Regulation of AMPA receptors through interacting proteins

The synapse is a specialized area of cell-cell contact that facilitates the transmission of electrical signals between neurons. It contains a presynaptic terminal where an action potential triggers the release of neurotransmitters that diffuse across the synaptic cleft and open ligand-gated ion-channels on the postsynaptic membrane. In the mammalian central nervous system, glutamate is the major neurotransmitter and mediates the majority of excitatory synaptic transmission. Glutamate binds to glutamate receptors, which are ligand-gated ion-channels and can be divided into three subtypes based on their physiological and pharmacological properties: the NMDA, AMPA and kainate receptors. This thesis is primarily concerned with AMPA receptors, which are formed from various combinations of four different subunits, GluR1–4. The synaptic targeting, clustering and immobilization of these ligand-gated receptor ion-channels play an important role in synapse formation, synaptic transmission and plasticity. Regulation of the receptor is mainly mediated via phosphorylation or associated proteins. Presented in this thesis are works on two interacting proteins of GluR1 subunit of the AMPA receptor, protein 4.1 and SAP97, and their roles in the regulation of AMPA receptor targeting.; Chapter one of this thesis focuses on the identification and characterization of the association of GluR1 with two members of the protein 4.1 family, 4.1G and 4.1N, and the role 4.1N may play in regulating GluR1 surface expression by linking the receptor to the actin cytoskeleton. 4.1G and 4.1N proteins are homologues of the erythrocyte membrane cytoskeletal protein 4.1 that have been shown to be important for maintaining the cell membrane integrity and surface stability of membrane receptors. Using the yeast two-hybrid system and a heterologous cell system we demonstrated that both 4.1G and 4.1N bind to a membrane proximal region of GluR1 C-terminus, and that a region within the carboxy-terminal domain of 4.1G or 4.1N is sufficient to mediate the interaction. We also found that 4.1N can associate with GluR1 in vivo and colocalizes with AMPA receptors at excitatory synapses. Disruption of the interaction of GluR1 with 4.1N, or disruption of actin filaments, decreased the surface expression of GluR1 in heterologous cells. Moreover, disruption of actin filaments in cultured cortical neurons dramatically reduced the level of surface AMPA receptors. These results suggest that protein 4.1N may link AMPA receptors to the actin cytoskeleton and therefore stabilize them on the cell surface.; Chapter two of this thesis investigates the role of CaMKII and SAP97, a PDZ domain-containing protein in the regulation of surface expression of GluR1. It was found that co-expression of a constitutively active form of CaMKII can increase GluR1 surface expression in heterologous cells. Interestingly mutation of the CaMKII phosphorylation site on GluR1 C-terminus did not eliminate the effect of CaMKII. However, mutation of the C-terminal PDZ ligand in GluR1 increased surface expression of the receptor and eliminated the regulation of GluR1 surface expression by CaMKII co-expression. To investigate the role of PDZ domain-containing proteins in this regulation, SAP97, a PDZ domain-containing protein, that was previously reported to bind to the PDZ ligand in GluR1, was co-expressed with GluR1. SAP97 overexpression attenuated GluR1 surface expression and overexpression of CaMKII relieved the inhibition of receptor surface expression by SAP97. These results indicate that SAP97 and CaMKII may cooperate in the regulation of AMPA receptor surface expression.