A novel interaction between AMPA receptor binding protein and neural plakophilin-related Armadillo protein

Glutamate receptors are the major neurotransmitter receptors at excitatory synapses in the central nervous system. The AMPA-type glutamate receptor is one class of ligand-gated cation channel that mediates the majority of fast synaptic transmission at glutamatergic synapses. The proper localization of neurotransmitter receptors in the postsynaptic membrane is crucial for efficient synaptic transmission, and the synaptic targeting of glutamate receptors depends on interactions between the C-terminal cytoplasmic domains of receptor subunits and proteins within the postsynaptic density (PSD). AMPA Receptor Binding Protein (ABP) and Glutamate Receptor Interacting Protein (GRIP) are homologous multi-PDZ domain proteins that bind the C-terminal domains of the AMPAR subunits GluR2 and GluR3. PDZ domains are protein interaction modules that are important in the assembly of multi-protein complexes. In order to discover additional binding partners of ABP, the yeast two-hybrid system was used to screen a rat brain cDNA library with the first three PDZ domains of ABP as bait. Neural Plakophilin-Related Armadillo Protein (NPRAP) was identified as a potential binding partner in one of the screens and the interaction between ABP and NPRAP was confirmed using several independent methods. Full length NPRAP and both ABP and GRIP were co-immunoprecipitated from heterologous cells, and the extreme C-terminal amino acids of NPRAP constituting a class I PDZ binding motif were shown to bind PDZ domain 2 within ABP.; NPRAP is a member of the Armadillo family of proteins which function in cell adhesion and contain Armadillo repeats that act as a protein interaction unit. NPRAP recruits ABP to adherens junctions in MDCK cells and can form complexes with both ABP and E-cadherin simultaneously. In addition, ABP can bind NPRAP and GIuR2 concurrently in heterologous cells. In cultured hippocampal neurons, endogenous NPRAP displayed a synaptic distribution and colocalized with both GRIP and N-cadherin. Exogenous NPRAP expressed from a Sindbis virus vector extensively distributed to dendritic spines and colocalized with AMPARs. When coexpressed with ABP in cultured neurons, NPRAP significantly altered the distribution of ABP. Together the results suggest that ABP and NPRAP interact in brain and possibly function as scaffolding proteins that connect AMPARs and N-cadherin.