Insertion of tetracysteine motifs into dopamine transporter extracellular domains

Abstract

The neuronal dopamine transporter (DAT) is a major determinant of extracellular dopamine (DA) levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC) activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC) to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly) dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3)H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

Figure 1. Schematic of DAT with target sites for tetracysteine mutagenesis.

Top: DAT model. Asteriks indicate extracellular Pro-Gly residues targeted for tetracysteine mutagenesis. Bottom: DAT sequences spanning across the Pro-Gly tetracysteine target sites. Pro-Gly dipeptides are highlighted in the shaded boxes.

Figure 2. DA uptake and PKC-mediated downregulation are preserved when a tetracysteine motif is introduced into EL2, but not EL3 or EL4.

DA uptake assay. Cells were transfected as described in Methods and [³H]DA uptake was assessed 48 hours post transfection. A. Introduction of tetracysteine motifs decreases DA uptake. Data are expressed as % wildtype activity ±S.E.M. *Significantly different from wildtype (p<.01, One-way ANOVA with Tukey's multiple comparison test, n = 3), **Significantly different from EL2-CCPGCC DAT (p<.001, One-way ANOVA with Tukey's multiple comparison test, n = 3). B. EL2-CCPGCC DAT is acutely downregulated by PKC activation, whereas EL3- and EL4-CCPGCC are not. Cells were treated with either vehicle or 100 nM PMA, 30 min, 37°C and [³H]DA uptake was measured. Data are expressed as % vehicle-treated DA uptake ±S.E.M. for each construct. *Significantly different from vehicle treated (p<.05, Student's t test, n = 3), **significantly different from vehicle treated (p<.02, Student's t test, n = 3).

Figure 3. The presence of a tetracysteine in DAT EL2 does not alter DAT maturation or surface expression.

Surface biotinylation assay. Cells were transfected with the indicated DAT constructs and cell surface proteins were biotinylated and isolated from intracellular proteins as described in Methods . A. Representative immunoblot displaying surface (S) and intracellular (I) wildtype and EL2-CCPGCC DAT. Mature (90 kDa) and immature (56 kDa) species are indicated. B. Averaged data. Data are expressed as %total mature DAT on cell surface ±S.E.M. (n = 4). C. Representative immunoblot displaying surface (S) and intracellular (I) EL3-CCPGCC and EL4-CCPGCC DAT. Total lysates are displayed in the far right hand lanes. Note that no mature protein is detected. Immature species (56 kDa) are indicated.

Figure 4. Cellular distribution of tetracysteine mutant DATs.

EL2-CCPGCC DAT is robustly expressed at the cell surface and internalizes in response to PKC activation. Immunofluorescence microscopy. Cells were transfected with the indicated constructs and were fixed and stained with rat anti-DAT antibodies as described in Methods . A representative panel of deconvolved images is presented. A single plane through each cell center is shown. A. Effect of PKC activation on EL2-CCPGCC DAT. Prior to fixation, cells were treated with either vehicle or 1 µM PMA (37°C, 30 min). B. Cellular distribution of EL3-CCPGCC and EL4-CCPGCC DAT. Note the lack of either protein at the cell surface.