James D. Foster

Mechanisms of dopamine transporter regulation in normal and disease states

The dopamine (DA) transporter (DAT) controls the spatial and temporal dynamics of DA neurotransmission by driving reuptake of extracellular transmitter into presynaptic neurons. Many diseases such as depression, bipolar disorder, Parkinsons disease (PD), and attention deficit hyperactivity disorder (ADHD) are associated with abnormal DA levels, implicating DAT as a factor in their etiology. Medications used to treat these disorders and many addictive drugs target DAT and enhance dopaminergic signaling by suppressing transmitter reuptake. We now understand that the transport and binding properties of DAT are regulated by complex and overlapping mechanisms that provide neurons with the ability to modulate DA clearance in response to physiological demands. These processes are controlled by endogenous signaling pathways and affected by exogenous transporter ligands, demonstrating their importance for normal neurotransmission, drug abuse, and disease treatments. Increasing evidence supports the disruption of these mechanisms in DA disorders, implicating dysregulation of transport in disease etiologies and suggesting these processes as potential points for therapeutic manipulation of DA availability.

Psychoactive Substrates Stimulate Dopamine Transporter Phosphorylation and Down-regulation by Cocaine-sensitive and Protein Kinase C-dependent Mechanisms

Dopamine transporters (DATs) undergo intracellular sequestration and functional down-regulation upon exposure to psychostimulant substrates. To investigate the potential mechanism underlying these responses, we examined the acute in vitro and in vivo effects of amphetamine and methamphetamine (METH) on phosphorylation and down-regulation of rat DAT using wild type and N-terminal truncation mutants. Phosphorylation of DAT assessed by 32PO4 metabolic labeling was increased up to 2-fold by in vitro treatment of rDAT LLC-PK1 cells with amphetamine or METH and was similarly increased in rat striatal tissue by in vitro application or in vivo injection of METH. The dopamine transport blocker (-)-cocaine did not affect DAT phosphorylation but prevented the phosphorylation increase induced by METH. Phosphorylation of DAT induced by METH was also prevented by the protein kinase C blocker bisindoylmaleimide I and was absent in an N-terminally truncated protein that lacks the first 21 residues including 6 serines that also represent the site of phorbol ester induced phosphorylation. Down-regulation of transport induced by METH was also cocaine- and protein kinase C-dependent but was retained in the N-terminal truncation mutant. These results demonstrate that transport or binding of METH stimulates DAT phosphorylation and down-regulation by a mechanism that requires protein kinase C but that METH-induced down-regulation can occur independently of direct transporter phosphorylation. The finding that DAT phosphorylation is stimulated by amphetamines reveals a previously unknown effect of these drugs that is not produced by cocaine and may be related to reinforcement.

SLC6 transporters: Structure, function, regulation, disease association and therapeutics☆

The SLC6 family of secondary active transporters are integral membrane solute carrier proteins characterized by the Na(+)-dependent translocation of small amino acid or amino acid-like substrates. SLC6 transporters, which include the serotonin, dopamine, norepinephrine, GABA, taurine, creatine, as well as amino acid transporters, are associated with a number of human diseases and disorders making this family a critical target for therapeutic development. In addition, several members of this family are directly involved in the action of drugs of abuse such as cocaine, amphetamines, and ecstasy. Recent advances providing structural insight into this family have vastly accelerated our ability to study these proteins and their involvement in complex biological processes.

Phorbol ester induced trafficking-independent regulation and enhanced phosphorylation of the dopamine transporter associated with membrane rafts and cholesterol.

We examined the mechanisms involved in protein kinase C (PKC)‐dependent down‐regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin‐mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl‐β‐cyclodextrin (MβC) prior to treatment with the PKC activator phorbol 12‐myristate, 13‐acetate (PMA). Con A blocked PMA‐induced surface reductions of DAT but only partially inhibited down‐regulation, while MβC partially blocked down‐regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC‐induced DAT down‐regulation occurs by a combination of trafficking and non‐trafficking processes. Using density‐gradient centrifugation, we found that DATs are distributed approximately equally between Triton‐insoluble, cholesterol‐rich membrane rafts and Triton‐soluble non‐raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA‐induced loss of cell surface DAT occurred only from non‐raft populations, demonstrating that non‐raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol‐dependent non‐trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft‐non‐raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non‐rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.

