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Gonzalo E Torres

Publications and source records attributed to Gonzalo E Torres.

11 recordsLinked to original sources
PubMed2006

The dopamine transporter proteome.

Dopamine (DA) uptake through the neuronal plasma membrane DA transporter (DAT) is essential for the maintenance of normal DA homeostasis in the brain. The DAT-mediated re-uptake system limits not only the intensity but also the duration of DA actions at presynaptic and postsynaptic receptors. This protein is the primary target for cocaine and amphetamine, both highly addictive and major substances of abuse worldwide. DAT is also the molecular target for therapeutic agents used in the treatment of mental disorders, such as attention deficit hyperactivity disorder and depression. Given the role played by the DAT in regulation of DA neurotransmission and its contribution to the abuse potential of psychostimulants, it becomes not only important but also necessary to understand the functional regulation of this protein. To investigate the cellular and molecular mechanisms associated with DAT function and regulation, our laboratory and others have embarked on a systematic search for DAT protein-protein interactions. Recently, a growing number of proteins have been shown to interact with DAT. These novel interactions might be important in the assembly, targeting, trafficking and/or regulation of transporter function. In this review, I summarize the main findings obtained from the characterization of DAT-interacting proteins and discuss the functional implications of these novel interactions. Based on these new data, I propose to use the term DAT proteome to explain how interacting proteins regulate DAT function. These novel interactions might help define new mechanisms associated with the function of the transporter.

Animals↗
PubMed2004

Effect of torsinA on membrane proteins reveals a loss of function and a dominant-negative phenotype of the dystonia-associated DeltaE-torsinA mutant.

Most cases of early-onset torsion dystonia (EOTD) are caused by a deletion of one glutamic acid in the carboxyl terminus of a protein named torsinA. The mutation causes the protein to aggregate in perinuclear inclusions as opposed to the endoplasmic reticulum localization of the wild-type protein. Although there is increasing evidence that dysfunction of the dopamine system is implicated in the development of EOTD, the biological function of torsinA and its relation to dopaminergic neurotransmission has remained unexplored. Here, we show that torsinA can regulate the cellular trafficking of the dopamine transporter, as well as other polytopic membrane-bound proteins, including G protein-coupled receptors, transporters, and ion channels. This effect was prevented by mutating the ATP-binding site in torsinA. The dystonia-associated torsinA deletion mutant (DeltaE-torsinA) did not have any effect on the cell surface distribution of polytopic membrane-associated proteins, suggesting that the mutation linked with EOTD results in a loss of function. However, a mutation in the ATP-binding site in DeltaE-torsinA reversed the aggregate phenotype associated with the mutant. Moreover, the deletion mutant acts as a dominant-negative of wild-type torsinA through a mechanism presumably involving association of wild-type and mutant torsinA. Taken together, our results provide evidence for a functional role for torsinA and a loss of function and a dominant-negative phenotype of the DeltaE-torsinA mutation. These properties may contribute to the autosomal dominant nature of the condition.

Adenosine Triphosphate↗
PubMed2004

Identification of PSD-95 as a regulator of dopamine-mediated synaptic and behavioral plasticity.

To identify the molecular mechanisms underlying psychostimulant-elicited plasticity in the brain reward system, we undertook a phenotype-driven approach using genome-wide microarray profiling of striatal transcripts from three genetic and one pharmacological mouse models of psychostimulant or dopamine supersensitivity. A small set of co-affected genes was identified. One of these genes encoding the synaptic scaffolding protein PSD-95 is downregulated in the striatum of all three mutants and in chronically, but not acutely, cocaine-treated mice. At the synaptic level, enhanced long-term potentiation (LTP) of the frontocortico-accumbal glutamatergic synapses correlates with PSD-95 reduction in every case. Finally, targeted deletion of PSD-95 in an independent line of mice enhances LTP, augments the acute locomotor-stimulating effects of cocaine, but leads to no further behavioral plasticity in response to chronic cocaine. Our findings uncover a previously unappreciated role of PSD-95 in psychostimulant action and identify a molecular and cellular mechanism shared between drug-related plasticity and learning.

Animals↗
PubMed2004

Hypertension and impaired glycine handling in mice lacking the orphan transporter XT2.

A family of orphan transporters has been discovered that are structurally related to the Na(+)-Cl(-)-dependent neurotransmitter transporters, including the dopamine transporter. One member of this family, the mouse XT2 gene, is predominantly expressed in the kidney and has 95% homology to rat ROSIT (renal osmotic stress-induced Na(+)-Cl(-) organic solute cotransporter). To study the physiological functions of this transporter, we generated XT2-knockout mice by gene targeting. XT2(-/-) mice develop and survive normally with no apparent abnormalities. To attempt to identify potential substrates for XT2, we screened urine from XT2-knockout mice by high-pressure liquid chromatography and mass spectrometry and found significantly elevated concentrations of glycine. To study glycine handling, XT2(+/+) and XT2(-/-) mice were injected with radiolabeled glycine, and urine samples were collected to monitor glycine excretion. After 2 h, XT2(-/-) mice were found to excrete almost twice as much glycine as the XT2(+/+) controls (P = 0.03). To determine whether the absence of the XT2 transporter affected sodium and fluid homeostasis, we measured systolic blood pressure by computerized tail-cuff manometry. Systolic blood pressure was significantly higher in XT2(-/-) mice (127 +/- 3 mmHg) than in wild-type controls (114 +/- 2 mmHg; P < 0.001). This difference in systolic blood pressure was maintained on high and low salt feeding. To examine whether the alteration in blood pressure and the defect in glycine handling were related, we measured systolic blood pressure in the XT2(-/-) mice during dietary glycine supplementation. Glycine loading caused systolic blood pressure to fall in the XT2(-/-) mice from 127 +/- 3 to 115 +/- 3 mmHg (P < 0.001), a level virtually identical to that of the wild-type controls. These data suggest that the XT2 orphan transporter is involved in glycine reabsorption and that the absence of this transporter is sufficient to cause hypertension.

