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, IP₃Rs 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 αT3-1 mouse pituitary cells expressing exogenous wild-type or mutant-type-I IP₃Rs (IP₃R1) to provide several lines of evidence that Ca²⁺ is also a trigger. Firstly, depletion of ER Ca²⁺ stores with thapsigargin blocked wild-type IP₃R1 ubiquitination. Secondly, ubiquitination was blocked by mutating Glu²¹⁰⁰ to Asp, which is known to markedly suppress Ca²⁺-binding to IP₃R1 and the potency of Ca²⁺ as a stimulus for channel opening. Thirdly, mutating Asp²⁵⁵⁰ to Ala, which inhibits Ca²⁺ flux through the channel pore, partially inhibited ubiquitination indicating that Ca²⁺ released via wild-type IP₃R1 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 IP₃R1, it strongly inhibited the ubiquitination of the Asp²⁵⁵⁰ to Ala mutant. Overall, these results show that Ca²⁺ plays an important role in triggering IP₃R ubiquitination. Additional experiments with IP₃R1 containing an Arg²⁶⁵ to Gln mutation, which decreases IP₃-binding affinity, confirmed that IP₃-binding also plays a role. Finally, the mutations at Glu²¹⁰⁰, Asp²⁵⁵⁰ and Arg²⁶⁵ inhibited IP₃R1 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.