Use of Bioluminescence Resonance Energy Transfer (Bret) for Studies of Protein-Protein Interactions Between the GLUD2 Receptor and Intracellular Interaction Partners

Abstract

The GluD2 receptor is a member of the ionotropic glutamate receptor (iGluR) superfamily and is expressed in cerebellar Purkinje cells (PCs). GluD2 receptors recognize the neurotransmitters D-serine, but lack excitatory ligand-gated ion channel activity. Recently, D-serine activity mediated via GluD2 has been shown to modulate cerebellar long-term-depression (LTD). The intracellular C-terminal domain (CTD) of GluD2 is required for D-serine regulation. A working hypothesis for this regulation is that D-serine binding to the extracellular ligand-binding domain is translated to intracellular rearrangement of the CTD and hereby potentially affects protein-protein interactions (PPIs) with intracellular scaffolding proteins or signaling enzymes involved in expression of cerebellar LTD. Various interacting partners of the GluD2 CTD have been identified; including the PDZ-domain containing scaffolding protein S-SCAM and Delphilin and the tyrosine kinase PTPMEG. Furthermore, the CTD contains multiple phosphorylation sites, which may regulate PPIs.In the present study, our aim is to study the potential regulation of GluD2 PPIs by phosphorylation and D-serine. For this purpose, we have established a bioluminescence resonance energy transfer (BRET) assay that enables measurement of GluD2 PPIs in intact cells by inserting the resonance energy donor Renilla luciferase 8 (Rluc8) at various positions in full-length GluD2 or the isolated CTD and acceptor molecule green fluorescence protein 2 (GFP2) to known GluD2 interaction partners.For PSD95, Delphilin, AP-4 and S-SCAM, it was possible to detect robust BRET between GluD2 and GFP2-tagged full-length interacting partners as well as truncated versions containing the isolated PDZ domain. Hereby, an experimental platform has been created for studying GluD2 PPIs in intact cells. Results from ongoing characterizations of the influence of CTD phosphorylation and D-serine ligand-binding on PPIs will be presented.