Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

Hanna K Buist,Joanne Sunderland,Brian O Smith,Colin Rickman,Boy Van Basten,Jessica Valli,Stephen J Yarwood,Bryon Ricketts,Ryan Hannam,Alex Davidson,Urszula Luchowska-Stańska,George S Baillie

doi:10.3390/cells10092307

Hanna K Buist, Joanne Sunderland + Show 10 more

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https://doi.org/10.3390/cells10092307

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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.

Highlights

The universal second messenger, 3,5 -cyclic adenosine monophosphate, was the first to be discovered in eukaryotic cells in 1958 by Earl Sutherland and colleagues [1]
Through the functional assessment of exchange protein directly activated by cyclic adenosine monophosphate (cAMP) 1 (EPAC1)∆Disheveled-EGL-plekstrin homology (DEP)-selective Affimers identified from the phage-display screens detailed in Table 1, Affimers with weak affinities and/or cross-reactivity negative control proteins were deselected
Successive phage-display screens were used to identify nine potential EPAC1-selective Affimer candidates, three of which were confirmed as being EPAC1 binders using a range of in vitro assays

Summary

Introduction

The universal second messenger, 3 ,5 -cyclic adenosine monophosphate (cAMP), was the first to be discovered in eukaryotic cells in 1958 by Earl Sutherland and colleagues [1]. Activation of Gs protein-coupled receptors (GsPCRs) promotes activation of intracellular adenylyl cyclases (AC), leading to the generation of cAMP inside the cell from ATP. The downstream signaling effects in response to cAMP production were originally attributed to activation of protein kinase A (PKA) isoforms [8]. Cyclic nucleotide-gated ion channels (HCN) and Popeye domain containing (POPDC) gene family are two additional recently discovered cAMP effectors [12,13,14]. PKA and EPAC are known to be anchored to specific intracellular sites by scaffold proteins, which act to maintain compartmentalization of cAMP signaling by anchoring PDEs adjacent to target effectors and, regulate the ability of cAMP to initiate downstream signaling events at discrete subcellular compartments or nanodomains [15]

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