Neurofilament-M interacts with the D1 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 approximately 50% 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.