The application's long-term objective is to delineate the molecular mechanisms underlying synaptic differentiation and modulation. Regulated targeting of ionotropic glutamate receptors to the synapse is a critical step in synaptogenesis and synaptic plasticity. Recent studies indicate that stargazin and its family members are essential for glutamate receptor targeting. Two distinct mechanisms are involved: interactions between stargazin and AMPA receptor subunits are required for surface delivery of the receptors; and interactions between stargazin and PSD-95 family proteins is required for targeting AMPA receptors to the synapse. Many questions remain regarding this novel regulation of AMPA receptor trafficking: 1) In which steps of the AMPA receptor targeting processes is stargazin involved? 2) Does stargazin interact with AMPA receptors directly or through other intermediate proteins? 3) If they interact directly, where are the functional domains in stargazin that interact with AMPA receptors, transport them to the plasma membrane and target them to the synapse? 4) Where are the functional domains in AMPA receptors that interact with stargazin? I propose to address these questions by focusing on the following aims. The first aim of this application is to investigate the roles of stargazin in different stages of AMPA receptor targeting. Stargazin may potentially control AMPA receptor targeting at different stages along its transport pathway-i.e. ER -> Golgi apparatus -> membrane fusion machinery -> internalization machinery. We will selectively block some of these steps and examine its impact on AMPA receptor trafficking. The second aim is to map the functional domains in stargazin and the AMPA receptor that mediate their interactions and control their targeting. There are potentially two types of functional domains in stargazin and the AMPA receptor, one responsible for bringing AMPA receptor and stargazin into same protein complex (the interaction domains), the other for bring the whole complex to plasma membrane (the trafficking domains). We will use Co-IP and FRET methods to map the interaction domains, and surface biotinylation and electrophysiological approaches to determine the trafficking domains. A large number of brain disorders are related to abnormal brain development and plasticity. The proposed studies will advance our understanding on molecular mechanisms of synaptogenesis and plasticity, thereby providing insights in these brain diseases, and their remediation.