Next Article in Journal
Overexpression of Msx1 in Mouse Lung Leads to Loss of Pulmonary Vessels Following Vascular Hypoxic Injury
Next Article in Special Issue
Functional Proteomic Profiling of Triple-Negative Breast Cancer
Previous Article in Journal
Comparison of a New 68Ga-Radiolabelled PET Imaging Agent sCD146 and RGD Peptide for In Vivo Evaluation of Angiogenesis in Mouse Model of Myocardial Infarction
Previous Article in Special Issue
Targeting Canonical and Non-Canonical STAT Signaling Pathways in Renal Diseases
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cells
Volume 10
Issue 9
10.3390/cells10092307
cells-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Hanna K. Buist
1,
Urszula Luchowska-Stańska
1,
Boy van Basten
1,
Jessica Valli
1,
Brian O. Smith
2,
George S. Baillie
3,
Colin Rickman
1,
Bryon Ricketts
4,
Alex Davidson
4,
Ryan Hannam
4,
Joanne Sunderland
4 and
Stephen J. Yarwood
1,*
1
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK
2
Institute of Molecular Cell and System Biology, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
3
Institute of Cardiovascular & Medical Science, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
4
Avacta Life Sciences, Unit 20, Ash Way, Thorp Arch Estate & Retail Park, Wetherby LS23 7FA, UK
*
Author to whom correspondence should be addressed.
Cells 2021, 10(9), 2307; https://doi.org/10.3390/cells10092307
Submission received: 28 July 2021 / Revised: 30 August 2021 / Accepted: 1 September 2021 / Published: 3 September 2021
(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Signaling and Regulated Cell Death)
Browse Figures
Versions Notes

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.
Keywords: EPAC1; cyclic AMP; affimer; microscopy; protein interactions; phage display EPAC1; cyclic AMP; affimer; microscopy; protein interactions; phage display

Share and Cite

MDPI and ACS Style

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.; et al. Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1. Cells 2021, 10, 2307. https://doi.org/10.3390/cells10092307

AMA Style

Buist HK, Luchowska-Stańska U, van Basten B, Valli J, Smith BO, Baillie GS, Rickman C, Ricketts B, Davidson A, Hannam R, et al. Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1. Cells. 2021; 10(9):2307. https://doi.org/10.3390/cells10092307

Chicago/Turabian Style

Buist, Hanna K., Urszula Luchowska-Stańska, Boy van Basten, Jessica Valli, Brian O. Smith, George S. Baillie, Colin Rickman, Bryon Ricketts, Alex Davidson, Ryan Hannam, and et al. 2021. "Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1" Cells 10, no. 9: 2307. https://doi.org/10.3390/cells10092307

APA Style

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., & Yarwood, S. J. (2021). Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1. Cells, 10(9), 2307. https://doi.org/10.3390/cells10092307

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop