Ubiquitination and Proteasomal Degradation of Endogenous and Exogenous Inositol 1,4,5-Trisphosphate Receptors in αT3-1 Anterior Pituitary Cells

Abstract

276, 3123-3129). In the current study, we sought to define the mechanism behind this adaptive response. We show that GnRH induces a rapid and dramatic increase in InsP 3 receptor polyubiquitination and that proteasome inhibitors block InsP 3 receptor down-regulation and cause the accumulation of polyubiquitinated receptors. Thus, the ubiquitin/proteasome pathway is active in ␣T3-1 cells, and GnRH regulates the levels of InsP 3 receptors via this mechanism. Given these findings and further characterization of this system, we also examined the possibility that ␣T3-1 cells could be used to examine the ubiquitination of exogenous InsP 3 receptors introduced by cDNA transfection. This was found to be the case, since exogenous wild-type InsP 3 receptors, but not bindingdefective mutant receptors, were polyubiquitinated in a GnRH-dependent manner, and agents that inhibited the polyubiquitination of endogenous receptors also inhibited the polyubiquitination of exogenous receptors. Further, we used this system to determine whether phosphorylation was involved in triggering InsP 3 receptor polyubiquitination. This was not the case, since mutation of serine residues 1588 and 1755 (the predominant phosphorylation sites in the type I receptor) did not inhibit polyubiquitination. In total, these data show that the ubiquitin/proteasome pathway is active in anterior pituitary cells, that this pathway targets both endogenous and exogenous InsP 3 receptors in GnRH-stimulated ␣T3-1 cells, and that, in contrast to the situation for many other substrates, phosphorylation does not trigger InsP 3 receptor polyubiquitination.

Gonadotropin-releasing hormone and the control of gonadotrope function

“…A similar GnRH-induced degradation of IP3 receptors has also been described [38]. In addition to such metabolic events, studies have shown that GnRH may also alter the degree of its effect on gonadotropin secretion through its multigenic regulatory action within the gonadotrophs.…”

RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation

“…This lack of complete inhibition could be due to the action of residual RNF170 after RNA interference or from the activity of another ligase or other ligases. Interestingly, whereas stimulus-induced IP 3 receptor degradation was only partially blocked by RNF170 depletion in ␣T3-1 cells, it was completely blocked in mHeLa cells. This suggests that, in mHeLa cells, in addition to catalyzing IP 3 receptor ubiquitination, RNF170 may govern the activity of an additional protein, perhaps an ERAD pathway component, that is required for IP 3 receptor degradation.…”

“…FLAG Inhibits IP 3 Receptor Ubiquitination and Degradation-To assess the role of RNF170 in IP 3 receptor processing, we used RNA interference to deplete endogenous RNF170. In ␣T3-1 cells, introduction by electroporation of a vector encoding a siRNA targeting RNF170 resulted in specific depletion of RNF170.…”

“…This, in concert with Ca 2ϩ binding, induces yet to be defined conformational changes in the tetrameric channel that permit Ca 2ϩ to flow from stores within the ER lumen into the cytosol (1). There are three IP 3 receptor types in mammals, IP 3 R1, IP 3 R2, and IP 3 R3, and although they differ considerably in their tissue distribution, they have broadly similar properties and are often coexpressed (1).…”

“…In recent years it has become clear that G-protein-coupled receptor-induced activation of endogenous IP 3 receptors can lead to their rapid degradation via the ubiquitin-proteasome pathway (UPP) (2). This IP 3 receptor "down-regulation" has been demonstrated in many mammalian cell types, including gonadotropin-releasing hormone (GnRH)-stimulated ␣T3-1 mouse pituitary gonadotropes (3), endothelin 1 (ET1)-stimulated Rat1 fibroblasts (4), and muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells (5) and mHeLa cells (6).…”