The effects of phosphorylation on the functional regulation of an expressed recombinant human dopamine transporter

doi:10.1016/0304-3940(96)13034-2

Neuroscience Letters

Volume 216, Issue 2, 27 September 1996, Pages 133-136

https://doi.org/10.1016/0304-3940(96)13034-2 Get rights and content

Abstract

Metabolic labeling experiments were performed using eukaryotic cells transfected with the human dopamine (DA) transporter cDNA. Autophosphorylation in the presence and absence of the transporter substrate DA, was analyzed. Dopamine transporter (DAT) was phosphorylated in the absence of DA and dephosphorylated in the presence of the substrate. The functional significance of this phenomenon was checked by incubating cells with phosphorylation promoting agents, all of which reduced substrate uptake and ligand binding significantly. It is shown that at least one site of phosphorylation on DAT is a serine residue. These experiments suggest that the state of phosphorylation of the DAT may play an important role in its biological function.

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DAT phosphorylation was reviewed extensively by Foster et al. (2006). DAT-Ser phosphorylation was identified using p-Ser antibody (Vrindavanam et al., 1996). It is, however, important to note that the cytoplasmic domains of DAT contain putative consensus sites for several protein kinases (Kilty et al., 1991; Shimada et al., 1991).
Presynaptic biogenic amine transporters mediate reuptake of released amines from the synapse, thus regulating serotonin, dopamine and norepinephrine neurotransmission. Medications utilized in the treatment of depression, attention deficit-hyperactivity disorder and other psychiatric disorders possess high affinity for amine transporters. In addition, amine transporters are targets for psychostimulants. Altered expression of biogenic amine transporters has long been implicated in several psychiatric and degenerative disorders. Therefore, appropriate regulation and maintenance of biogenic amine transporter activity is critical for the maintenance of normal amine homoeostasis. Accumulating evidence suggests that cellular protein kinases and phosphatases regulate amine transporter expression, activity, trafficking and degradation. Amine transporters are phosphoproteins that undergo dynamic control under the influence of various kinase and phosphatase activities. This review presents a brief overview of the role of amine transporter phosphorylation in the regulation of amine transport in the normal and diseased brain. Understanding the molecular mechanisms by which phosphorylation events affect amine transporter activity is essential for understanding the contribution of transporter phosphorylation to the regulation of monoamine neurotransmission and for identifying potential new targets for the treatment of various brain diseases.
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After extensive washing they were resuspended in 40 μl Laemmli buffer, denatured, separated by electrophoresis in 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Bearing in mind that DAT is mostly phosphorylated on serine residues (Cervinski et al., 2005; Vrindavanam et al., 1996), blots were immunoreacted for phosphoserine using a mouse anti-phosphoserine monoclonal antibody (1:1000, Sigma). [3H]-DA uptake assays were performed as previously described (Salvatore et al., 2003; Zhu et al., 2005) with minor modifications, by using freshly obtained striatal synaptosomes resuspended in ice-cold assay buffer (see above).
Several studies report that the striatal dopamine (DA) uptake declines with age, but the underlying mechanisms are still unclear. The use of molecular, biochemical and morphological techniques, and antibodies which detect the glycosylated (80 kDa) and non-glycosylated (50 kDa) DA transporter (DAT) forms in the rat mesostriatal system, reveals that DAT is pre- and post-translationally damaged during aging. In middle age (18 months), the glycosylated DAT form decreases in the plasma membrane of striatal terminals, and the non-glycosylated form is accumulated in the endoplasmic reticulum–Golgi complex. Thereafter, in aged rats (24 months), DAT synthesis is also affected as the decrease in both DATmRNA and total DAT protein levels suggests. However, the evidence of a decrease in both DAT expression in the endosomal (vesicle-enriched) compartment and the phosphorylated DAT fraction from middle age, as well as its compartmental redistribution towards the terminal plasma membrane, with an increase in the membrane DAT/total DAT ratio in striatal synapotosomes, in aged rats, indicate that DA-cells activate compensatory mechanisms directed at maintaining DAT function during normal aging.
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DAT functions are regulated by presynaptic receptors, protein kinases and membrane trafficking and changes in DAT levels can clearly alter motor activity (Marshall and Grosset, 2003; Schenk et al., 200; Uhl, 2003). Agents that alter protein kinase C (PKC), inositol triphosphate (PI3) kinase and mitogen and signal‐regulated kinase (MEK1 and 2) alter DAT function (Vrindavanam et al., 1996; Carvelli et al., 2002; Uhl, 2003). Transporters can also function in reverse and they possess channel‐like activity (Torres et al., 2003).
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Phosphatidylinositol 3-kinase, protein kinase C, and MEK1/2 kinase regulation of dopamine transporters (DAT) require N-terminal DAT phosphoacceptor sites
