Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a pivotal role in vertebrate intracellular signaling. They are a family of three very similar proteins that form tetrameric ion channels in endoplasmic reticulum (ER) membranes. For the last 15 years, I have been studying the molecular biology of IP3Rs and for the last 10 years or so, have been examining how they are degraded by the ubiquitin-proteasome pathway (UPP). During these studies, it became obvious that IP3Rs are degraded by the ERAD pathway (the facet of the UPP that accounts for the disposal of aberrant ER proteins) and that given their unique properties, IP3Rs might be particularly useful tools for identifying the proteins and mechanisms that mediate ERAD in mammalian cells. Thus, in the last grant cycle we sought to identify and characterize the proteins and mechanisms that mediate IP3R ERAD, and discovered that the ubiquitin conjugates that become attached to activated IP3Rs are unexpectedly complex, and that a completely novel, and very large (>1MDa) complex composed of two uncharacterized ER membrane proteins, termed SPFH1 and 2, mediates the ERAD of activated IP3Rs and participates in the degradation of model ERAD substrates. Interestingly, we have also very recently found that RNF170, an uncharacterized, putative ubiquitin ligase, interacts strongly with the SPFH1/2 complex. The current proposal is designed to extend this work. As in the past, the bulk of the work will be done with endogenous proteins in mammalian cells, ensuring that the results obtained closely mirror the physiological situation.
The Specific Aims of the current proposal are to (i) identify and characterize the proteins that govern the levels of ubiquitin conjugates on activated IP3Rs, and (ii) investigate the structure and function of the SPFH1/2 complex. These goals are significant for three main reasons. First, the studies in Aim 1 on the fine details of IP3R ubiquitination, should help solve the recently revealed mystery of how and why UPP substrates are modified with diverse ubiquitin conjugates. Second, Aim 2 should generate a clearer understanding of the structure and function of the recently discovered, but poorly understood, SPFH1/2 complex. Third, our focus on endogenous proteins in mammalian cells means that the results obtained will accurately model the in vivo situation, and will provide information on the ERAD pathway under near- physiological conditions. Results from this work should have a significant impact on our understanding of both ERAD and the UPP in general, and will hopefully facilitate efforts aimed at developing drugs targeting these pathways.

Public Health Relevance

The degradation of proteins via the proteasome, which is governed by another protein called ubiquitin, is a fundamental cellular process that controls the levels of many key proteins, and has enormous potential as a therapeutic target in diseases such as cystic fibrosis, neuropathies, diabetes and cancer. The studies I propose will investigate in mammalian cells some of the mechanisms that underpin this process with the aim of obtaining a clearer understanding of this area.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-CB-N (03))
Program Officer
Sechi, Salvatore
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Upstate Medical University
Schools of Medicine
United States
Zip Code
Hirose, Masahiko; Kamoshita, Maki; Fujiwara, Katsuyoshi et al. (2013) Vitrification procedure decreases inositol 1,4,5-trisphophate receptor expression, resulting in low fertility of pig oocytes. Anim Sci J 84:693-701
Wojcikiewicz, Richard J H; Pearce, Margaret M P; Sliter, Danielle A et al. (2009) When worlds collide: IP(3) receptors and the ERAD pathway. Cell Calcium 46:147-53
Hanson, C Jane; Bootman, Martin D; Distelhorst, Clark W et al. (2008) Bcl-2 suppresses Ca2+ release through inositol 1,4,5-trisphosphate receptors and inhibits Ca2+ uptake by mitochondria without affecting ER calcium store content. Cell Calcium 44:324-38
Soulsby, M D; Wojcikiewicz, R J H (2007) Calcium mobilization via type III inositol 1,4,5-trisphosphate receptors is not altered by PKA-mediated phosphorylation of serines 916, 934, and 1832. Cell Calcium 42:261-70
Means, Shawn; Smith, Alexander J; Shepherd, Jason et al. (2006) Reaction diffusion modeling of calcium dynamics with realistic ER geometry. Biophys J 91:537-57
Kelley, Grant G; Kaproth-Joslin, Katherine A; Reks, Sarah E et al. (2006) G-protein-coupled receptor agonists activate endogenous phospholipase Cepsilon and phospholipase Cbeta3 in a temporally distinct manner. J Biol Chem 281:2639-48
Alzayady, Kamil J; Wojcikiewicz, Richard J H (2005) The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination. Biochem J 392:601-6
Soulsby, Matthew D; Wojcikiewicz, Richard J H (2005) The type III inositol 1,4,5-trisphosphate receptor is phosphorylated by cAMP-dependent protein kinase at three sites. Biochem J 392:493-7
Alzayady, Kamil J; Panning, Margaret M; Kelley, Grant G et al. (2005) Involvement of the p97-Ufd1-Npl4 complex in the regulated endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors. J Biol Chem 280:34530-7
Soulsby, Matthew D; Alzayady, Kamil; Xu, Qun et al. (2004) The contribution of serine residues 1588 and 1755 to phosphorylation of the type I inositol 1,4,5-trisphosphate receptor by PKA and PKG. FEBS Lett 557:181-4

Showing the most recent 10 out of 16 publications