Neurotransmitter receptor targeting to postsynaptic and presynaptic sites requires correct sorting, insertion/retention at the plasma membrane, and clustering. To date, the molecular mechanisms underlying targeting of receptors to dendrites vs. axons remain largely unknown. The underlying hypothesis of this proposal is that neurotransmitter receptors may interact via targeting signals with adaptor proteins mediating recruitment to specialized cargo vesicles or, alternatively, with scaffolding proteins, which may cause compartmentalized retention and/or stabilization. The experimental approach is to identify proteins that bind to targeting signals contained within the different splice variants of the metabotropic glutamate receptor 1 (mGluR1). Preliminary studies have identified a signal sequence in the intracellular tail of the mGluRlb isoform required for exclusion from distal dendrites and for axonal targeting. This signal is also required for apical targeting in Madin-Darby canine kidney (MDCK) cells. Aim 1 will identify proteins involved in mGluRlb targeting using the yeast two-hybrid system. A brain cDNA library will be screened with the intracellular tail of mGluRlb as bait. Binding partners will be tested for interaction with a mutant bait lacking the targeting signal. Preliminary studies, have identified a novel protein, termed mGRAB, attesting to the feasibility of this approach. In Aim 2, a novel technology, Tandem Affinity Purification (TAP), will be used to isolate native complexes of proteins interacting with mGluRlb in MDCK cells. The receptor-interacting protein complex will be analyzed by mass spectrometry to identify individual proteins. Preliminary results have identified three proteins, alpha-actinin-4, myosin IC and vimentin as potential components of the mGluRlb associated complex. Understanding the molecular mechanisms underlying regulation of mGluR trafficking has important implications for the understanding of the molecular mechanisms underlying synaptic activity in normal and pathological conditions. Moreover, mGluRs are an important therapeutic target for the treatment of neurological disorders, such as Parkinson's disease, and of psychotic symptoms and dysfunctions of cognitive processes associated with schizophrenia. This application is ideally suited to the R21 format in that it employs high-risk experiments based on new technologies that may lead to major advances in this field.