 receptor co-localized with DARPP-32 in fibres of the entopeduncular nucleus and the pars reticulata of the substantia nigra. In the globus pallidus, however, D 1 receptor immunoreactivity was barely detectable, while DARPP-32 immunolabelling of axons and axon terminals was intense. These data suggest that the striatal somata containing both the D 1 receptor and DARPP-32 project to the entopeduncular nucleus and substantia nigra, whereas somata containing only DARPP-32 immunoreactivity project to the globus pallidus. Thus, the differences in expression of the D 1 receptor and of DARPP-32 within striatal cell bodies are likely reflected in their projections. The co-localization of the D 1 receptor and DARPP-32 is consistent with the known regulation of DARPP-32 phosphorylation by D 1 receptor activation. The demonstration of a large population of striatal neurons that contain DARPP-32 but apparently do not contain D 1 receptors substantiates the premise that these cells have an alternative signal transduction pathway. Subsequent studies are needed to search for a signal transduction pathway for these neurons analogous to the dopamine D 1 receptor pathway.

Nitric oxide/cGMP pathway stimulates phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, in the substantia nigra.

DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000, has been shown to be phosphorylated on threonine-34, both in vitro with high efficiency by cAMP-dependent and cGMP-dependent protein kinases and in vivo by dopamine acting through cAMP-dependent protein kinase. In the present study, we investigated the nitric oxide (NO)/cGMP pathway for its ability to regulate the state of phosphorylation of DARPP-32 in slices of rat substantia nigra. DARPP-32 was phosphorylated on threonine-34 in these slices by sodium nitroprusside (SNP), an NO donor. The effect of SNP was abolished by preincubation of the slices with hemoglobin, indicating that the effect of SNP was due to released NO. The same concentration of SNP produced a 4-fold elevation of the cGMP level but did not alter the level of cAMP. The effect of SNP on DARPP-32 phosphorylation was mimicked by low concentrations of 8-bromo-cGMP and 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate, activators of cGMP-dependent protein kinase, but not by low concentrations of 8-bromo-cAMP, an activator of cAMP-dependent protein kinase. The data indicate a physiological role for the NO/cGMP pathway in the regulation of DARPP-32 phosphorylation in nerve terminals of striatonigral neurons. The results provide further evidence that the state of phosphorylation of DARPP-32 represents an important mechanisms for integration of information arriving at striatonigral neurons via a variety of neuronal pathways.