CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D2-like receptors

To identify proteins interacting with the C-terminal cytoplasmic tail of the dopamine D-3 receptor (D3R), we used the two-hybrid system to screen a rat brain cDNA library. We isolated three partial cDNAs encoding, respectively, for the MUPP1 multi-PDZ protein, for the N-terminal region of radixin, for GIPC and for a 160-amino acid open reading frame sharing high homology with the human CLIC6, also identified as parchorin in rabbit. In the two-hybrid system, CLIC6 was also able to interact with the D2R and D4R. The interaction between D3R and CLIC6 was confirmed by the use of a GST-D3R C-terminus fusion protein and COS cell extracts transiently expressing epitope-tagged CLIC6. In adult rat brain, CLIC6 mRNA expression was restricted to the choroid plexus, the striatal proliferative subventricular zone and the cerebellum where it is co-expressed with the D3R in the Purkinje cells of the lobules IX and X. CLIC6 mRNA was also detected in the pituitary in the posterior lobe and in cells co-expressing the D2R at the border between the intermediate and anterior lobes. In transfected HEK293 cells, D3R and CLIC6 co-localized at the plasma-membrane. No effect of CLIC6 transfection was observed on either intracellular chloride concentration or D3R/D2R-mediated response. In two-hybrid system, CLIC6 also interacted with MUPP1 and radixin but not GIPC, suggesting it could take part in a complex with D-2-like receptors, not only by direct interaction with their C-termini, but also through interactions with scaffolding proteins. (C) 2003 Elsevier B.V. All rights reserved.