Phosphoinositide Signaling Regulates the Surface Localization of the δ2 Ionotropic Glutamate Receptor

Abstract

The δ2 glutamate receptor (GluD2) is a member of the ionotropic glutamate receptor family. Since GluD2 remains an orphan receptor, it has not been shown to conduct ions. It is highly expressed in the parallel fiber-Purkinje cell (PF-PC) synapse and its role in cerebellar physiology is increasingly appreciated. GluD2 has been linked to the induction of cerebellar long-term depression (LTD) and presynaptic terminal differentiation. A single point mutation in the second transmembrane domain (A654T), named Lurcher (GluD2Lc), confers constitutive activity to the receptor.We have used the Lurcher mutant as a model to examine whether phosphoinositides regulate GluD2. We show that decreasing PIP2 levels in the membrane, through Gq-coupled receptor activation or pretreatment with a PI4K inhibitor, potentiates GluD2Lc currents. Conversely, increasing PIP2 levels by co-expressing PIP5K leads to decreased GluD2Lc currents. On the other hand, utilizing PI3K inhibitors wortmannin or LY294002, and presumably decreasing PIP3 levels, reduces GluD2Lc currents, while co-expression of PI3K leads to potentiation of GluD2Lc currents.A chemiluminescence-based assay that quantifies surface localization of the GluD2 and GluD2Lc receptors, showed that manipulations of the membrane phosphoinositide levels evoke changes in the cell surface localization of both wild-type and mutant receptors. These changes in surface localization of the receptor correspond to the effects we have observed by monitoring GluD2Lc currents, suggesting that current measurements from this mutant receptor serve as a good reporter for the localization of the wild-type δ2 receptor. These results are consistent with the interpretation that increased PIP2 levels decrease, while increased PIP3 levels increase localization of the receptor at the cell surface. Signals that affect the levels of these phosphoinositides simultaneously are likely to regulate the surface localization of GluD2 based on the net change of the two opposing effects.