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, 22 (16), 7045-54

The Multiple LIM Domain-Containing Adaptor Protein Hic-5 Synaptically Colocalizes and Interacts With the Dopamine Transporter

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The Multiple LIM Domain-Containing Adaptor Protein Hic-5 Synaptically Colocalizes and Interacts With the Dopamine Transporter

Ana M Carneiro et al. J Neurosci.

Abstract

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.

Figures

Fig. 1.
Fig. 1.
The C-terminal domain of DAT interacts with Hic-5in vitro. A, Interaction between the tail of monoamine transporters and Hic-5. pG694a yeast cells were transformed with pAS2.1 and pGAD10/Hic-5 (1), pAS2.1/CDAT and pGAD10 (2), pAS2.1/NDAT and pGAD10/Hic-5 (3), pAS2.1/CDAT and pGAD10/Hic-5 (4), pAS2.1/CNET and pGAD10/Hic-5 (5), or pAS2.1/CSERT and pGAD10/Hic-5 (6). Positive transformant yeast cells were selected in media lacking tryptophan, leucine, and adenine. CDAT, CNET, and CSERT represent the C terminus of DAT, NET, and SERT, respectively, whereas NDAT represents the N terminus of DAT. B, GST fusion protein precipitation assay using the complete C terminus of DAT or NET fused to GST. Aliquots containing GST fusion fragments were incubated with 1 mg of mouse whole-brain lysate and analyzed by Western blot using a polyclonal anti-Hic-5 antibody.
Fig. 2.
Fig. 2.
The full-length DAT interacts with Hic-5 in HEK293 cells. HEK293 cells were transfected with the HA-tagged human DAT and the myc-tagged mouse Hic-5 individually or in combination.A, Immunoprecipitations (IP) with the anti-HA antibody and Western blot detection (IB) with a polyclonal anti-Hic-5 antibody. Hic-5-myc is immunoprecipitated with the anti-HA antibody only when coexpressed with DAT-HA.B, Immunoprecipitations using the anti-myc antibody and Western blot detection with the rat anti-DAT antibody. DAT-HA is immunoprecipitated with the anti-myc antibody only when it is coexpressed with Hic-5-myc.
Fig. 3.
Fig. 3.
Domains involved in the interaction between Hic-5 and DAT. A, The multiple LIM domain-containing C terminus of Hic-5 mediates the interaction with DAT. Left panel, Schematic diagram of the myc-tagged multiple LD motif-containing N terminus (NH-myc) and myc-tagged multiple LIM domain-containing C terminus of Hic-5 (COOH-myc). Right panel, Protein lysates of cells transfected with the indicated constructs were immunoprecipitated with the anti-myc antibody. DAT was coprecipitated only when coexpressed with the C terminus of Hic-5. B, Hic-5 binds to amino acids 571–580 of DAT. Top panel, Schematic diagram of the fragments containing 10 aa pieces of the last 60 residues of the C terminus of DAT as GST fusion proteins.DATC represents the C terminus of DAT. Bottom panel, GST-fused fragments were incubated with lysates from HEK293 cells transfected with Hic-5, precipitated with GST beads, and analyzed by Western blot with a polyclonal anti-Hic-5 antibody.IB, Immunoblotting; IP, immunoprecipitation.
Fig. 4.
Fig. 4.
Hic-5 overexpression downregulates DAT uptake activity by decreasing the cell-surface levels of the transporter.A, [3H]DA uptake activity in cells transfected with DAT alone (■) or in combination with Hic-5 (▪). Each point corresponds to the mean ± SEM of three independent experiments. B, Biotinylation experiments in cells transfected with DAT alone or in combination with Hic-5. Transfected HEK293 cells were incubated with sulfo-NHS-SS-biotin, and labeled proteins were analyzed by Western blot using the rat anti-DAT antibody. Results are representative of three independent experiments.C, Averaged quantitation of Hic-5 overexpression on DAT surface density. Immunoblots from five separate biotinylation experiments were scanned densitometrically, and mean values were plotted ±SEM. Data are expressed as a percentage of control. Theasterisk indicates a statistically significant reduction in biotinylated DAT protein (p < 0.05; Student's t test).
