AFFINITY LABELS FOR INOSITOL POLYPHOSPHATE RECEPTORS

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Brain cells respond to neurotransmitters and psychoactive drugs through a multitude of cell-specific receptor proteins. To understand how the brain integrates diverse signals in healthy and pathological conditions, attention has focused on two second messenger systems, cyclic AMP and the phosphoinositides. The second messenger D-myo-inositol 1,4,5- trisphosphate (Ins(1,4,5)P3, hereafter IP3) interacts stereospecifically with membrane receptors to promote the release of Ca2+ from intracellular stores. Receptor proteins which recognize IP3 and mediate its role in calcium release have been characterized very recently. The higher inositol polyphosphates, principally Ins(1,3,4,5)P4 (IP4) and Ins(1,2,3,4,5,6)P6 (IP6), also have specific intracellular receptors which remain to be fully characterized. Chemical affinity labels, photoaffinity labels, and immobilized affinity matrices for proteins which recognize IP3, IP4, and IP6 will be chemically synthesized. Each of the three inositol polyphosphates will be prepared with selective functionalization of the C-1 or C-2 phosphate as a phosphodiester derivative of a tethered primary amine. Four 1-O- aminoalkyl tethers will be examined which vary in chain length and rigidity. The primary amine groups will be converted to two chemically- reactive affinity labels, and three different photoactivatable derivatives will also be prepared. A novel Ferrier rearrangement route to P-1 tethered D-myo-IP4 and [3H]D-myo-IP4 will be developed. The pharmacological and biological properties of the analogs will also be examined by collaborating labs. For example, IP3 analogs will be competed with [3H]Ins(1,4,5)P3 for binding to crude or purified IP3 receptors (IP3Rs) from rat brain, rat cilia, or catfish cilia. Reconstituted receptor liposomes will be used to show calcium release when stimulated by the analogs. Competitive binding assays will be carried out with the IP4 and IP6 derivatives. Affinity chromatography with immobilized IP3, IP4, and IP6 derivatives will be used for receptor purification. The correct D-myo forms are expected to show improved specificity over the already successful racemic IP3 and IP4 ligands. The use of the affinity probes for receptor purification and sequencing of covalently-modified polypeptides in the ligand binding site of rat brain IP3R, IP4R, and IP6R and of two new olfactory IP3Rs will be conducted jointly with Stony Brook researchers. Immunogenic IPn conjugates will be synthesized, and antisera will be used to develop IPn ELISAs. Fluorescent IPn probes will be prepared for histochemical studies.