Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1
Hanna K. Buist,
Urszula Luchowska-Stańska,
Boy van Basten,
Jessica Valli,
Brian O. Smith,
George S. Baillie,
Colin Rickman,
Bryon Ricketts,
Alex Davidson,
Ryan Hannam,
Joanne Sunderland,
Stephen J. Yarwood
Affiliations
Hanna K. Buist
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK
Urszula Luchowska-Stańska
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK
Boy van Basten
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK
Jessica Valli
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK
Brian O. Smith
Institute of Molecular Cell and System Biology, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
George S. Baillie
Institute of Cardiovascular & Medical Science, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
Colin Rickman
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK
Bryon Ricketts
Avacta Life Sciences, Unit 20, Ash Way, Thorp Arch Estate & Retail Park, Wetherby LS23 7FA, UK
Alex Davidson
Avacta Life Sciences, Unit 20, Ash Way, Thorp Arch Estate & Retail Park, Wetherby LS23 7FA, UK
Ryan Hannam
Avacta Life Sciences, Unit 20, Ash Way, Thorp Arch Estate & Retail Park, Wetherby LS23 7FA, UK
Joanne Sunderland
Avacta Life Sciences, Unit 20, Ash Way, Thorp Arch Estate & Retail Park, Wetherby LS23 7FA, UK
Stephen J. Yarwood
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK
An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149–811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.
