The phosphoinositide system is a major intracellular second messenger system in the brain which may be especially relevant to Psychiatry since it is believed to be a site of action of Lithium in Manic Depressive Illness. Neurotransmitter or hormonal stimulation of cell surface receptors generates inositol (1,4,5) trisphosphate (IP3), which binds to a receptor on the endoplasmic reticulum (the 1P3 receptor or IP3R), causing the release of intracellular calcium. The primary sequence of the rat and mouse IP3R have been described, identifying the IP3R as a large (313 kD) glycoprotein with most likely 8 membrane spanning domains. We have been engaged in the molecular cloning of cDNAs for the human IP3R, and we and others have recently discovered in the mouse that the IP3R is a member of a family of genes expressed in the brain and the periphery, with the original member of the family termed Type I and the others II, III and IV. In the proposed studies we will further characterize the IP3R and its gene family in mouse and human.
In Specific Aim #1 we will complete molecular cloning of the human IP3R-1. We have discovered that there are variants of the receptor formed by alternative splicing in a neuron specific manner. This alternative splicing appears to alter phosphorylation of the receptor by cyclic AMP dependent protein kinase, which is important for regulation. We will study this regulation using receptors expressed in mammalian cells and site directed mutagenesis.
In Specific Aim #2 we will complete the cloning and characterization of new IP3 receptor related cDNAs. We will study their developmental and tissue distribution using RNA analysis techniques and in situ hybridization in the mouse. In a collaborative study we will determine the single channel conductances of these new receptors. We will develop specific antisera to the individual receptors to study the subunit composition of the receptor and the subcellular distribution of the different receptors. Finally, in Specific Aim #3 we will pursue cloning of the genomic DNA for the human IP3R and its related family of genes. We will determine the intron and exon structure of the alternatively spliced regulatory region of IP3R-I and the chromosomal localization of the receptors. In future studies, we will find polymorphic simple sequence repeats for linkage studies. These studies should shed light on basic mechanisms of brain second messenger systems, and help clarify the action of Lithium in Manic Depressive Illness.
| Nucifora Jr, F C; Li, S H; Danoff, S et al. (1995) Molecular cloning of a cDNA for the human inositol 1,4,5-trisphosphate receptor type 1, and the identification of a third alternatively spliced variant. Brain Res Mol Brain Res 32:291-6 |
