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Evaluation of Conformational Changes in hER-HBD by Pharmacological Dissection of Hormone Dissociation Rates in a Homogeneous Hormone-Binding Assay

  • Johan Häggblad
  • Bo Carlsson
  • Jean-Pierre Raynaud
Conference paper

Summary

Hormone dissociation rates can be used as a surrogate parameter to monitor changes in conformational states, as the hormone binding affinity is directly related to the dissociation rate and is dependent on receptor conformation. High-resolution studies of dissociation rates are tedious because the bound radioligand has to be separated from unbound. In contrast, homogeneous radioligand binding assays are rapid and have high temporal resolution.The homogeneous assay format allows rapid mixing, followed by continuous measurement of bound radioactivity. We describe a homogeneous hormone-binding assay for human estrogen-receptor hormone-binding domain (hER-HBD) produced in a yeast expression system. The binding assay was used for quantitation of dissociation rates for [3H]-estradiol (E2) by addition of different estrogens and anti-estrogens. Our results revealed that different compounds caused significantly different monophasic dissociation rates (expressed as min) for [3H]-E2: DES (0.046 min−1) > >TAM (0.040) >E3 (0.032) ≈ E2 (0.029). However, a closer examination of the concentration dependence showed that at higher concentrations certain nonsteroidal compounds induced biexponential dissociation curves. We propose that, depending on the concentration and identity of the chaser used, different conformational states of the hER-HBD are induced. These different states have different affinities for the hormone.

Keywords

Dissociation Rate Conformational State Estradiol Concentration Exponential Decay Model Receptor Conformation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Salomonsson M, Häggblad J, O’Malley B, Sitbon G (1994) The human estrogen receptor hormone binding domain dimerizes independently of ligand activation. J Steroid Biochem Mol Biol 48:447–452PubMedCrossRefGoogle Scholar
  2. 2.
    Haggblad J, Carlsson B, Kivelä P, Siitari H (1994) Scintillating microtitration plates as platform for determination of [3H]-estradiol binding constants for the hER-HBD. Biotechniques, in press.Google Scholar

Copyright information

© Springer-Verlag New York, Inc. 1996

Authors and Affiliations

  • Johan Häggblad
  • Bo Carlsson
  • Jean-Pierre Raynaud

There are no affiliations available

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