Kinetic analysis of estrogen receptor homo- and heterodimerization in vitro.
The coexistence of ERalpha and ERbeta suggests that active receptor complexes are present as homo- or heterodimers. In addition each of three forms of active receptors may trigger different cellular responses. A real-time biosensor based on surface plasmon resonance was used as instrument to determine binding kinetics of homo- and heterodimerization of estrogen receptor alpha and beta. Partially purified full-length estrogen receptor alpha was expressed intracellularly as a C-terminal fusion to a hexa-histidine tag using the baculovirus-expression system. Purified estrogen receptor alpha and beta without tags were used as partners in the dimerization process. An association rate constant of 3.6 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the homodimer formation of ERalpha and 5.7 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the heterodimer formation was found assuming a pseudo first-order reaction kinetic. The equilibrium dissociation constant for homodimerization of ERalpha was 2.2 x 10(-8) to 5.4 x 10(-8) and 1.8 x 10(-8) to 2.6 x 10(-8)M for the heterodimer formation. The homo- and heterodimer formation was characterized by a slow association kinetics and kinetic rate constants were within the same range.
Jisa
Elisabeth
E
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Jungbauer
Alois
A
D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D019281:Dimerization; D004958:Estradiol / Q000378:metabolism; D047628:Estrogen Receptor alpha; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D047628:Estrogen Receptor alpha; D008024:Ligands; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D004958:Estradiol