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Abstract
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.
Highlights
IP3 to mobilize Ca2ϩ [4, 8]
Basic Characterization of IP3R Down-regulation in Chinese hamster ovary (CHO)-K1 Cells— Our previous studies on IP3R degradation were carried out in WB rat liver epithelial cells stimulated with angiotensin II [8, 30]
Acetylcholine was the least potent agonist with half-maximal effects at 50 M and only a 40% down-regulation even at 1 mM. These data, together with the finding that the carbachol effects on down-regulation are completely blocked by atropine (Fig. 2), suggest that IP3R down-regulation is mediated by endogenous muscarinic receptors in CHO-K1 cells
Summary
Materials—Carbachol, acetylcholine, atropine, ALLN, and ALLM were purchased from Sigma. To resolve IP3R domains that contained ubiquitin, we employed a Myc-tagged Ub plasmid (kindly given by Dr Ron Kopito) that was transfected together with the type I. The plates were treated with 1 mM carbachol, lysed as described above in NEMcontaining medium, and immunoprecipitated overnight with CT-1 Ab. After washing twice in solubilization buffer, an aliquot of the immunoprecipitate (80%) was digested with caspase-3 in 200 l of a buffer containing 50 mM HEPES (pH 7.2), 1 mM dithiothreitol, 1 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and 1 g recombinant caspase-3. The uncut control sample and the Myc reimmunoprecipitated samples were processed on 10% SDS-PAGE and immunoblotted with IP3R Abs to locate the ubiquitinated fragments
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