In vitro interaction of estradiol receptor with Ca2+-calmodulin.
Estradiol receptor (ER) activity requires interaction with hormone and specific DNA sequence. We now report that this receptor also interacts with calmodulin (CaM), the major intracellular mediator of Ca2+ action in eucaryotic cells. This interaction has been observed using both CaM-Sepharose and [125I]CaM. Crude and purified [3H]ER complex show high affinity interaction with CaM-Sepharose [dissociation constant (Kd) 0.12 and 0.16 nM, respectively]. Unoccupied receptor shows a similar high affinity interaction. Tamoxifen-ER complex also binds to CaM-Sepharose. Several findings show that this CaM-ER interaction is very specific: lack of this interaction has been observed in the presence of trifluoperazine, an inhibitor of protein binding to CaM; the receptor binds neither Sepharose, nor parvalbumin-Sepharose; competition of interaction of [3H]ER complex with CaM-Sepharose is observed by cold ER complex; rat liver glucocorticoid receptor does not bind to CaM-Sepharose. The interaction of purified receptor with 125I-labeled CaM has been detected by various techniques: centrifugation through sucrose gradient of CaM incubated with receptor shows that CaM binds to a protein forming a complex sedimenting at 5 S. This complex is shifted to the 7.5 S region by a monoclonal antireceptor antibody. Incubation of CaM with receptor followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography of the immunoprecipitated receptor shows that [125I]CaM coprecipitates with the receptor. Competition of this interaction by an excess of cold CaM is observed. Interaction of the receptor with CaM is also observed by the overlay technique.(ABSTRACT TRUNCATED AT 250 WORDS)
Castoria
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G
II Cattedra di Patologia Generale, Istituto di Patologia Generale e Oncologia, I Facoltà di Medicina e Chirurgia, Università di Napoli, Italy.
Migliaccio
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A
Nola
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E
Auricchio
F
F
D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D011959:Receptors, Estradiol / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D012685:Sepharose
D002147:Calmodulin; D011959:Receptors, Estradiol; D011960:Receptors, Estrogen; D012685:Sepharose; D002118:Calcium