Estrogen receptor microarrays: subtype-selective ligand binding.
We present the first example of a nuclear hormone receptor microarray, using for illustration the ligand-binding domains of the two estrogen receptors, ERalpha-LBD and ERbeta-LBD. The proteins are printed and allowed to attach to aldehyde slides; the efficiency of attachment depends on whether the LBD is liganded with agonists (low attachment) versus liganded with antagonists or unliganded (high attachment). This suggests that attachment is orientation specific and involves principally a single lysine residue. The attached ERs retain good ligand-binding activity that can be assessed using an estradiol-fluorophore conjugate, and specific and ER subtype-selective binding of ligands can be determined conveniently in competitive binding assays. This powerful new, high-throughput technique to study ligand binding to ER-LBDs can be extended to other nuclear hormone receptors and adapted to assay the recruitment of coregulator proteins.
Kim
Sung Hoon
SH
Department of Chemistry, University of Illinois, 600 South Matthews Avenue, Urbana, Illinois 61801, USA.
Tamrazi
Anobel
A
Carlson
Kathryn E
KE
Daniels
Jonathan R
JR
Lee
In Young
IY
Katzenellenbogen
John A
JA
D000447:Aldehydes / Q000737:chemistry; D001667:Binding, Competitive; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D008024:Ligands; D008958:Models, Molecular; D040081:Protein Array Analysis / Q000379:methods*; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism*
D000447:Aldehydes; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D005456:Fluorescent Dyes; D008024:Ligands; D011960:Receptors, Estrogen