Functional Characterization of CRF Receptors Expressed in E.coli

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 389-408-Signaling Originating from Membrane Receptors
Basic/Translational
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-405
Marilyn H Perrin*1, Roberto Jappelli2, Kathy A Lewis3, Joan M Vaughan3, Judit Erchegyi3, Jean E Rivier4, Wylie Vale5 and Roland Riek6
1The Salk Institute, La Jolla, CA, 2the Salk Institute, La Jolla, CA, 3The Salk Institute, 4Salk Inst, San Diego, CA, 5the salk institute, 6ETH, Zurich
Corticotropin releasing factor receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response.  The two CRF receptors, CRFR1 and CRFR2 exhibit specificity in ligand binding.  Of interest is the selective binding of the small molecule antagonist, antalarmin to CRFR1. In order to dissect the receptors’ binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β, were expressed in E.coli.  The properties of bacteria expressing different CRFR2β-PhoA gene fusion products were found to be in agreement with the predicted hepta-helical membrane topology model.  The large majority of receptor proteins became integrated in the bacterial membrane.  Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α.  Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions recapitulated, in part, the complex pharmacology observed in eukaryotic cells.  Pachymedusa dacnicolor, PD-sauvagine, a new CRF agonist bound to the two receptors expressed both in mammalian cells and in E.coli.  Interestingly, the small molecule antagonist, antalarmin, displaced labeled PD-sauvagine bound to CRFR1α expressed in mammalian cells as well as in E.coli.  The binding site for antalarmin is in the third and fifth transmembrane domains of CRFR1α.  The observation that antalarmin displaced labeled PD-sauvagine bound to CRFR1α when expressed in E.coli suggests that there is a subset of receptors whose transmembrane segments three and five are correctly folded.  Use of this new CRF ligand together with the E.coli expression system may be useful for further structural studies.

Disclosure: JER: , Sentia Medical Sciences, Inc., Principal Investigator, Salk Institute. Nothing to Disclose: MHP, RJ, KAL, JMV, JE, WV, RR

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm