Endocrine Abstracts (2006) 11 P730

A ras recruitment screen for glucocorticoid receptor-interacting proteins using the entire receptor as bait

K Ecker, F Wolf, S Geley & A Helmberg

Medical University, Division of Molecular Pathophysiology, Innsbruck, Austria.

Glucocorticoid hormones play a key role in glucose, fat and protein metabolism and dampen immune and inflammatory reactions. Synthetic equivalents are widely used as therapeutic drugs. Upon hormone binding, the glucocorticoid receptor (GR) detaches from a cytoplasmic multiprotein complex, translocates to the nucleus and regulates the transcription of target genes either directly or indirectly through interference with other transcription regulators.

While many GR interaction partners have been identified using transcription-based assays, less effort has been invested in unbiased screening for protein-protein interactions.

One classical tool to detect protein-protein interactions, the yeast two-hybrid system, is inherently ill-equipped to handle transcription factors, as it relies on the reconstitution of a transcription factor activity by the sought interaction.

To overcome these limitations, we used an alternative yeast two hybrid system developed by A. Aronheim, the so called ‘reverse ras recruitment system’. Here, successful interaction between bait and prey reconstitutes defective Ras/Adenylate cyclase signaling in the yeast mutant cdc25-2. Using the entire human GR as bait, we are screening a HeLa cDNA library. Positive candidates identified in this screen are expressed as FLAG -tagged fusion proteins and validated in mammalian cells by coimmunoprecipitation experiments. Potential colocalization of receptor and interaction partner is monitored by fluorescence microscopy and live cell imaging by expressing preys as YFP- and the receptor as CFP-tagged fusion proteins. So far, screening of 200 000 clones yielded 40 (0.02%) primary interaction candidates. The isolation of proteins that have previously been described to make contact with the GR, such as Bip, proves the usefulness of this approach. Several novel interaction partners were identified, some of which are currently more closely investigated.

Support: FWF (P16013)

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