Cellular Prion Protein Engages the <i>N</i> -Methyl- <scp>d</scp> -Aspartate Receptor through N- and C-Terminal Domains

Nonpathogenic cellular prion protein (PrP^C) is expressed by neurons and other cells, regulating neurite outgrowth, cell survival, myelin maintenance, and immunity, yet the PrP^C-protein interaction network and signaling pathways that underlie PrP^C function remain incompletely understood. PrP^C is glycophosphatidylinositol-anchored in lipid rafts and reportedly interacts with membrane-bound proteins at the cell surface, including the <i>N</i>-methyl-d-aspartate receptor (NMDA-R), triggering cell-signaling responses. PrP^C 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 PrP^C 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 PrP^C that bind to the NMDA-R. One site was located in the N-terminal disordered region of PrP^C. This site is in a lysine-rich segment that incorporates the sequence previously identified as the biologically active PrP^C-derived peptide, P3. The second site was located in the C-terminal structured region of PrP^C within the α1 helix and β1 strand. PrP^C bound GluN1-GluN2B complexes as well as GluN1 in isolation. Notably, the N-linked glycans in PrP^C inhibited binding to GluN1. Mutation of PrP^C to incorporate a third glycosylation site further inhibited binding to GluN1. These results demonstrate binding sites in PrP^C that may mediate interaction with the NMDA-R when PrP^C is membrane-anchored to the cell of origin, released in extracellular vesicles, or shed from the cell surface by proteases.