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

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.

Differential Expression of Ezrin and CLP36 in the Two Layers of Syncytiotrophoblast in Rats

“…37,38) A homeodomain LIM protein, hydrogen peroxide-inducible clone-5 (Hic-5), also interacts with dopamine transporter (DAT) and reduces the membrane localization of DAT in synapses. 39) Therefore, our results suggest that CLP36 may regulate the localization of apical and basal-side membrane proteins in rat SynI.…”

Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

“…PICK1 is known to bind to the distal region of the carboxy terminus (Torres et al, 2001;Bjerggaard et al, 2004), although its role in DAT trafficking is not well defined. Hic-5 binds to the DAT carboxy terminus in the 571-580 region, closely upstream of the FREKLAYAIA domain (Carneiro et al, 2002), and a recent study revealed a likely role for Hic-5 in PKC-mediated SERT downregulation (Carneiro and Blakely, 2006). A recent study also elegantly demonstrated PKC-modulated binding of CamKII to the DAT carboxy terminus that is necessary for amphetamine-induced DA efflux via DAT (Fog et al, 2006).…”

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane

“…DAT function is also regulated by its interaction with the PDZcontaining-domain PKC-interacting protein-1 (PICK1; Ref. 15), the focal adhesion protein HIC-5 (16), receptor for activated C-kinase1 (RACK1), the SNARE component syntaxin-1A (17), parkin (18), the DA receptors D 2 and D 3 (19,20), synaptogyrin-3 (21), the GTPase Rin (22), and the G protein ␤␥ subunits (23).…”