When worlds collide: IP(3) receptors and the ERAD pathway

Abstract

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca(2+) store, those who study protein synthesis tend to see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP(3)) receptors are activated, since in addition to acting as release channels for stored ER Ca(2+), IP(3) receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP(3) receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP(3) receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP(3) receptors resemble aberrant proteins, which triggers their binding to the SPFH1/2 complex, their ubiquitination and extraction from the ER membrane and finally, their degradation by the proteasome. This degradation of activated IP(3) receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca(2+) stores to IP(3) and may protect cells against deleterious effects of over-activation of Ca(2+) signaling pathways.

Figure 1. IP₃ receptor structure and activation

A. Cryo-electron microscopy image of tetrameric IP₃R1 purified from mouse cerebellum (modified from reference 8). The scale bar =100Å, and the region thought to span the ER membrane and contain the channel pore is indicated by the double lines. Models obtained by other groups are broadly similar to the image shown, but are not identical [1]. B. Model of channel opening; for clarity, only two IP₃R1 subunits are shown (modified from reference 6; see text for description). The domains indicated are the SD (yellow, amino acids 1–223), deletion of which creates a protein that binds IP₃, but which cannot not form functional channels; the LBD (orange and red, amino acids 224–575), which is composed of 2 halves linked by a putative hinge; the channel domain (blue, amino acids 2276–2749), which contains 6 TM helices linked by 3 lumenal loops and 2 cytosolic loops, and a coiled-coil (CC) region that participates in tetramer assembly; and the intervening coupling domain (green, amino acids 576–2275), which contains several regulatory sites. The channel pore is formed by TM helices 5 and 6 and the intervening lumenal loop [2,6,7]. The arrows indicate the putative movements that occur after IP₃ binding that cause channel opening.

Figure 2. Simplified model of the ERAD pathway

ERAD of membrane or luminal proteins containing some kind of aberration (yellow stars) can be thought of as a 4-step process consisting of (1) recognition, (2) retrotranslocation, (3) ubiquitination, and (4) proteasomal degradation (see text for details).

Figure 3. Ubiquitination sites and ubiquitin chain linkages on IP₃R1

A A schematic representation of mouse IP₃R1, with the 11 ubiquitination sites (K916, 962, 1571, 1771, 1884, 1885, 1886, 1901, 1924, 2118 and 2257) indicated by “Ks” in the main diagram or by arrows in the expanded regions. Also indicated are sites at which trypsin preferentially cleaves IP₃R1 and which are thought to be surface-exposed loops (arrowheads), a glycine rich region, a caspase-3 cleavage site, ATP- and calmodulin-binding sites, the “Ca²⁺-sensor”, and sites of PKA-mediated phosphorylation [2]. Note that the ubiquitination sites are near exposed loops, or regulatory sites. B Relative amounts of ubiquitin in chains linked via K48 or K63 or other lysines. C Depicted is a single IP₃R1 subunit ubiquitinated in a manner that approximates to the average modification; the subunit is tagged at 6 sites with a total of 8 ubiquitin moieties, with 1 ubiquitin moiety containing a K48 linkage and 1 containing a K63 linkage.

Figure 4. The SPFH1/2 complex and its role in IP₃ receptor ERAD

A. SPFH1 and SPFH2 associate very rapidly with activated IP₃R1, as observed when αT3-1 cells are exposed to GnRH, and anti-IP₃R1 immunoprecipitates are probed for ubiquitin, p97, SPFH1 and SPFH2. B. The basic molecular architecture of SPFH1 and SPFH2 is shown, with the N-terminal TM domains indicated by black boxes, the SPFH domains by gray boxes, the glycans by the asterisks, and the locations of the various motifs by amino acid number. C. A 3-dimensional reconstruction of the SPFH1/2 complex, determined at a resolution of ~33Å and contoured at a volume corresponding to a calculated molecular mass of ~2MDa (scale bar = 100Å). Putative side (1), top (2) and bottom (3) views are shown, with the membrane-spanning and lumenal regions indicated by arrows and arrowheads, respectively. D. Summary model of how IP₃ receptor ERAD occurs. Upon binding of the co-agonists IP₃ and Ca²⁺, IP₃ receptor tetramers undergo a conformational change that both opens the Ca²⁺ channel and triggers the association of the SPFH1/2 complex, which recruits the E2 and E3 enzymes that catalyze the ubiquitination of IP₃ receptors in the coupling domain. Ubiquitinated receptors are then extracted from the membrane and delivered to the proteasome through the action of the cytosolic p97-Ufd1-Npl4 complex.