Glucose-6-Phosphatase Structure, Regulation, and Function: An Update

Abstract Work on the glucose-6-phosphatase system has intensified and diversified extensively in the past 3 years. The gene for the catalytic unit of the liver enzyme has been cloned from three species, and regulation at the level of gene expression is being studied in several laboratories worldwide. More than 20 sites of mutation in the catalytic unit protein have been demonstrated to underlie glycogenesis type 1a. Inhibition of glucose-6-P hydrolysis by several newly identified competitive and time-dependent, irreversible inhibitors has been demonstrated and in several instances the predicted effects on liver glycogen formation and/or breakdown and on blood glucose production have been shown. Refinements in and additions to the presently dominant “substrate transport-catalytic unit” topological model for the glucose-6-phosphatase system have been made. A new model alternative to this, based on the “combined conformational flexibility-substrate transport” concept, has emerged. Experimental evidence for the phosphorylation of glucose in liver by high-K m, glucose enzyme(s) in addition to glucokinase has continued to emerge, and new in vitro evidence supportive of biosynthetic functions of the glucose-6-phosphatase system in this role has appeared. High levels of multifunctional glucose-6-phosphatase have been shown present in pancreatic islet β cells. Glucose-6-P has been established as the likely insulin secretagog in β cells. Interesting differences in the temporal responses of glucose-6-phosphatase in kidney and liver have been demonstrated. An initial attempt is made here to meld the hepatic and pancreatic islet β-cell glucose-6-phosphatase systems, and to a lesser extent the kidney tubular and small intestinal mucosal glucose-6-phosphatase systems into an integrated, coordinated mechanism involved in whole-body glucose homeostasis in health and disease.

Regulation of the Dopamine Transporter by Phosphorylation

The dopamine transporter (DAT) is a neuronal phosphoprotein and target for psychoactive drugs that plays a critical role in terminating dopaminergic transmission by reuptake of dopamine from the synaptic space. Control of DAT activity and plasma membrane expression are therefore central to drug actions and the spatial and temporal regulation of synaptic dopamine levels. DATs rapidly traffic between the plasma membrane and endosomal compartments in both constitutive and protein kinase C-dependent manners. Kinase activators, phosphatase inhibitors, and transported substrates modulate DAT phosphorylation and activity, but the underlying mechanisms and role of phosphorylation in these processes are poorly understood. Complex adaptive changes in DAT function potentially related to these processes are also induced by psychostimulant and therapeutic transport blockers such as cocaine and methylphenidate. This chapter provides an overview of the current state of knowledge regarding DAT phosphorylation and its relationship to transporter activity and trafficking. A better understanding of how dopaminergic neurons regulate DAT function and the role of phosphorylation may lead to the identification of novel therapeutic targets for the treatment and prevention of dopaminergic disorders.

Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux

Background: DAT activity is regulated by protein kinases. Results: We identify Thr53 as a DAT phosphorylation site in rat striatum by mass spectrometry and a phospho-specific antibody; Thr53 mutation reduced dopamine influx and ablated transporter-mediated efflux. Conclusion: Phosphorylation of DAT Thr53 is involved in transport activity. Significance: These results identify Thr53 phosphorylation of DAT in vivo and elucidate associated functional properties. In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr53. In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr53 in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr53 occurred with a stoichiometry of ∼50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr53 to prevent phosphorylation led to reduced dopamine transport Vmax and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.

The biochemistry and molecular biology of the glucose-6-phosphatase system.

Progress has continued to be made over the past 4 years in our understanding of the glucose-6-phosphatase (G6Pase) system. The gene for a second component of the system, the putative glucose-6-P transporter (G6PT), was cloned, and mutations in this gene were found in patients diagnosed with glycogen storage disease type 1b. The functional characterization of this putative G6PT has been initiated, and the relationship between substrate transport via the G6PT and catalysis by the systems catalytic subunit continues to be explored. A lively debate over the feasibility of various aspects of the two proposed models of the G6Pase system persists, and the functional/structural relationships of the individual components of the system remain a hot topic of interest in G6Pase research. New evidence supportive of physiologic roles for the biosynthetlc functions of the G6Pase system in vivo also has emerged over the past 4 years.

Proline-directed phosphorylation of the dopamine transporter N-terminal domain

Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with protein kinase C (PKC)- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues, we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCalpha, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCalpha-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses.

Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding

Background: Dopamine transporter (DAT) activity is regulated by PKC. Results: We identify Ser-7 as a PKC phosphorylation site on DAT and show that phosphorylation conditions and Ser-7 mutation alter cocaine analog binding characteristics. Conclusion: Ser-7 phosphorylation affects cocaine analog binding by altering DAT conformational equilibrium. Significance: Ser-7 phosphorylation of DAT may impact recognition and action of cocaine. As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.