Fig. 5.
Fig. 5.
Hic-5 colocalizes with DAT in HEK293 cells. The distribution pattern of DAT and Hic-5 when expressed individually or in combination in HEK cells is shown. Immunostaining was performed using the rat anti-DAT or the rabbit anti-Hic-5 antibodies and secondary antibodies: Texas Red anti-rat and FITC-conjugated anti-rabbit antibodies. Note that there is no increase in the intensity of the DAT signal in the presence of Hic-5. Individual cells displayed are representative of the entire population of cells from five independent experiments. Scale bars, 25 μm.
Fig. 6.
Fig. 6.
Dominant-negative effect of the multiple LIM domain-containing C-terminal half of Hic-5. A, [3H]DA uptake activity in cells transfected with the indicated constructs. The COOH-myc fragment blocks the downregulation of DAT uptake activity by Hic-5, whereas no effect on uptake activity was observed when the COOH-myc was coexpressed with DAT (∗∗∗p ≤ 0.001). B, The expression of COOH-myc abolishes the colocalization of DAT and Hic-5 in HEK293 cells. Immunostaining was performed as described using the rat anti-DAT antibody and the polyclonal anti-Hic-5 antibody. Note that the Hic-5 antibody does not detect the Hic-5 fragment containing the LIM region. Scale bar, 25 μm.
Fig. 7.
Fig. 7.
Hic-5 is expressed in neuronal and non-neuronal cells from rat midbrain cultures. Dissociated cells from rat ventral midbrains were plated on glass coverslips and immunostained with the polyclonal anti-Hic-5 antibody. A, Primary non-neuronal cells show prominent immunoreactivity at focal structures; theboxed area in A is magnified as theleft panel in D. B, D, right panel, Hic-5 antibody showed a punctated immunoreactivity in neuronal cells; the boxed area in B is magnified as the right panel in D. C, A monoclonal anti-Hic-5 antibody showed a similar staining pattern in neurons. Scale bars: A, 15 μm; C, 25 μm;D, 2.5 μm.
Fig. 8.
Fig. 8.
Localization of Hic-5 in rat midbrain culture neurons. A, Neuronal cultures were stained with the rabbit anti-Hic-5 (green) and monoclonal anti-FAK (red) antibodies. FAK and Hic-5 proteins colocalized at the cell bodies and tips of neurites (arrow).B, Neuronal cultures were stained with the anti-Hic-5 (green) and monoclonal anti-syntaxin (red) antibodies. Hic-5 and syntaxin colocalize at presynaptic sites (arrowheads). Scale bars, 15 μm.
Fig. 9.
Fig. 9.
Colocalization of endogenous Hic-5 and DAT in rat dopamine neurons. Double labeling of midbrain primary culture neurons with the anti-Hic-5 (green) and the anti-DAT (red) antibodies is shown. DAT immunoreactivity is observed as clusters along the neurite processes where it colocalizes with Hic-5 (top panels). DAT and FAK are coexpressed in dopamine neurons. Double labeling of midbrain primary culture neurons with anti-FAK (green) and anti-DAT (red) antibodies is also shown (bottom panels). Scale bars, 10 μm.
Fig. 10.
Fig. 10.
Hic-5 is expressed in several brain areas and forms a protein complex with DAT in the striatum. A, Analysis of Hic-5 and DAT proteins by Western blot in several mouse brain areas and spinal cord. Hic-5 is expressed in all brain areas examined, whereas DAT protein is detected only in the striatum.B, Coimmunoprecipitation of DAT and Hic-5 with the anti-Hic-5 antibody. DAT is coprecipitated by the Hic-5 antibody only in the striatum, where both proteins are expressed, but not in the cerebellum or skeletal muscle (Sk. muscle). DAT is not immunoprecipitated when an irrelevant antibody is used (inset). IP, Immunoprecipitation;C, cerebellum; S, striatum,M, muscle.

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