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J. Biol. Chem., Vol. 281, Issue 6, 3722-3730, February 10, 2006

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

From the Department of Pathology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107

myo-Inositol 1,4,5-trisphosphate receptor (IP₃R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP₃Rs and study the role of Ca²⁺ and substrate recognition properties of the degradation system using exogenously expressed IP₃R constructs. Employing caspase-3 for IP₃R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca²⁺ to Ca²⁺-depleted Chinese hamster ovary (CHO) cells initiates IP₃R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP₃ and a minimal content of Ca²⁺ in the endoplasmic reticulum lumen is required to initiate IP₃R degradation. Transient transfection of IP₃R constructs into CHO cells indicated the selective degradation of only the SI(+) splice variant of the type I IP₃R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI(+) IP₃R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca²⁺ dependence and structural requirements of the IP₃R proteasomal degradation pathway.

Received for publication, September 12, 2005 , and in revised form, November 14, 2005.

^* This study was supported by National Institutes of Health Grant DK34804. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Rm. 230A, JAH, 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-503-1222; Fax: 215-923-6813; E-mail: suresh.joseph{at}mail.tju.edu.

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