Elsevier

Cell Calcium

Volume 46, Issue 3, September 2009, Pages 147-153
Cell Calcium

Review
When worlds collide: IP3 receptors and the ERAD pathway

https://doi.org/10.1016/j.ceca.2009.05.002Get rights and content

Abstract

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca2+ 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 (IP3) receptors are activated, since in addition to acting as release channels for stored ER Ca2+, IP3 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 IP3 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 IP3 receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP3 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 IP3 receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca2+ stores to IP3 and may protect cells against deleterious effects of over-activation of Ca2+ signaling pathways.

Section snippets

IP3 receptors and their activation

IP3 receptors are large (∼2700 amino acid) endoplasmic reticulum (ER) membrane proteins which form tetrameric channels that govern the release of Ca2+ stored within the ER lumen of vertebrate cells (Fig. 1) [1], [2], [3]. They are named for their ability to bind to and be opened by the second messenger IP3, which is generated at the plasma membrane in response to cell surface receptor activation. Thus, IP3 receptors are pivotal in signaling pathways that couple extracellular hormones,

IP3 receptor down-regulation

In 1991 it was discovered in mammalian cell lines that in response to activation of certain IP3-generating cell surface receptors, IP3 receptors are “down-regulated”, i.e. there is a rapid and dramatic decline in cellular IP3 receptor content [9]. Typically, this decline is >50%, with half-maximal effect at 30–60 min [10], [11], [12], [13], [14], but is particularly marked in αT3-1 anterior pituitary cells, in which gonadotropin-releasing hormone (GnRH) receptor activation down-regulates IP3R1

The UPP

The UPP is currently the focus of intense interest, since it is now known to be the major route of protein degradation in eukaryotic cells and mediates the selective destruction of many important proteins, including signaling pathway proteins and regulators of the cell cycle and transcription [26]. In addition, it is responsible for “quality control” in the ER; i.e., the selective degradation of misfolded proteins, and of unused subunits of multimeric protein complexes, in a process known as

ERAD

In addition to being a Ca2+ store, the ER is of course, also the synthesis site of membrane and secreted proteins, which account for ∼1/3 of all proteins [27]. It has emerged in recent years that a sophisticated system, ERAD, exists in eukaryotes for the disposal of proteins that do not fold properly or which cannot find their normal binding partners (Fig. 2) [27]. Intriguingly, some ER-resident proteins that are stable under normal conditions are also processed in this manner, the prototype

Are IP3 receptors ERAD substrates?

Evidence that IP3 receptors are UPP substrates came from experiments showing that IP3 receptors are polyubiquitinated, and that proteasome inhibitors block their down-regulation [12], [16], [18], [25], [40]. Obviously, their location in the ER immediately suggested that IP3 receptors could be targeted by the ERAD pathway and subsequent studies supported this view – an E2 that ubiquitinates IP3 receptors is ubc7 [40], an enzyme implicated in both yeast and mammalian ERAD pathways [31], [36], and

IP3 receptor ubiquitination is surprisingly complex

A fundamental unanswered question for most UPP substrates concerns where they are ubiquitinated and with what, and only recently, with the advent of mass spectrometry-based technologies [33], [43], [44], [45], has it been possible to address this question. Application of this approach to IP3R1 isolated from GnRH-stimulated αT3-1 cells, showed that at least 11 of IP3R1's 167 lysines can be sites of ubiquitination (Fig. 3A), that of the attached ubiquitin moieties, at least ∼40% are

Special delivery

To contemplate how ubiquitinated IP3 receptors might be degraded by the proteasome is quite daunting. To enter the catalytic core of the proteasome, proteins must first be unfolded [27], [28], yet IP3 receptor subunits have 6 TM domains and in their native state are tightly associated into tetramers ∼1 MDa in size (Fig. 1A). Two new pieces of data appear to speak to this issue. First, in contrast to many model ERAD substrates [49], [50], IP3 receptors are not released into the cytosol prior to

The SPFH1/2 complex and selection of activated IP3 receptors for ERAD

Several proteins, including p97, associate with IP3 receptors in an activation-dependent manner [41] and most recently it has been demonstrated that SPFH1 and SPFH2 (erlin 1 and erlin 2; see Section 9) [51], also have this property [42], [52]. Fig. 4A–C shows the essential features of these two proteins – that they associate rapidly with IP3 receptors in a manner that precedes maximal IP3 receptor ubiquitination and association of p97, that they are type II ER membrane glycoproteins, and that

SPFH domain-containing proteins

SPFH1 and SPFH2 belong to a family of ∼100 mammalian proteins that contain an “SPFH” domain, an ∼250 amino acid motif named because of minor sequence similarities in the proteins Stomatin, Prohibitin, Flotillin (reggie), and HflC/K [51]. SPFH domain-containing proteins share some similarities, including localization to cholesterol-rich, detergent-resistant membranes (DRMs), and assembly into large (>1 MDa) oligomeric structures [51]. To date, however, no universal function has been attributed to

Conclusions and perspectives

That a fraction of activated IP3 receptors are hived off for ERAD is both surprising and intriguing. The cell is inactivating IP3 gated channels in a very radical manner – by degradation as opposed to a reversible modification. This could represent a finely tuned mechanism to suppress Ca2+ signaling. Alternatively, it could be more by accident than design – it is possible that during the activation process, IP3 receptors “accidentally” expose regions (e.g. hydrophobic patches) that makes them

Acknowledgements

The authors wish to apologize to those whose work was omitted due to space constraints, and wish to thank the National Institutes of Health, the Pharmaceutical Research and Manufacturers of America Foundation, and the American Heart Association for financial support.

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    Present address: Department of Biology, Stanford University, Palo Alto, CA 94305, USA.

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