Ubiquitination and proteasomal degradation of endogenous and exogenous inositol 1,4,5-trisphosphate receptors in αT3-1 anterior pituitary cells

In αT3-1 mouse anterior pituitary gonadotropes, chronic activation of gonadotropin-releasing hormone (GnRH) receptors causes inositol 1,4,5-trisphosphate (InsP₃) receptor down-regulation (Willars, G. B., Royall, J. E., Nahorski, S. R., El-Gehani, F., Everest, H. and McArdle, C. A. (2001) J. Biol. Chem. 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 InsP₃ receptor polyubiquitination and that proteasome inhibitors block InsP₃ receptor down-regulation and cause the accumulation of polyubiquitinated receptors. Thus, the ubiquitin/proteasome pathway is active in αT3-1 cells, and GnRH regulates the levels of InsP₃ receptors via this mechanism. Given these findings and further characterization of this system, we also examined the possibility that αT3-1 cells could be used to examine the ubiquitination of exogenous InsP₃ receptors introduced by cDNA transfection. This was found to be the case, since exogenous wild-type InsP₃ receptors, but not binding-defective 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 InsP₃ 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 InsP₃ receptors in GnRH-stimulated αT3-1 cells, and that, in contrast to the situation for many other substrates, phosphorylation does not trigger InsP₃ receptor polyubiquitination.