Attention deficit/hyperactivity disorder-derived coding variation in the dopamine transporter disrupts microdomain targeting and trafficking regulation.

Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain-imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine- and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at presynaptic and postsynaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a hDAT (human DAT) coding variant, R615C, located in the distal C terminus of the transporter, a region previously implicated in constitutive and regulated transporter trafficking. Here, we demonstrate that, whereas wild-type DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively and demonstrate insensitivity to the endocytic effects of AMPH and PKC (protein kinase C) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered CaMKII (calcium/calmodulin-dependent protein kinase II) and flotillin-1 interactions. Using C-terminal peptides derived from wild-type DAT and the R615C variant, we establish that the DAT 615C C terminus can act dominantly to preclude AMPH regulation of wild-type DAT. Mutagenesis of DAT C-terminal sequences suggests that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.

Dopamine receptors in human adipocytes: expression and functions.

INTRODUCTION: Dopamine (DA) binds to five receptors (DAR), classified by their ability to increase (D1R-like) or decrease (D2R-like) cAMP. In humans, most DA circulates as dopamine sulfate (DA-S), which can be de-conjugated to bioactive DA by arylsulfatase A (ARSA). The objective was to examine expression of DAR and ARSA in human adipose tissue and determine whether DA regulates prolactin (PRL) and adipokine expression and release. METHODS: DAR were analyzed by RT-PCR and Western blotting in explants, primary adipocytes and two human adipocyte cell lines, LS14 and SW872. ARSA expression and activity were determined by qPCR and enzymatic assay. PRL expression and release were determined by luciferase reporter and Nb2 bioassay. Analysis of cAMP, cGMP, leptin, adiponectin and interleukin 6 (IL-6) was done by ELISA. Activation of MAPK and PI3 kinase/Akt was determined by Western blotting. RESULTS: DAR are variably expressed at the mRNA and protein levels in adipose tissue and adipocytes during adipogenesis. ARSA activity in adipocyte increases after differentiation. DA at nM concentrations suppresses cAMP, stimulates cGMP, and activates MAPK in adipocytes. Acting via D2R-like receptors, DA and DA-S inhibit PRL gene expression and release. Acting via D1R/D5R receptors, DA suppresses leptin and stimulates adiponectin and IL-6 release. CONCLUSIONS: This is the first report that human adipocytes express functional DAR and ARSA, suggesting a regulatory role for peripheral DA in adipose functions. We speculate that the propensity of some DAR-activating antipsychotics to increase weight and alter metabolic homeostasis is due, in part, to their direct action on adipose tissue.