Molecular Characterization of Novel Binding Interactions for Major Histocompatibility Complex Class I Immune Proteins in Mouse Hippocampus

Abstract

Major Histocompatibility Complex Class I (MHCI) molecules play critical roles in the adaptive immune response. Although the central nervous system is an immune- privileged site, MHCI proteins are expressed in the mammalian brain and, unexpectedly, are required for the normal establishment, function, and plasticity of glutamatergic synapses. In fact, MHCI is one of the few known inhibitors of NMDAR-mediated transmission in hippocampus, but the mechanisms underlying this association are unknown. We sought to characterize the binding interactions of MHCI in the mouse hippocampus in order to elucidate how MHCI regulates NMDAR-mediated transmission. The MHCI cytoplasmic domain has been largely dismissed as inessential for MHCI activity; however, bioinformatics analyses conducted by our lab revealed that the MHCI cytoplasmic tail contains highly conserved amino acids embedded within cryptic PDZ binding motifs, hinting at undiscovered functionality. Preliminary pull-down experiments suggested that two post-synaptic proteins, PSD-95 and spinophilin, can bind to the MHCI cytoplasmic tail. Co-immunoprecipitation experiments corroborated that MHCI binds spinophilin in hippocampal lysate, supporting spinophilin as a candidate mediator of MHCI neuronal function. Site-directed mutagenesis of highly conserved residues within the MHCI cytoplasmic tail suggested that the “GDYA” motif may be important for MHCI binding to spinophilin, while “TSDL” was identified as a putative interaction site for PSD-95. Finally, the MHCI cytoplasmic domain was investigated for its ability to outcompete full-length MHCI for binding to hippocampal protein mediators. Elucidating the mechanisms by which MHCI regulates NMDAR activity will provide new insight into the etiologies of synaptic disorders such as autism and schizophrenia.