Detection of a raft-located estrogen receptor-like protein distinct from ER alpha.
17Beta-estradiol (17beta-E2) elicits at the cell membrane rapid actions that remain insensitive to the inhibitory effect of ICI 182,780, a pure estrogen antagonist, and therefore cannot be attributed to the classic nuclear receptors. We addressed the question of the identity of the protein involved in these rapid actions. We first examined the responses of several cell lines for intracellular calcium mobilization, an effect not inhibited by ICI 182,780, tamoxifen and raloxifen. We then demonstrated the presence of binding sites in the membranes, by incubating them with antibodies directed against different domains of ER alpha, and by flow cytometry analysis. The membrane proteins were eluted by affinity chromatography using E2 conjugated to bovine serum albumin as a ligand. Western blots of the elution fractions using an antibody directed against the ligand binding site of ER alpha showed the existence of a protein of approximately 50 kDa. The protein was concentrated in the lipid rafts, together with another heavier form of approximately 66 kDa. The 50 kDa protein was immunoprecipitable, and co-immunoprecipitation experiments showed that it was associated with the Gbeta(1-4) protein, but not with caveolin-1. The protein was expressed in ER alpha-null cells, like HO-23 and Cos-7 cells. Therefore, in the lipid rafts, there exists a protein, similar to, but molecularly distinct from ER alpha.
Heberden
Christine
C
Laboratoire de Nutrition et Sécurité Alimentaire, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
Reine
Fabienne
F
Grosse
Brigitte
B
Henry
Céline
C
Zagar
Yvrick
Y
Chaumaz
Gilles
G
Lieberherr
Michèle
M
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002458:Cell Fractionation; D002460:Cell Line; D004958:Estradiol / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D051381:Rats
D047628:Estrogen Receptor alpha; D004958:Estradiol; D002118:Calcium