title: Ubiquitination and proteasomal degradation of endogenous and exogenous inositol 1,4,5-trisphosphate receptors in alpha T3-1 anterior pituitary cells.
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.
authors.affiliation: Department of Pharmacology, State University of New York Upstate Medical University, Syracuse 13210-2339, USA. wojcikir@mail.upstate.edu
authors.forename: Richard J H
authors.initials: RJ
authors.lastname: Wojcikiewicz
authors.forename: Qun
authors.initials: Q
authors.lastname: Xu
authors.forename: Jack M
authors.initials: JM
authors.lastname: Webster
authors.forename: Kamil
authors.initials: K
authors.lastname: Alzayady
authors.forename: Chen
authors.initials: C
authors.lastname: Gao
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000502:physiology*; D005029:Ethylenediamines / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D009097:Multienzyme Complexes / Q000502:physiology*; D010766:Phosphorylation; D010903:Pituitary Gland, Anterior / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D005029:Ethylenediamines; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009097:Multienzyme Complexes; D018160:Receptors, Cytoplasmic and Nuclear; D025801:Ubiquitin; D007987:Gonadotropin-Releasing Hormone; D019284:Thapsigargin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D005990:Glycerol; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine