Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1
| Autor: | 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 |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: | |
| Zdroj: | Cells, Vol 10, Iss 9, p 2307 (2021) |
| Druh dokumentu: | article |
| ISSN: | 10092307 2073-4409 |
| DOI: | 10.3390/cells10092307 |
| Popis: | 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. |
| Databáze: | Directory of Open Access Journals |
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