Abstract
In alphaT3-1 mouse anterior pituitary gonadotropes, chronic activation of gonadotropin-releasing hormone (GnRH) receptors causes inositol 1,4,5-trisphosphate (InsP(3)) 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 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 alphaT3-1 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 alphaT3-1 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 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 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 alphaT3-1 cells, and that, in contrast to the situation for many other substrates, phosphorylation does not trigger InsP(3) receptor polyubiquitination.
Highlights
Hormone-induced secretion from anterior pituitary cells is modulated at many different levels, and among these is regulation of the activity and abundance of receptors involved in signal transduction [1, 2]
The data presented show that gonadotropin-releasing hormone (GnRH)-induced InsP3 receptor down-regulation in ␣T3-1 cells is mediated by the ubiquitin/proteasome pathway, that transiently expressed exogenous InsP3 receptors are polyubiquitinated to endogenous receptors, and that InsP3 binding, but not PKAmediated InsP3 receptor phosphorylation, is a key event in the process that leads to polyubiquitination
These data raise the possibility that other proteins, perhaps those involved signal transduction [26, 40, 41], might be targeted by the ubiquitin/proteasome pathway in anterior pituitary cells upon G-protein-coupled receptor (GPCR) activation
Summary
GnRH, gonadotropin-releasing hormone; InsP3, inositol 1,4,5-trisphosphate; InsP3RHAwt, wild-type type I InsP3 receptor; InsP3RHA⌬, type I InsP3 receptor lacking residues 316 – 352; InsP3RHAA/A, type I InsP3 receptor with serine 3 alanine mutations at positions 1588 and 1755; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; ALLN, N-acetyl-Leu-Leu-norleucinal; HA, hemagglutinin; Protein A, Protein A-Sepharose CL-4B; TPEN, N,N,NЈ,NЈtetrakis(2-pyridylmethyl)ethylenediamine); PKA, protein kinase A; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase. We used ␣T3-1 cells to develop conditions for the analysis of exogenous InsP3 receptor ubiquitination, and by expressing mutant receptors, we show that InsP3 binding is important in triggering this event but that phosphorylation is not
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
