DESCRIPTION: The synaptic cytoskeleton plays a critical role in the formation and maintenance of synapses in the central nervous system. Recent studies have identified a new protein motif called a PDZ domain which may be important in the proper targeting of proteins to cell-cell junctions. PDZ domains within cytoskeleton associated proteins mediate the interaction of the cytoskeleton with the C-termini of a variety of membrane proteins. The PDZ domains of the SAP/PSD family of proteins have recently been implicated in the synaptic targeting of NMDA receptors and K+-channels in neurons. The investigator has recently identified a novel PDZ domain containing protein, GRIP (Glutamate Receptor Interacting Protein), that specifically interacts with the C-termini of the GluR2 and GluR3 subunits of AMPA receptors. GRIP appears to link AMPA receptors to the synaptic cytoskeleton and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain. In this research proposal it is planned to further characterize the structure and function of GRIP and related proteins and determine their role in the synaptic targeting of AMPA receptors. Specifically, the regional and subcellular distribution of GRIP will be examined using immunocytochemical and in-situ hybridization techniques. The structural domains of GRIP and GluR2 that are involved in their interaction will be determined by a combination of techniques, including the yeast two hybrid system, fusion protein binding studies and cell transfection techniques. In addition, other proteins (GRASPs -GRIP associated Proteins) that interact with GRIP and form a PDZ domain-based cytoskeleton at synapses will be identified. The functional effect of GRIP on the AMPA receptor ion channel will also be examined using patch clamp techniques. Dominant negative constructs of GRIP and GluR2 will be used to disrupt the interaction of AMPA receptors with GRIP in neurons in culture. Finally, gene targeting techniques will be used to knockout the GRIP gene to determine its role in the mechanisms underlying synaptic transmission in the brain. Such knowledge is essential for understanding the function of the brain