The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination

The IP3R (inositol 1,4,5-trisphosphate receptor) forms tetrameric Ca2+ channels in ER (endoplasmic reticulum) membranes, where channel activity is largely under the control of the co-agonists IP3 and Ca2+. In cells stimulated using extracellular ligands that persistently elevate phosphoinositidase C activity, IP(3)Rs are rapidly ubiquitinated and then degraded by the proteasome through as yet undefined mechanisms. Whereas binding of IP3 has been suggested to be a key event in the triggering of IP3R ubiquitination the role of Ca2+ in this process remains unknown. In the present study we use alpha T3-1 mouse pituitary cells expressing exogenous wild-type or mutant-type-I IP(3)Rs (IP(3)R1) to provide several lines of evidence that Ca2+ is also a trigger. Firstly, depletion of ER Ca2+ stores with thapsigargin blocked wild-type IP(3)R1 ubiquitination. Secondly, ubiquitination was blocked by mutating Glu(2100) to Asp, which is known to markedly suppress Ca2+-binding to IP(3)R1 and the potency of Ca2+ as a stimulus for channel opening. Thirdly, mutating Asp(2550) to Ala, which inhibits Ca2+ flux through the channel pore, partially inhibited ubiquitination indicating that Ca2+ released via wild-type IP(3)R1 contributes to triggering ubiquitination. Fourthly, and consistent with this conclusion, although suppression of increases in cytoplasmic Ca2+ concentration did not inhibit the ubiquitination of wild-type IP(3)R1, it strongly inhibited the ubiquitination of the Asp(2550) to Ala mutant. Overall, these results show that Ca2+ plays an important role in triggering IP3R ubiquitination. Additional experiments with IP(3)R1 containing an Arg(265) to Gln mutation, which decreases Ip(3)-binding affinity, confirmed that IP3-binding also plays a role. Finally, the mutations at Glu(2100) , ASp(2550) and Arg(265) inhibited IP(3)R1 degradation to an extent that paralleled their inhibitory effects on ubiquitination. We conclude that IP3R ubiquitination and degradation are triggered by the concerted action of IP3- and Ca2+-binding.