The focus of my research in recent years has been the intracellular signaling that occurs in response to cell surface receptor activation. In particular, I have studied inositol 1, 4, 5-trisphosphate (InsP3) receptors, proteins that form channels in endoplasmic reticulum (ER) membranes and which, in response to InsP3 binding, mobilize Ca2+ stored within the ER. My long-term objectives are (i) to understand the role of InsP3 receptors in intracellular signaling, (ii) to define mechanisms that regulate InsP3 receptors and other signaling proteins, and (iii) to establish the biological significance of InsP3 receptor regulation. Since 1991, I have studied InsP3 receptor down-regulation, a novel adaptive response to cell surface receptor activation that rapidly reduces cellular InsP3 receptor content and, thus, the sensitivity of ER Ca2+ stores to InsP3. Recently, it has been shown that InsP3 receptor down-regulation is mediated by the ubiquitin / proteasome pathway, a crucial pathway for the degradation of many cellular proteins that is currently being considered as a therapeutic target. Ubiquitination of InsP3 receptors is the event that initiates their degradation.
The Specific Aims of the current proposal are (1) to define the site(s) of ubiquitination in InsP3 receptors using biochemical techniques and InsP3 receptor mutagenesis, (2) to identify the enzymes responsible for InsP3 receptor ubiquitination using biochemical techniques and transfection of cDNAs encoding these enzymes, (3) to define the signaling events that accelerate InsP3 receptor ubiquitination using InsP3 receptor mutagenesis, and (4) to characterize InsP3 receptor ubiquitination and down-regulation in rat brain. Accomplishment of these Aims will define the events that cause InsP3 receptor ubiquitination and will provide information on it's biological significance. The health relevance of this work is threefold. First, it will lead to a better understanding of mechanisms that cells use to adapt to extracellular stimuli; such adaptation is the basis for many physiological modifications to cell function and of tolerance to the effects of many therapeutic and recreational drugs. Second, it will establish whether muscarinic agonists used in the treatment of Alzheimer's disease cause InsP3 receptor ubiquitination and down-regulation in vivo. Third, it will help to map the ubiquitin / proteasome pathway and, thus, will provide a better understanding of drugs designed to interfere with this process.
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