Characterization of estrogen-binding sites associated with the endoplasmic reticulum of rat uterus.
Microsomes prepared from rat uterine homogenates harbor high-affinity (Ka = 10(10) M-1), low-capacity binding sites for estrogens. Previous work from our laboratory has demonstrated that these estrophiles are located on endoplasmic reticulum and are not cytosolic contaminants of the membrane preparation. Subfractionation of microsomes into granular and agranular membranes and polysomes revealed approximately equal distribution of estrogen-binding activity among each of these constituents. These binding sites were fully extractable with 0.6 M KCl. Microsomal estrophiles solubilized under conditions of low ionic strength and complexed with estradiol migrated as 8S forms on continuous sucrose gradients. In the presence of 0.4 M KCl, the solubilized binding sites exhibit a sedimentation coefficient of 4S. Extracted binding sites do not undergo heat-induced transformation from a 4S to 5S species. The monoclonal antibody JS34/32 interacted with the endoplasmic reticulum-associated estrogen-binding sites when present in 50-fold molar excess, but not at lower antibody to binding site ratios. In comparison, the rat uterine cytosolic estrogen receptor formed complexes with JS34/32 at antibody to receptor ratios as low as 2:1. These results suggest that the endoplasmic reticulum possesses estrogen-binding sites with biochemical properties that differ from those of the classically described cytosolic (loosely associated nuclear) estrogen receptor.
Evans
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AC
Department of Physiology and Endocrinology, Medical College of Georgia, Augusta.
Muldoon
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TG
D000818:Animals; D002458:Cell Fractionation; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism; D005260:Female; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure; D009994:Osmolar Concentration; D011132:Polyribosomes / Q000378:metabolism; D011189:Potassium Chloride; D051381:Rats; D011919:Rats, Inbred Strains; D011960:Receptors, Estrogen / Q000378:metabolism*; D012995:Solubility; D014599:Uterus / Q000648:ultrastructure*
D011960:Receptors, Estrogen; D004958:Estradiol; D011189:Potassium Chloride; D004247:DNA