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The Journal of Neuroscience, July 15, 2002, 22(14):5920-5930

Neurofilament-M Interacts with the D₁ Dopamine Receptor to Regulate Cell Surface Expression and Desensitization

Ok-Jin Kim¹, Marjorie A. Ariano², Robert A. Lazzarini³, Michael S. Levine⁴, and David R. Sibley¹

¹ Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-1406, ² Department of Neuroscience, The Chicago Medical School, North Chicago, Illinois 60064, ³ Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, and ⁴ Mental Retardation Research Center, University of California, Los Angeles, California 90095

We used the yeast two-hybrid assay to identify novel proteins that interact with the D₁ dopamine receptor. The third cytoplasmic loop (residues 217-273) of the rat D₁ 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 D₂, D₃, or D₄ receptors, but showed weak interaction with that of the D₅ receptor. Coexpression of full-length NF-M with the D₁ receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D₁ receptor-mediated cAMP accumulation. NF-M had no effect on the expression of other dopamine receptor subtypes. Using a D₁ receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D₁ receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol. Interestingly, the D₁ 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 D₁ receptor in the rat brain was examined by epifluorescence microscopy. These experiments showed that ~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 D₁ receptor expression compared with control mice. These results suggest that NF-M interacts with the D₁ receptor in vivo and may modify its expression and regulation.

Key words: D₁ receptor; neurofilament-M; yeast two-hybrid; interaction; desensitization; colocalization

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Copyright © 2002 Society for Neuroscience  0270-6474/02/22145920-11$05.00/0

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