Mutation in hotfoot-4J mice results in retention of δ2 glutamate receptors in ER

The orphan glutamate receptor δ2 is selectively expressed in Purkinje cells and plays a critical role in cerebellar function. Recently, the ataxia of hotfoot-4J (ho-4J) mice was shown to be caused by a 170-amino acid deletion in the N-terminal region of δ2 receptors. To understand δ2 receptor function, we characterized these mutant receptors (δ2^ho) in Purkinje cells. Immunohistochemical staining showed that δ2^ho receptors of the ho-4J homozygotes were abundantly expressed but localized to the Purkinje cell soma; in wild-type mice, δ2 receptors were predominantly present at distal dendrites of Purkinje cells. In addition, δ2^ho receptors of the ho-4J mice were sensitive to endoglycosidase H, a finding suggesting that δ2^ho receptors were not transported beyond the endoplasmic reticulum (ER) or cis-Golgi apparatus. To gain further insights into the mechanisms of this phenomenon, we characterized δ2^ho receptors in transfected HEK293 cells. δ2^ho receptors expressed in HEK293 cells were also sensitive to endoglycosidase H. Immunohistochemical staining showed that δ2^ho receptors colocalized with proteins retained in the ER. Furthermore, δ2^ho receptors were not labelled by membrane-impermeable biotinylation reagents. Coimmunoprecipitation assays showed that the intermolecular interaction of δ2^ho receptors was significantly weaker than those of wild-type δ2 receptors, a finding suggesting that the ho-4J region is involved in oligomerization of δ2 receptors. Thus, δ2^ho receptors were retained in the ER, probably by the quality control mechanism that detects unstable oligomers. We conclude that the absence of δ2 receptors on the cell surface by failed transport from the ER of Purkinje cells causes ataxia.