My laboratory researches IP3 receptors. The answers to the obvious questions of what IP3 receptors are and what they do are summarized in Figures 1 and 2.
What are they? They are a family of large proteins (~300kDa) that span the ER membrane (fig. 1). There are three of them (termed types I, II and III receptors), they are 60-70% identical at the amino acid level, and they have the same overall structure.
What do they do? They play a crucial role in intracellular signaling. They tetramerize to form pores in the ER membrane that allow Ca2+ to escape from the ER into the cytosol. As you can see in the movie (fig. 1), when IP3 (red) binds to the IP3 receptor tetramer (green), the pore opens and Ca2+ stored in the ER flows down its concentration gradient into the cytosol. This 'mobilized' Ca2+ then triggers many cell functions, ranging from secretion to gene expression. Also, you can see that the released Ca2+ is returned to the ER via ATP-dependent pumps (purple). So, IP3 receptor complexes act like gates through which Ca2+ flows. IP3 is the key to the gate.
Specifically, we currently work on IP3 receptor degradation by the ubiquitin / proteasome pathway.
Degradation by the ubiquitin / proteasome pathway.
IP3 receptor down-regulation is a remarkable phenomenon by which IP3 receptors are rapidly degraded when cells are stimulated. It is a classic adaptive response that enables cells to adjust to their external environment; persistent cell activation decreases the levels of IP3 receptors - proteins that mediate cell signaling - thus dampening cell activation. Thus, a cell can survive in the face of a constant stimulus.
I was lucky enough to discover IP3 receptor down-regulation in 1991 (1) and have worked on it ever since (2). I soon showed that IP3 down-regulation (which can deplete cells of 90% of their IP3 receptors in 1 hour) was due to an acceleration of receptor proteolysis, occurred in a wide range of cell lines and primary cultures and could account for the degradation of type I, II and III receptors (2,3).
We were amazed to find, subsequently, that degradation was via the ubiquitin / proteasome pathway (2,4). Take a look at the figure below. This is our current model and shows that when IP3 receptors are activated they become polyubiquitinated and are then degraded by the proteasome, a giant 26S proteolytic complex.
This finding places us in a very hot area of Cell Biology, as interest in the ubiquitin / proteasome pathway has expanded enormously in the last few years. Indeed, the 2004 Nobel Prize in Chemistry was awarded to the three scientists who first mapped this pathway. Basically, the ubiquitin / proteasome pathway does two things – it accounts for the degradation of misfolded proteins (particularly those found in the ER lumen and membrane), and proteins such as cell cycle regulators and transcription factors that have to be rapidly degraded at certain times. With regard to IP3 receptors, they are ER membrane protein complex and seem to be degraded by the same pathway that accounts for the degradation of misfolded ER proteins. Most recently we have shown that Ubc7 is the E2 that ubiquitinates IP3 receptors (5), that an ER membrane protein called SPFH1/2 complex mediates ubiquitination (6,7), that a complex involving p97 mediates the extraction of IP3 receptors from the ER membrane (8), and that binding of both IP3 and Ca2+ is required to trigger IP3 receptor ubiquitination (9). Further, and amazingly, also we have found that IP3 receptors are conjugated with both Lys48- and Lys63-linked ubiquitin chains, with the different chains types segregated to different IP3 receptor subunits and performing different functions (10,11). Much of this work has revolved around mass spectrometry to identify IP3 receptor-associated proteins followed by RNAi to characterize them. We have funding to pursue this area until at least 2013 and I expect that my students will make some interesting discoveries in the next few years! Watch this space and how our latest model of IP3 receptor (Fig.2) processing develops (12).
- Wojcikiewicz, R.J.H. and Nahorski, S.R. (1991) Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action: Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding. J. Biol. Chem. 266, 22234-22241.
- Wojcikiewicz, R.J.H. (2004) Regulated ubiquitination of proteins in GPCR-initiated signalling pathways. Trends Pharmacol. Sci. 25, 35-41.
- Wojcikiewicz, R.J.H., Furuichi, T., Nakade, S., Mikoshiba, K. and Nahorski, S.R. (1994) Muscarinic receptor activation down-regulates the type I inositol 1,4,5-trisphosphate receptor by accelerating its degradation. J. Biol. Chem. 269, 7963-7969.
- Oberdorf, J.A., Webster, J.M., Zhu, C.C., Luo, S.G. and Wojcikiewicz, R.J.H. (1999) Down-regulation of types I, II and III inositol 1,4,5 trisphosphate receptors is mediated by the ubiquitin / proteasome pathway. Biochem. J. 339, 453-461.
- Webster, J.M., Tiwari, S., Weissman, A.M. and Wojcikiewicz, R.J.H. (2003) Inositol 1,4,5 trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the Endoplasmic Reticulum-Associated Degradation pathway, and is inhibited by chelation of intracellular Zn2+. J. Biol. Chem. 278, 38238-38246.
- Pearce, M.M., Wang, Y., Kelley, G.G. and Wojcikiewicz, R.J.H. (2007) SPFH2 mediates the ERAD of IP3 receptors and other substrates in mammalian cells. J. Biol. Chem. 282, 20104-20115.
- Pearce, M.M.P., Wormer, D.B., Wilkens, S. and Wojcikiewicz, R.J.H. (2009) An ER membrane complex composed of SPFH1 and SPFH2 mediates the ER-associated degradation of IP3 receptors. J. Biol. Chem. 284, 10433-10445.
- Alzayady, K., Panning, M.M., Kelley, G.G. and Wojcikiewicz, R.J.H. (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, 34530-34537.
- Alzayady, K. and Wojcikiewicz, R.J.H. (2005) The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination. Biochem J. 392, 601-606.
- Sliter, D., Kirkpatrick, D.S., Alzayady, K., Kubota, K., Gygi, S.P. and Wojcikiewicz, R.J.H. (2008) Mass spectral analysis of type I inositol 1,4,5-trisphosphate receptor ubiquitination. J. Biol. Chem. 283, 35319-35328.
- Sliter D.A., Aguiar, M., Gygi, S.P. and Wojcikiewicz, R.J.H. (2010) Activated inositol 1,4,5-trisphosphate receptors are modified by homogenous LYS48- and LYS63-linked ubiquitin chains, but only LYS48-linked chains are required for degradation. J. Biol. Chem. (submitted)
- Wojcikiewicz, R.J.H., Pearce, M.M.P., Sliter, D. and Wang. Y. (2009) When worlds collide: IP3 receptors and the ERAD pathway. Cell Calcium 46, 147-153.