Inositol 1,4,5-trisphosphate receptor down-regulation is activated directly by inositol 1,4,5-trisphosphate binding: Studies with binding- defective mutant receptors

Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5- trisphosphate (InsP₃) receptors and, thus, lead to InsP₃ receptor down- regulation. To gain insight into this process, we examined whether or not InsP₃ receptor degradation is a direct consequence of InsP₃ binding by analyzing the downregulation of exogenous wild-type and binding-defective mutant InsP₃ receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP₃ binding appears to mediate down- regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP₃-induced Ca²⁺ release in permeabilized cells, presumably by increasing the number of InsP₃-sensitive Ca²⁺ channels, (iii) have minimal effects on Ca²⁺ mobilization and InsP₃ formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down- regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SHSY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP₃ binding directly activates InsP₃ receptor degradation. Because InsP₃ binding induces a conformational change in the InsP₃ receptor, these data suggest that this change provides the signal for accelerated proteolysis.