Ligand binding directly stimulates ubiquitination of the inositol 1,4,5-trisphosphate receptor

Down-regulation of the Ins(1,4,5)P-3 receptor is an adaptive response to th e activation of certain phosphoinositidase C-linked cell-surface receptors. It is manifested as a profound decline in cellular Ins(1,4,5)P-3 receptor content, occurs with a half-time of 0.5-2 h and is due to accelerated prote olysis. It has been shown that this process is mediated by the ubiquitin/pr oteasome pathway and is therefore initiated by Ins(1,4,5)P-3 receptor ubiqu itination. To investigate the role of ligand binding in Ins(1,4,5)P-3 recep tor ubiquitination, we expressed 'exogenous' wild-type and ligand-binding-d efective mutant type I Ins(1,4,5)P-3 receptors in human neuroblastoma SH-SY 5Y cells, in which muscarinic receptor activation elicits Ins(1,4,5)P-3 rec eptor down-regulation. We found (1) that exogenous wild-type Ins(1,4,5)P-3 receptors are efficiently ubiquitinated in response to muscarinic receptor stimulation, (2) that exogenous ligand binding-defective mutant Ins(1,4,5)P -3 receptors are resistant to ubiquitination, (3) that this resistance is n ot caused by the removal of potential ubiquitin-conjugating sites in the mu tated region, and (4) that in heterotetramers of exogenous mutant receptors and 'endogenous' receptors, only the latter are targeted for ubiquitinatio n. These results indicate that the binding of Ins(1,4,5)P-3 directly stimul ates ubiquitination of the Ins(1,4,5)P-3 receptor and that the targeting of Ins(1,4,5)P-3 receptors for ubiquitination is a highly specific process. W e therefore propose that an Ins(1,4,5)P-3-binding-induced conformational ch ange in the receptor exposes a degradation signal that leads to ubiquitinat ion.