Mass Spectrometric Analysis of Type 1 Inositol 1,4,5-Trisphosphate Receptor Ubiquitination

Abstract

Inositol 1,4,5-trisphosphate (IP 3 ) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP 3 -induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP 3 levels, IP 3 receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP 3 receptor (IP 3 R1) activated by endogenous signaling pathways and processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin conjugates. Our findings are (i) that at least 11 of the 167 lysines in IP 3 R1 can be ubiquitinated and that these are clustered in the regulatory domain and are found in surface regions, (ii) that at least ϳ40% of the IP 3 R1-associated ubiquitin is monoubiquitin, (iii) that both Lys 48 and Lys 63 linkages are abundant in attached ubiquitin chains, and (iv) that Lys 63 linkages accumulate most rapidly. Additionally, we find that not all IP 3 R1 subunits in a tetramer are ubiquitinated and that nontetrameric IP 3 R1 complexes form as degradation proceeds, suggesting that ubiquitinated subunits may be selectively extracted and degraded. Overall, these data show that endogenous IP 3 R1 is tagged with an array of ubiquitin conjugates at multiple sites and that both IP 3 R1 ubiquitination and degradation are highly complex processes.Inositol 1,4,5-trisphosphate (IP 3 ) 4 receptors (IP 3 Rs) are ϳ300-kDa endoplasmic reticulum (ER) membrane proteins that tetramerize to form Ca 2ϩ channels that are gated by the co-agonists IP 3 and Ca 2ϩ and that govern Ca 2ϩ release from the ER (1-3). There are three homologous mammalian IP 3 R types (termed IP 3 R1, IP 3 R2, and IP 3 R3) that can form either homo-or heterotetrameric channels. Each can be divided into an N-terminal ligand-binding domain, a large central regulatory domain that contains several modulatory sites, and a C-terminal channel domain that contains six membrane-spanning helices and the channel pore (see Fig.

RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation

“…It is thought that the intralumenal regions of the erlin1/2 complex interact with intralumenal regions of activated IP 3 receptors and that the erlin1/2 complex acts as a "recognition factor" that selects activated IP 3 receptors for degradation via the ERAD pathway (2,15). IP 3 receptors are ubiquitinated at specific, exposed lysine residues in their cytosolic domains (29,30), and although it has yet to be defined how the erlin1/2 complex and RNF170 interact, it is reasonable to think that this interaction juxtaposes the cytosolic RING domain of RNF170 with the cytosolic regions of activated IP 3 receptors that become ubiquitinated.…”

Characterizing Ubiquitination Sites by Peptide-based Immunoaffinity Enrichment

“…The two main strategies have been identification of K-GG peptides following in vitro ubiquitination of a purified substrate or immunoprecipitation of ubiquitinated substrates directly from cells. Analyses from cells have the advantage that modified proteins occur in biological context and can be induced by relevant stimuli, as in the cases of epidermal growth factor receptor, inositol 1,4,5-trisphosphate receptor, and dopamine transporter ubiquitination following treatment with epidermal growth factor, gonadotropin-releasing hormone, or protein kinase C activation, respectively (36,42,43). In the case of caspase-8, ubiquitination was shown to occur on Lys-461 in response to activation of death receptors by an extrinsic stimulus (44).…”

Isoform-specific Regulation of the Inositol 1,4,5-Trisphosphate Receptor by O-Linked Glycosylation

“…Ubiquitination was shown to occur on all InsP 3 R subtypes at approximately the same site and with a similar functional consequence (40,41), whereas phosphorylation of the InsP 3 R is isoform-specific (19). It is now clear that O-GlcNAc modification is an additional and complementary subtype-specific regulation of the InsP 3 R.…”

“…The InsP 3 R is post-translationally modified through a variety of other pathways, including phosphorylation (19), N-glycosylation (39), and ubiquitination (40). Ubiquitination was shown to occur on all InsP 3 R subtypes at approximately the same site and with a similar functional consequence (40,41), whereas phosphorylation of the InsP 3 R is isoform-specific (19).…”