BIOSYNTHESIS AND DEGRADATION OF IP3 RECEPTORS

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Ca2+ mobilization from internal stores is important in the acute regulation of cell metabolism and in regulating key steps in cell growth, cell division and programmed cell death. Binding of the intracellular messenger D-myo-Inositol 1,4,5-trisphosphate (IP3) by a specific receptor/Ca2+ ion channel (IP3R) mediates the mobilization of Ca2+ from intracellular stores. Although the structural and functional aspects of this receptor are under active investigation, there has been no systematic study of the biosynthesis and degradation of this class of important intracellular ion-channels and this represents the focus of this grant proposal. Our initial studies have shown that chronic treatment of WB cells with the Ca2+ mobilizing agonist Angiotensin II causes down-regulation of IP3R isoforms by activating the ubiquitin/proteasome pathway. We have also identified selective interactions of nascent IP3R isoforms with the Ca2+-binding molecular chaperones calnexin and calreticulin. These studies will be extended using several experimental models including cultured cells, cell-free translation systems and expression of specific IP3R cDNAs in transfected cells. The specific aims of this proposal are: [1]. To characterize early steps in the assembly of IP3Rs - The kinetics and mechanism of oligomerization will be studied. [2]. To study the association of IP3Rs with molecular chaperones - The consequences of chaperone association for IP3R assembly and function will be assessed. [3].To characterize the structural features of IP3R isoforms that are important for regulated degradation - Mutant IP3Rs will be tested as degradation substrates in various model systems. [4]. To define the role of the ubiquitin/proteasome pathway in the regulated turnover of IP3R - The regulation of proteasomal activity in ER membranes will be investigated. Our long-term goal is to understand the factors that regulate the levels of individual IP3R isoforms. The proposed studies are also intended to provide fundamental insights into the mechanisms of ion-channel assembly and the regulated degradation of polytopic membrane proteins in the endoplasmic reticulum.