PROTEASOMAL DEGRADATION OF INOSITOL TRISPHOSPHATE RECEPTORS

Abstract

The present study delineates the mechanism of Inositol trisphosphate receptor (IP3R) degradation in response to carbachol (Cch) in CHO-K1cells. We have used these cells to identify the sites of ubiquitination on IP3Rs, the role of Ca2+in IP3R degradation and the substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination at the N-terminal region and monoubiquitination in the C-terminal region. The addition of extracellular Ca2+ to Ca2+ –depleted CHO cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggests that both a sustained elevation of IP3 and a minimal content of Ca2+ in the ER lumen are required for initiating IP3R degradation. The addition of an epitope tag at the N- or C- terminus of IP3Rs blocks IP3R degradation but does not interfere with ubiquitination. This suggests that both termini need to be accesible for proper unfolding and/ or degradation. A pore defective mutant of IP3R (D2550A) was not degraded in response to Cch stimulation. These data begin to define some of the structural and functional requirements of IP3R degradation in the endoplasmic reticulum.