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

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)
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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.
Original languageEnglish
Article number2307
JournalCells
Volume10
Issue number9
DOIs
Publication statusPublished - 3 Sept 2021

Keywords

  • Affimer
  • Cyclic AMP
  • EPAC1
  • Microscopy
  • Phage display
  • Protein interactions

ASJC Scopus subject areas

  • General Medicine

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