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The dopamine transporter (DAT) modulates dopamine neurotransmission and is a primary target for psychostimulant influences on locomotion and reward. Selective DAT expression by dopaminergic neurons has led to use of cocaine analog DAT radioligands to assess rates of progression of dopamine neuronal degeneration in Parkinson's disease. We have documented that DAT is a phosphoprotein that is regulated by phosphorylation through pathways that include protein kinase C cascades. We now extend this work using drugs selective for phosphatidylinositol 3-kinase (PI3K), protein kinase C, MEK1/2, p38 kinase, and Ca²⁺/calmodulin kinase II. We compare the drug effects on wild type DAT to the effects on 20 DAT mutants and a DAT deletion. PI3K and MEK1/2 modulators exert strong effects on DAT expression patterns and dopamine uptake V_max. PKC principally modulates V_max. Neither p38 nor Ca²⁺/calmodulin kinase II agents exert significant influences on wild type DAT. Several mutants and a DAT with an N-terminal deletion display alterations that interact with the effects of kinase modulators, especially S7A for PKC effects; T62A, S581A, and T612A for PI3K effects; and S12A and T595A mutants for MEK1/2 effects. ³²P-Labeling studies confirm several of these effects of kinase pathway modulators on DAT phosphorylation. DAT expression and activities can be regulated by kinase cascades that require phosphoacceptor sites most concentrated in its N terminus. These results have a number of implications for DAT regulation and mandate caution in using DAT radioligand binding to infer changes in dopaminergic neuronal integrity after treatments that alter activities of these kinase pathways.
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Catecholamines are translocated across plasma membranes by transporters that belong to two large families with mainly neuronal or extraneuronal locations. In mammals, neuronal uptake of catecholamines involves the dopamine transporter (DAT) at dopaminergic neurons and the norepinephrine transporter (NET) at noradrenergic neurons. Extraneuronal uptake of catecholamines is mediated by organic cation transporters (OCTs), including the classic corticosterone-sensitive extraneuronal monoamine transporter. Catecholamine transporters function as part of uptake and metabolizing systems primarily responsible for inactivation of transmitter released by neurons. Additionally, the neuronal catecholamine transporters, recycle catecholamines for rerelease, thereby reducing requirements for transmitter synthesis. In a broader sense, catecholamine transporters function as part of integrated systems where catecholamine synthesis, release, uptake, and metabolism are regulated in a coordinated fashion in response to the demands placed on the system. Location is also important to function. Neuronal transporters are essential for rapid termination of the signal in neuronal-effector organ transmission, whereas non-neuronal transporters are more important for limiting the spread of the signal and for clearance of catecholamines from the bloodstream. Besides their presynaptic locations, NET and DAT are also present at several extraneuronal locations, including syncytiotrophoblasts of the placenta and endothelial cells of the lung (NET), stomach and pancreas (DAT). The extraneuronal monoamine transporter shows a broad tissue distribution, whereas the other two non-neuronal catecholamine transporters (OCT1 and OCT2) are mainly localized to the liver, kidney, and intestine. Altered function of peripheral catecholamine transporters may be involved in disturbances of the autonomic nervous system, such as occurs in congestive heart failure and hypernoradrenergic hypertension. Peripheral catecholamine transporters provide important targets for clinical imaging of sympathetic nerves and diagnostic localization and treatment of neuroendocrine tumors, such as neuroblastomas and pheochromocytomas.
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The dopamine transporter mediates uptake of dopamine into neurons and is a major target for various pharmacologically active drugs and environmental toxins. Since its cloning, much information has been obtained regarding its structure and function. Binding domains for dopamine and various blocking drugs including cocaine are likely formed by interactions with multiple amino acid residues, some of which are separate in the primary structure but lie close together in the still unknown tertiary structure. Chimera and site-directed mutagenesis studies suggest the involvement of both overlapping and separate domains in the interaction with substrates and blockers, whereas recent findings with sulfhydryl reagents selectively targeting cysteine residues support a role for conformational changes in the binding of blockers such as cocaine. The dopamine transporter can also operate in reverse, i.e. in an efflux mode, and recent mutagenesis experiments show different structural requirements for inward and outward transport. Strong evidence for dopamine transporter domains selectively influencing binding of dopamine or cocaine analogs has not yet emerged, although the development of a cocaine antagonist at the level of the transporter remains a possibility.

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The effects of phosphorylation on the functional regulation of an expressed recombinant human dopamine transporter

Abstract

FEBS Lett.

Neurobiology

Eur. J. Pharmacol.

Neurosci. Lett.

Prog. Neuropsychopharmacol. Biol. Psychiatry

Neurotransmitter transporters: recent progress

Annu. Rev. Neurosci.

The metabolism, storage and release of catecholamines

Rec. Prog. Horm. Res.

Inhibitors of arachidonic acid metabolism: effects on rat striatal dopamine release and uptake

J. Pharmacol. Exp. Ther.