Brain cells respond to neurotransmitters and psychoactive drugs through cell-specific receptor proteins. Studies on the integration of diverse signals in healthy and pathological conditions have focused on nitric oxide, cyclic nucleotide, and phosphoinositide pathways. The second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, or IP3) binds stereospecifically to domains of membrane receptors to cause aggregation and opening of calcium channels. Several IP3 receptors (IP3R) have been cloned and expressed; establishing functional domains in homologous proteins is ongoing. Receptors and functions have been identified for other inositol polyphosphates and phosphoinostides; to date, however, the structures of proteins and binding domains specifically recognizing IP4, IP5, IP6, and the diphosphates IP7 and lP8 remain undetermined. Photoaffinity labels and immobilized affinity matrices will be developed in this proposal for proteins that bind specific inositol polyphosphates (IPn) from IP3 to IP7. These affinity probes will be prepared either from D-glucose or myo-inositol by stereospecific chemical synthesis of analogs selectively functionalized with a phosphodiester bearing an aminopropyl tether. For binding assays and visualization in electrophoretic gels, high specific activity tritium labels will be incorporated. Furthermore, synthesis of phosphatidyl inositol trisphosphate (PtdIns(3,4,5)P3, or PIP3) analogs, will be pursued to provide radioligands for the study of this new ligand. In addition, new synthetic routes to the inositol polyphosphate diphosphates such as IP7 will be developed. For each ligand prepared, the covalently-modified binding sites will be determined by protein digestion and amino acid sequencing. The structural biology of IPn - binding domain interactions will be explored using tools of molecular biology and biophysical chemistry. The phosphoinositide pathway has a pervasive impact in cellular biology, including calcium signaling, hormone signal transduction, odor and taste perception, neurotransmitter action, energy metabolism, protein phosphorylation, and protein trafficking. The affinity reagents and the binding domain data obtained from these reagents will be important in defining the mechanisms of these ubiquitous and varied molecular switches.
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