Inositol 1,4,5-trisphosphate (InsP/3) constitutes an intracellular second messenger that mediates the release of Ca2+ from intracellular stores of the endoplasmic reticulum. The inositol phosphate pathway appears to be involved with the mental disorder, manic depressive psychosis where InsP/3 levels are greatly reduced after lithium treatment. InsP/3 acts by interacting with a family of highly conserved receptors localized to the endoplasmic reticulum (ER) which contain an intrinsic calcium release channel. The InsP/3 receptor family shares both structural and sequence similarities with the ryanodine receptor, the primary Ca2+ release channel of the sarcoplasmic reticulum (SR) in cardiac and skeletal muscle. The InsP/3 receptor family also appears to be involved in cardiac calcium dynamics. The type 1 InsP/3 receptor is localized to regions at or near the intercalated disc of cardiomyocytes. The current proposal describes experiments that will define and characterize the elements which confer the receptors specialized structure and function. These experiments will be pursued using a combination of cloned receptors, and cell lines expressing multiple homologues of the InsP/3 receptor.
The specific aims of this proposal are: 1.) What are the topological orientations of the multiple membrane spanning regions in the endoplasmic reticulum? The location and orientation of the receptors multiple membrane spanning regions will be determined using a series of receptor chimeras and an in vitro transcription/translation/proteolysis protection assay. Together with the data from specific aim 2 a model defining the receptors calcium channel will be proposed. 2.) What sequences or domains are responsible for the subunit assembly of the receptor into the tetrameric state? The sequences involved in receptor subunit assembly into the tetrameric structure will be elucidated by expressing mutagenized receptor constructs into COS cells and assessing subunit assembly by sucrose gradient sedimentation and chemical cross linking. 3.) What are the subcellular localizations and expression patterns for the InsP/3 receptor in cells which express multiple forms of the receptor? Immunofluorescence microscopy and quantitative polymerase chain reaction (PCR) analysis will be performed on the PC12 cell line asking whether the different forms of receptor expressed within the same cell are differentially targeted and/or expressed. If distinct receptor homologues are differentially targeted to different regions of the endoplasmic reticulum, what are the sequences involved? These sequences can be identified using a systematic exchange of transmembrane domains between receptor homologues. Do cell lines expressing multiple types of the receptor form homotetramers or heterotetrameric assemblies? 4.) What are the properties of calcium release from intracellular stores of cells expressing transfected InsP/3 receptor cDNA's? Tissue cultured cell lines transfected with full length and mutagenized receptor expression plasmids will be assayed for InsP/3 mediated calcium release in experiments designed to examine receptor/channel function.
