Mass spectrometric analysis of type 1 inositol 1,4,5-trisphosphate receptor ubiquitination

Abstract

Inositol 1,4,5-trisphosphate (IP3) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP3-induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP3 levels, IP 3 receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP3 receptor (IP 3R1) 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 IP3R1 can be ubiquitinated and that these are clustered in the regulatory domain and are found in surface regions, (ii) that at least ∼40% of the IP3R1-associated ubiquitin is monoubiquitin, (iii) that both Lys48 and Lys63 linkages are abundant in attached ubiquitin chains, and (iv) that Lys63 linkages accumulate most rapidly. Additionally, we find that not all IP 3R1 subunits in a tetramer are ubiquitinated and that nontetrameric IP3R1 complexes form as degradation proceeds, suggesting that ubiquitinated subunits may be selectively extracted and degraded. Overall, these data show that endogenous IP3R1 is tagged with an array of ubiquitin conjugates at multiple sites and that both IP3R1 ubiquitination and degradation are highly complex processes. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.