The proposed study is designed to examine the G protein and its downstream effectors activated by the metabotropic glutamate receptors (mGluR6), which are present on the dendrites of the rod and ON cone bipolar cells. More specifically, the investigator intends to: (1) identify mRNAs in retina enriched for bipolar cells to initially identify the GC (GC) and phosphodiesterase (PDE) with RT-PCR; (2) use in situ hybridization to identify the subset of isoforms and splice-variants that localize to ON bipolar cells, and carry out double immunostaining with confocal and electron microscopy to identify the subset of isoforms and proteins that co-localize postsynaptically with mGluR6; (3) test the function of the selected proteins in """"""""knock-out"""""""" mice by examining the ERG b-wave; (4) test for the binding between mGluR6 and Go and between Go and selected protein effectors by using the two-hybrid interaction trap, GTS pulldown, and co-immunoprecipitation; and (5) identify Go's effector using a constitutively-active Go-alpha as bait in two-hybrid screening, which will be performed in parallel with aims 1-4 because it may identify novel effectors. The molecular approach outlined may characterize multiple isoforms and interactions irrelevant to the mGluR6 cascade, but they may be relevant to other aspects of retinal signaling. To determine the relevant isoforms, the study is designed to examine the broad picture with a highly specific microscopic assay and then to test the ultimate candidate with a simple physiological assay.
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