Identification and characterization of an affimer affinity reagent for the detection of the cAMP sensor, EPAC1

Buist, H. K. et al. (2021) Identification and characterization of an affimer affinity reagent for the detection of the cAMP sensor, EPAC1. Cells, 10(9), 2307. (doi: 10.3390/cells10092307)

Abstract

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.

• Item Type: Articles
• Additional Information: This research was jointly funded by Medical Research Scotland (grant number: PhD-868- 2015) and Avacta Life Sciences, through a studentship awarded to H.K.B. U.L.-S. was a British Heart Foundation research scholar (grant number FS/17/12/32703). B.v.B. was funded by a James Watt Research Scholarship, funded by Heriot-Watt University.
• Keywords: EPAC1, cyclic AMP, affimer, microscopy, protein interactions, phage display.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Baillie, Professor George and Yarwood, Dr Stephen and Smith, Dr Brian
• Authors: Buist, H. K., Luchowska-Stańska, U., van Basten, B., Valli, J., Smith, B. O., Baillie, G. S., Rickman, C., Ricketts, B., Davidson, A., Hannam, R., Sunderland, J., and Yarwood, S. J.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health; College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Cells
• Publisher: MDPI
• ISSN: 2073-4409
• ISSN (Online): 2073-4409
• Published Online: 03 September 2021
• Copyright Holders: Copyright © 2021 The Authors
• First Published: First published in Cells 10(9): 2307
• Publisher Policy: Reproduced under a Creative Commons License