Neurofilament-M Interacts With The D-1 Dopamine Receptor To Regulate Cell Surface Expression And Desensitization

We used the yeast two-hybrid assay to identify novel proteins that interact with the D-1 dopamine receptor. The third cytoplasmic loop (residues 217-273) of the rat D-1 receptor was used as bait to identify clones encoding interacting proteins from a rat brain cDNA library. This identified two clones encoding the C terminus of rat neurofilament-M (NF-M) (residues 782-846). The NF-M clone did not interact with the third cytoplasmic loops of the rat D-2, D-3, or D-4 receptors, but showed weak interaction with that of the D-5 receptor. Coexpression of full-length NF-M with the D-1 receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D-1 receptor-mediated cAMP accumulation. NF-M had no effect on the expression of other dopamine receptor subtypes. Using a D-1 receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D-1 receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol. Interestingly, the D-1 receptors that were expressed at the cell surface in the presence of NF-M were resistant to agonist-induced desensitization. Cellular colocalization of NF-M and the D-1 receptor in the rat brain was examined by epifluorescence microscopy. These experiments showed that similar to50% of medium-sized striatal neurons expressed both proteins. Colocalization was also observed in pyramidal cells and interneurons within the frontal cortex. Similar immunohistochemical analyses using NF-M-deficient mice showed decrements in D-1 receptor expression compared with control mice. These results suggest that NF-M interacts with the D-1 receptor in vivo and may modify its expression and regulation.