Mechanism of Proteasomal Degradation of Inositol Trisphosphate Receptors in CHO-K1 Cells

Abstract

2؉dependence and structural requirements of the IP 3 R proteasomal degradation pathway.The activation of inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 2 by IP 3 initiates Ca 2ϩ mobilization from the ER and triggers the Ca 2ϩ signal that underlies alterations in cell function elicited by a diverse array of cell surface stimuli (1, 2). A commonly observed characteristic of cells is that they adapt their responses when chronically stimulated. In the case of cell surface receptors, this is usually the result of phosphorylation and/or internalization. Wojcikiewicz et al. (3,4) were the first to note that chronic stimulation of cultured SHSY-5Y neuroblastoma cells with carbachol for 6 h causes 90% loss of type I IP 3 R protein by a mechanism involving the marked acceleration of IP 3 R degradation. Subsequently, similar effects on IP 3 R degradation have been described in many different experimental systems with many different agonists (5-16). Downregulation of IP 3 R protein is associated with a decrease in the ability of IP 3 to mobilize Ca 2ϩ (4,8). More recently it has been shown that the frequency of elementary Ca 2ϩ puffs that trigger propagating Ca 2ϩ waves in cells is decreased by chronic agonist stimulation (14). The physiological significance of IP 3 R down-regulation is unknown. However, down-regulation of receptors would be expected to inhibit the global Ca 2ϩ signal elicited by all Ca 2ϩ mobilizing agonists (heterologous desensitization). If this is the only component of the signal transduction system that is down-regulated, then an imbalance in signaling pathways would exist and may have profound consequences for cellular responses. A physiologically relevant example is the response of pituitary cells to GnRH receptor agonists (17). Mammalian GnRH receptors lack a C-terminal tail and therefore do not bind arrestin. Consequently these receptors desensitize and internalize only very slowly. However, sustained activation of GnRH receptors does lead to desensitization of gonadotrophin hormone secretion, which suggests the presence of adaptive mechanisms distal to the cell surface receptor. Treatment with GnRH receptor agonists produce a rapid and pronounced degradation of IP 3 Rs, and this is associated with a marked reduction in the IP 3 -mediated Ca 2ϩ signal (15, 18). The mechanism of IP 3 R degradation has not been established. Previous studies have shown that chronic elevation of IP 3 and IP 3 binding to the receptor are required to facilitate IP 3 R degradation (19,20). It has been proposed that the sustained elevation of IP 3 causes the IP 3 R to adopt a conformation that exposes sites that become ubiquitinated. The proteasome pathway then degrades the ubiquitinated IP 3 receptor. The regions of the IP 3 R involved in ubiquitination have not been determined. Pretreatment of cells with the SERCA pump inhibitor thapsigargin has been found to inhibit agonist-mediated IP 3 R degradation, suggesting that Ca 2ϩ also plays a role in IP 3 R degradation (4). In the present study we have examined IP 3 R degra...

Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis

“…Thus, a caspase 3 cleavage of IP 3 R or an interaction between IP 3 R and cytochrome c generate an unregulated Ca 2 þ -release state, which potentiates the cell death (Boehning et al, 2003;Bhanumathy et al, 2006). In our work, 2-APB modulated the IP 3 R. This compound is an antagonist of the IP 3 R or of other calcium channels, such as those implied in capacitative calcium entry (Iwasaki et al, 2001).…”

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

“…In that study (35), examining rat IP 3 R1 overexpressed in CHO-K1 cells, it was concluded that the region C-terminal to Asp 1891 was monoubiquitinated and that the region N-terminal to Asp 820 was polyubiquitinated. Our data, in contrast, did not consistently detect any ubiquitination sites N-terminal to Asp 820 and found multiple sites C-terminal of Asp 1891 .…”

Inositol Trisphosphate Receptor Ca2+Release Channels

“…InsP 3 R isoform expression levels can be modified during development and differentiation (129,242,340,394,419,450,460) and in response to various normal and pathological stimuli (20,61,70,218,226,250,305,403,418,460,502,526). Furthermore, InsP 3 R protein expression levels can be downregulated by a use-dependent mechanism that involves InsP 3 -and Ca 2ϩ -dependent channel ubiquitination, and subsequent degradation involving the proteasome (9,10,35,515).…”