Differential cellular localization of estrogen receptor alpha in uterine and mammary cells.
The classical alpha isoform of the estrogen receptor (ER) has been reported to localize almost exclusively in the nucleus. However, studies on non-genomic steroid effects have also suggested the existence of ERs residing at the cell surface. In this work, we present immunological data supporting extra-nuclear ERalpha localization in uterine (SHM) and mammary (MCF-7) cell lines. Immunocytological studies performed on SHM cells revealed that native ERs mainly localize as a perinuclear cytoplasmic ring. The receptors were rapidly translocated to the nucleus by 17beta-estradiol. In addition to nuclear ERs, a peripheral reservoir of ERalpha immunoreactivity, most probably associated with the plasma membrane, was detected in MCF-7 cells. These results were confirmed by the detection of membrane estrogen binding sites using fluorescent estrogen-BSA derivatives and ligand binding assays to intact cells, where [3H]-estradiol could be partly displaced by impeded estrogen conjugates. Partial inhibition of radioligand binding by an antibody against the steroid binding domain of the ERalpha suggests that the isoform faces the extracellular media in MCF-7 cells. Moreover, ERalpha-like proteins ( approximately 67 kDa) were found to be associated in isolated membrane subfractions from the cells. However, immunocytology of COS-1 (ER-negative) and SHM cells transfected with the complete cDNA coding for the cloned ERalpha and beta isoforms showed exclusive nuclear localization of the overexpressed ERs. The non-classical distribution of native ERalpha-like proteins in each cell line, suggests an alternative mode of ERalpha cellular localization/function. Cell type-dependent processing may account for the differential localization shown by native and expressed receptors in the systems considered.
Monje
P
P
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000, Bahía Blanca, Argentina.
Zanello
S
S
Holick
M
M
Boland
R
R
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014599:Uterus / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*
D047628:Estrogen Receptor alpha; D020033:Protein Isoforms; D011960:Receptors, Estrogen; D004958:Estradiol