Neto1 associates with the NMDA receptor/amyloid precursor protein complex

Neuropilin tolloid‐like 1 (Neto1), is a CUB domain‐containing transmembrane protein that was recently identified as a novel component of the NMDA receptor complex. Here, we have investigated the possible association of Neto1 with the amyloid precursor protein (APP)695/GluN1/GluN2A and APP695/GluN1/GluN2B NMDA receptor trafficking complexes that we have previously identified. Neto1^HA was shown to co‐immunoprecipitate with assembled NMDA receptors via GluN2A or GluN2B subunits; Neto1^HA did not co‐immunoprecipitate APP695^FLAG. Co‐immunoprecipitations from mammalian cells co‐transfected with APP695^FLAG, Neto1^HA and GluN1/GluN2A or GluN1/GluN2B revealed that all four proteins co‐exist within one macromolecular complex. Immunoprecipitations from native brain tissue similarly revealed the existence of a GluN1/GluN2A or GluN2B/APP/Neto1 complex. Neto1^HA caused a reduction in the surface expression of both NMDA receptor subtypes, but had no effect on APP695^FLAG‐ or PSD‐95α^c‐Myc enhanced surface receptor expression. The Neto1 binding domain of GluN2A was mapped using GluN1/GluN2A chimeras and GluN2A truncation constructs. The extracellular GluN2A domain does not contribute to association with Neto1^HA but deletion of the intracellular tail resulted in a loss of Neto‐1^HA co‐immunoprecipitation which was paralleled by a loss of association between GluN2A and SAP102. Thus, Neto1 is concluded to be a component of APP/NMDA receptor trafficking complexes.