In the brain, neurons respond to many neurotransmitters and psychoactive compounds through specific surface receptors which are coupled to the production of second messengers. To understand how the brain integrates diverse signals in normal and pathological conditions, studies have focused on elucidating second messenger action, mechanisms of regulation, and interactions. Two systems,the cyclic nucleotides and the phosphoinositides (PI) have received the most attention. One messenger of the (PI) system, D-myo-inositol 1,4,5-triphosphate (Ins(1,4,5)P3) (IP3), interacts with membrane receptors to stimulate the release of calcium from intracellular stores. Recently, an IP3 receptor which mediates calcium release was purified, reconstituted and cloned. Higher inositol polyphosphates (IPs), including IP4, IP5 and IP6 are also synthesized in mammalian neurons, and increases of several IP's are elicited by transmitter action. These IPs function independently and synergistically with IP3 to modulate calcium levels and other ion channels. In initial studies, isolation of several inositol polyphosphate receptor binding proteins from rat brain was demonstrated. Still, neither the complete cellular functions nor the intracellular target receptors for inositol polyphosphates have been fully characterized. Using a recently developed strategy, affinity matrices and photoaffinity labels have been used to label and isolate inositol polyphosphate receptor proteins. In the current proposal, the IP receptors previously identified will be isolated for biochemical characterization and localization, generation of specific antibodies, functional reconstitution studies, and receptor peptide sequencing to begin cloning. The regional and subcellular localization of receptors will be determined by purification from isolated regions using IP4-affinity resin, photoaffinity labeling with [125I]ASAIP4, and antibody localization. Purified receptor proteins will be reconstituted into lipid vesicles and planar lipid bilayers to assess the physiological activities of these receptors. Finally, peptides will be generated from purified receptor protein so that partial amino acid sequences can be obtained. From the sequence of these peptides, oligonucleotide probes will be designed to initiate molecular cloning studies. The reconstitution and cloning of these inositol polyphosphate receptors should provide significant insight into the physiological function of these proteins. In addition, these receptor proteins, antibodies, and receptor cDNAs should provide useful probes to study the regulation of these proteins in normal neuronal signaling, in development, and in the pathological conditions produced in human neuronal diseases.
