Discoverx Corporation

Chin Yee Loh

Thomas S Wehrman

William Raab

Keith R Olson

Mahesh Mathrubutham

Methods and genetic constructs are provided for detecting the binding of nuclear hormone receptors to a coactivator/corepressor. The methods employ enzyme fragment complementation using fragments of &#x3b2;-galactosidase as the detection system. Cells are transformed to express the large fragment of &#x3b2;-galactosidase fused to a member of the complex with NHR for initiation of transcription and have it localized in the nucleus and to express the small fragment of &#x3b2;-galactosidase fused to the nuclear hormone receptor for binding to the member upon stimulation with a ligand.

Assays for nuclear hormone receptor binding

A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of &#x3b2;-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of &#x3b2;-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the &#x3b2;-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.

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Kensington, CA, United States of America

Chin Yee Loh