Role of cellular prion protein in trafficking of GluA2 -containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the neuronal lipid raft

Abstract

GluA2-containing α−amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate (AMPA) receptors are distributed in both lipid raft and non-raft compartments of neuronal plasma membrane (PM). However, the underlying molecular mechanisms involved in differential distribution and trafficking of AMPA receptors as well as their functional differences remain poorly understood. Cellular prion protein (PrPc) is a ubiquitously expressed Glycosyl-phosphatidylinositol (GPI)-anchored lipid raft localized protein that is almost entirely exposed to the extracellular compartment. We detected an interaction between PrPc and GluA2 in mouse hippocampi and cortices. Lipid raft isolation using brain tissues dissected from wild type and PrPc-knockout mice revealed that GluA2 was almost abrogated in the lipid rafts of knockout mice. We therefore hypothesize that an interaction between PrPc and the Ca2+ impermeable GluA2 subunit of AMPA receptors is responsible for trafficking of GluA2-containing AMPA receptors to neuronal lipid rafts. Co-immunoprecipitation (Co-IP), glutathione S-transferase (GST) assay, immunofluorescence microscopy, molecular docking stimulation and electron microscopy experiments further confirmed the interaction between GluA2 and PrPc. Subcellular fractionation and isolation experiments revealed that PrPc-GluA2 interaction occurs co-translationally at the microsomal level and is necessary for GluA2-containing AMPA receptor trafficking to the lipid rafts. Using a variety of cellular molecular techniques, we discovered for the first time that the N-terminal polybasic region of PrPc interacts with N-terminal domain of GluA2 (Y142-K172). Based on these two interacting domains, two TAT-fused interfering peptides (and scrambled controls) were designed and synthesized to disrupt the PrPc-GluA2 interaction and to study their biochemical, physiological and behavioral consequences. Disrupting PrPc-GluA2 interaction resulted in an alteration from Ca2+ impermeable AMPA receptor to Ca2+ permeable forms in the cellular membrane. Moreover, we discovered that PrPc-GluA2 interaction modulates the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2)-dependent GluA2 Q/R site editing. Disrupting this interaction resulted in increased adenosine level and decrease in Adenosine A1 receptor (A1R) expression. Any disruption of this interaction triggered GluA2-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) interaction in non-raft compartments and then induced subunit endocytosis to nucleus. These observations were also confirmed in PrPc-knockout (PrPc-KO) mouse hippocampus when compared with the wild type mouse. We also noticed a significant behavioral change in wild type mice after intraperitoneal injection of the TAT-fused interfering/disrupting peptides. Together, these results showed a previously unknown functional interaction between PrPc and GluA2 subunit of AMPA receptor with physiological and behavioral consequences.