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. Neto1HA was shown to co-immunoprecipitate with assembled NMDA receptors via GluN2A or GluN2B subunits; Neto1HA did not co-immunoprecipitate APP695FLAG. Co-immunoprecipitations from mammalian cells co-transfected with APP695FLAG, Neto1HA 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. Neto1HA caused a reduction in the surface expression of both NMDA receptor subtypes, but had no effect on APP695FLAG- or PSD-95αc-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 Neto1HA but deletion of the intracellular tail resulted in a loss of Neto-1HA 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.
Neto1 is shown to be a constituent of APP/NMDA receptor trafficking complexes. Since Neto1 is shown to associate with GluN2 expressed alone and, APP with GluN1-1a alone, it is proposed that trafficking complexes are formed in the endoplasmic reticulum and contribute to regulation of NMDA receptor surface expression. Alternative scenarios are depicted since it is to be determined if respective associations are direct.