Phosphoinositide polyphosphates (PIPns) are biosynthesized via the interplay of phosphatases and kinases and constitute key signaling molecules in cellular communication. The isolation, identification, and characterization of proteins that bind and metabolize PIPns have been realized using chemically-synthesized affinity probes, including photoaffinity labels, solid-support immobilized ligands, biotinylated probes, antigenic ligands, and fluorescently-labeled probes. We propose to expand the repertoire and uses of PIPE affinity probes by developing molecular sensors for intracellular events in the phosphoinositide signaling pathway. Thus, we will address six areas: (i) synthesis of affinity probes for PIPn-binding domain (PIPnBD) studies; (ii) optimization of cell permeant PIPns, (iii) development of novel fluorescence and antibody- based assays for measuring PIPn kinase and phosphatase activity (iv) discovery of new PIPn-binding proteins using classical biochemistry and in vitro expression cloning, (v) use of peptide libraries to identify novel peptides that act as PIPn selective mimics, and (vi) selected biophysical studies of novel PIPnBD-PIPn complexes. At The University of Utah, we will focus on preparation of affinity probes, development of assays, and biophysical study of selected complexes. At Utah State University, we will explore uses of antibody and affinity probes in cell biology and protein discovery. With collaborators elsewhere, new PIPn binding proteins will be identified and characterized, and uses of unique reagents will be developed.
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