Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells.
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.
Bhanumathy
Cunnigaiper D
CD
Department of Pathology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107, USA.
Nakao
Steven K
SK
Joseph
Suresh K
SK
D017398:Alternative Splicing; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018679:Cholinergic Agonists / Q000494:pharmacology; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
D015220:Calcium Channels; D018679:Cholinergic Agonists; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D025801:Ubiquitin; D019284:Thapsigargin; D002217:Carbachol; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium