Differential subcellular distribution and transcriptional activity of sigmaE3, sigmaE4, and sigmaE3-4 isoforms of the rat estrogen receptor-alpha.
E3, E4, and E3-4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERalpha primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERalpha or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERalpha-GFP was predominantly nuclear, and E3-GFP and E4-GFP were present both in cytoplasm and nucleus, whereas E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERalpha-GFP and E3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of E4-GFP and E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and E3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear E4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, E3-4 exhibited a very weak estrogen-dependent transactivation activity, whereas E3 had an inhibitory effect on full-length ER action. Conversely, E4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERalpha. In a gel mobility shift assay, phosphorylated E4 was able to form a specific complex with a consensus ERE, while E3 and E3-4 never did bind by themselves. The observed inhibitory action of E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERalpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that E3 and E4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.
Pasqualini
C
C
Developpement, Evolution et Plasticité du System Nerveux, Institut de Neurobiologie Alfred Fessard, Centre Nationale de la Recherche Scientifique, 91198 Gif-sur-Yvette cedex, France. Catherine.Pasqualini@iaf.cnrs-gif.fr
Guivarc'h
D
D
Barnier
J V
JV
Guibert
B
B
Vincent
J D
JD
Vernier
P
P
D017398:Alternative Splicing; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha; D005260:Female; D017930:Genes, Reporter; D015151:Immunoblotting; D008297:Male; D018613:Microscopy, Confocal; D010957:Plasmids; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D020033:Protein Isoforms; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D004958:Estradiol