An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

doi:10.1073/pnas.0607385103

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Departments of Radiology and Bioengineering, Bio-X Program, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, James H. Clark Center, 318 Campus Drive, East Wing, First Floor, Stanford, CA 94305-5427

Communicated by Michael E. Phelps, University of California School of Medicine, Los Angeles, CA, August 24, 2006 (received for review May 17, 2006)

Abstract

Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER–LBD) in complex with different ligands, we hypothesized that the hER–LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER–LBD fusion proteins that could lead to split Renilla/firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER–LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER–LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER–LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.

Footnotes

*To whom correspondence may be addressed. E-mail: paulmur8{at}stanford.edu or sgambhir{at}stanford.edu
Author contributions: R.P. and S.S.G. designed research; R.P. performed research; R.P. and S.S.G. analyzed data; and R.P. and S.S.G. wrote the paper.
The authors declare no conflict of interest.
Abbreviations:

RLUC,

Renilla luciferase;

FLUC,

firefly luciferase;

N-RLUC,

N-fragment of RLUC gene;

C-RLUC,

C-fragment of RLUC gene;

ER,

estrogen receptor;

hER,

human ER;

LBD,

ligand-binding domain;

SERM,

selective ER modulator;

DES,

diethylstilbestrol;

4-OHT,

4-hydroxytamoxifen;

E2,

17β-estradiol;

H12,

helix 12;

ICI,

ICI182,780.