Cellular Prion Protein Engages the N‑Methyl‑d‑Aspartate Receptor through N- and C‑Terminal Domains

Nonpathogenic cellular prion protein (PrPC) is expressed by neurons and other cells, regulating neurite outgrowth, cell survival, myelin maintenance, and immunity, yet the PrPC-protein interaction network and signaling pathways that underlie PrPC function remain incompletely understood. PrPC is glycophosphatidylinositol-anchored in lipid rafts and reportedly interacts with membrane-bound proteins at the cell surface, including the N-methyl-d-aspartate receptor (NMDA-R), triggering cell-signaling responses. PrPC may also be glycosylphosphatidylinositol (GPI)-anchored in extracellular vesicles or released from cells by proteases to interact with plasma membrane proteins in target cells. To identify PrPC binding sites for the NMDA-R in an unbiased manner, we generated extracts from HEK293T cells transfected with the GluN1 and GluN2B NMDA-R subunits and performed a targeted series of co-immunoprecipitation experiments, peptide arrays, and protein structure analyses. We identified two sites in PrPC that bind to the NMDA-R. One site was located in the N-terminal disordered region of PrPC. This site is in a lysine-rich segment that incorporates the sequence previously identified as the biologically active PrPC-derived peptide, P3. The second site was located in the C-terminal structured region of PrPC within the α1 helix and β1 strand. PrPC bound GluN1–GluN2B complexes as well as GluN1 in isolation. Notably, the N-linked glycans in PrPC inhibited binding to GluN1. Mutation of PrPC to incorporate a third glycosylation site further inhibited binding to GluN1. These results demonstrate binding sites in PrPC that may mediate interaction with the NMDA-R when PrPC is membrane-anchored to the cell of origin, released in extracellular vesicles, or shed from the cell surface by proteases.