Amino Acids↗
PubMed2003

Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice.

Brain dopaminergic transmission is a critical component in numerous vital functions, and its dysfunction is involved in several disorders, including addiction and Parkinson's disease. Responses to dopamine are mediated via G protein-coupled dopamine receptors (D1-D5). Desensitization of G protein-coupled receptors is mediated via phosphorylation by members of the family of G protein-coupled receptor kinases (GRK1-GRK7). Here we show that GRK6-deficient mice are supersensitive to the locomotor-stimulating effect of psychostimulants, including cocaine and amphetamine. In addition, these mice demonstrate an enhanced coupling of striatal D2-like dopamine receptors to G proteins and augmented locomotor response to direct dopamine agonists both in intact and in dopamine-depleted animals. The present study indicates that postsynaptic D2-like dopamine receptors are physiological targets for GRK6 and suggests that this regulatory mechanism contributes to central dopaminergic supersensitivity.

Amphetamine↗
PubMed2002

Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter.

The dopamine transporter (DAT) is a presynaptic plasma membrane protein responsible for the termination of dopaminergic neurotransmission in the central nervous system. While most studies have focused on structure/function analysis, much less information is available regarding the assembly and the trafficking of this protein. To address this problem, we performed a mutational analysis of the DAT protein, combined with biochemical, immunological, and functional approaches. In mammalian cells co-expressing differentially tagged DAT molecules, HA-tagged DAT co-purified with 6His-tagged DAT demonstrating a physical interaction between transporter proteins. Evidence for the functional oligomerization of DAT was obtained using dominant-negative mutants of DAT. Two loss-of-function mutant transporters (Y335A and D79G) that were targeted to the cell surface inhibited wild-type DAT uptake activity without affecting the membrane targeting of the wild-type transporter. Moreover, non-functional amino and carboxyl termini-truncated mutants of DAT inhibited wild-type DAT function by interfering with the normal processing of the wild-type transporter to the cell membrane. Mutations in the leucine repeat of the second transmembrane domain of the transporter could eliminate the dominant-negative effect of all these mutants. In addition, a small fragment comprising the first two transmembrane domains of DAT inhibited wild-type transporter function but not when the leucine repeat motif was mutated. Taken together, our results suggest that the assembly of DAT monomers plays a critical role in the expression and function of the transporter.

Blotting, Western↗
PubMed2002

The multiple LIM domain-containing adaptor protein Hic-5 synaptically colocalizes and interacts with the dopamine transporter.

The Na+/Cl--dependent dopamine transporter (DAT) is critical in terminating dopaminergic transmission by removing the transmitter away from the synapse. Several lines of evidence suggest that transporter-interacting proteins may play a role in DAT function and regulation. In this report, using the yeast two-hybrid system, we have identified a novel interaction between DAT and the multiple Lin-11, Isl-1, and Mec-3 (LIM) domain-containing adaptor protein Hic-5. This association involves the N-terminal portion of the intracellular tail of DAT and the LIM region of Hic-5. In human embryonic kidney 293 cells, Hic-5 colocalizes with DAT at polarized sites and reduces DAT uptake activity through a mechanism involving a decrease in the cell-surface levels of the transporter. A fragment of Hic-5 containing the LIM domains is sufficient to bind DAT but lacks the ability to inhibit transporter activity. In addition, the LIM fragment prevents the effect of the full-length Hic-5 on DAT localization and function. In the brain, Hic-5 protein is expressed in the cerebral cortex, hippocampus, hypothalamus, cerebellum, and striatum, suggesting a role for this protein in the nervous system. The association of the endogenous Hic-5 and DAT proteins was confirmed biochemically by coimmunoprecipitation from brain striatal extracts. Moreover, immunostaining of rat midbrain neurons in culture revealed a presynaptic colocalization of Hic-5 and DAT. Because Hic-5 has been shown to interact with several signaling molecules, including the nonreceptor protein tyrosine kinases focal adhesion kinase and Fyn, this raises the possibility that this adaptor protein may link DAT to intracellular signaling pathways.

Animals↗
PubMed2002

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex.

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP-VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I-K44A impaired GFP-VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I-K44A generated vesicles. A fusion protein containing the VAChT C-terminal tail precipitated the AP-2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C-terminal tail of VAChT interacts with the micro subunit of AP-2 in a di-leucine (L485A/L486A) dependent fashion. These observations suggest that the di-leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.

Adaptor Proteins, Vesicular Transport↗