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-trisph osphate (InsP(3)) receptors and, thus, lead to InsP(3) receptor down-regula tion. To gain insight into this process, we examined whether or not InsP(3) receptor degradation is a direct consequence of InsP(3) binding by analyzi ng the downregulation of exogenous wild-type and binding-defective mutant I nsP(3) receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulatio n of these cells with carbachol showed that wild-type exogenous receptors c ould be down-regulated but that the binding-defective mutant exogenous rece ptors were not. Thus, InsP(3) binding appears to mediate down-regulation. T o 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 InsP(3)-induced Ca2+ release in permeabilized cells, presumably by increasing the number o f InsP(3)-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobil ization and InsP(3) formation in intact cells, (iv) form heteromers with en dogenous receptors, and (v) do not alter the down-regulation of endogenous receptors, In total, these data show that the introduction of exogenous rec eptors into SH-SY5Y cells does not compromise intracellular signaling or th e down-regulatory process. We can thus conclude that InsP(3) binding direct ly activates InsP(3) receptor degradation. Because InsP(3) binding induces a conformational change in the InsP(3) receptor, these data suggest that th is change provides the signal for accelerated proteolysis.