Abstract
In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for targeted degradation of proteins. We show that, in F9 cells and in transfected COS-1 cells, the nuclear retinoid receptors, retinoic acid receptor gamma2 (RARgamma2), RARalpha1, and retinoid X receptor alpha1 (RXRalpha1) are degraded in a retinoic acid-dependent manner through the ubiquitin-proteasome pathway. The degradation of RARgamma2 is entirely dependent on its phosphorylation and on its heterodimerization with liganded RXRalpha1. In contrast, RARalpha1 degradation can occur in the absence of heterodimerization, whereas it is inhibited by phosphorylation, and heterodimerization reverses that inhibition. RXRalpha1 degradation is also modulated by heterodimerization. Thus, each partner of RARgamma/RXRalpha and RARalpha/RXRalpha heterodimers modulates the degradation of the other. We conclude that the ligand-dependent degradation of RARs and RXRs by the ubiquitin-proteasome pathway, which is regulated by heterodimerization and by phosphorylation, could be important for the regulation of the magnitude and duration of the effects of retinoid signals.
Highlights
Retinoic acid (RA),1 the most potent biologically active form of vitamin A, modulates a wide variety of biological processes including proliferation and differentiation of many cell types
RAR␥ is indispensable for RA-induced differentiation of F9 embryonal carcinoma (EC) cells into primitive endoderm [8, 10, 23], while RAR␣ is required for parietal endodermal differentiation that occurs in the presence of RA and cAMP, with primitive endodermal differentiation being a prerequisite for parietal endodermal differentiation [8, 24]
RA-induced Degradation of retinoic acid receptor ␥2 (RAR␥2) in Transfected COS-1 Cells Depends on Its Heterodimerization with RXR␣—To investigate whether retinoids decrease mRAR␥2 protein levels, transiently transfected COS-1 cells were treated for 15 h with tRA (10Ϫ7 M) or vehicle
Summary
Chemicals—Calpain inhibitor II (ALLN) was from Roche Molecular Biochemicals, and MG-132 was from France Biochem. MRAR␥2 deleted for the AF-2 AD core (amino acids 399 – 407/RAR␥2⌬AF-2) [40] was constructed in pSG5 by a double PCR amplification [41], generating a MscI–SacI fragment containing the appropriate mutation. MRXR␣ mutated at its heterodimerization surface (mRXR␣D364A/ E384A/E399A/Y402A/E406A/R462A/E439A) was constructed in pSG5 by successive double PCR amplifications generating BamHI–BglII fragments containing the appropriate mutations. Rabbit polyclonal antibodies against the F region of RAR␥ (RP␥(F)) were previously described [42]. Whole cell extracts were prepared from F9 cells or transfected COS-1 cells by four cycles of freeze-thaw in 50–200 l of 10 mM Tris-HCl (pH 8) containing 0.6 M KCl, 1.5 mM EDTA, and a protease inhibitor mixture and resolved by SDS-10% polyacrylamide gel electrophoresis. Whole cell extracts were incubated with Protein A-Sepharose beads cross-linked with the appropriate antibodies [5], and the immunoprecipitated proteins were detected by immunoblotting and chemiluminescence. The RT-PCR blots were probed with cognate 32P-labeled cDNA fragments and analyzed by autoradiography
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