A cell-based, quantitative and isoform-specific assay for exchange proteins directly activated by cAMP

Yingmin Zhu,Pei Luo,Xiaodong Cheng,Fang Mei

doi:10.1038/s41598-017-06432-4

Abstract

Extensive functional studies of the exchange protein directly activated by cAMP (EPAC) family of signaling molecules have demonstrated that EPAC proteins play a fundamental role in several physiological and pathophysiological responses, therefore are attractive drug targets. In this report, the development of a cell-based, medium to high throughput screening assay that is capable of monitoring EPAC-mediated activation of cellular Rap1 in an isoform-specific manner is described. This assay adapts a conventional ELISA format with immobilized RalGDS-RBD as a bait to selectively capture GTP-bound active Rap1. As a result, it fills an urgent need for a cell-based EPAC assay that can be conveniently performed using microtiter plates for the discovery and/or validation of isoform-specific EPAC agonists and antagonists.

Highlights

Exchange proteins directly activated by cAMP (EAPC1 and EPAC2) mediate the intracellular functions of cAMP by acting as guanine nucleotide exchange factors for the Ras-like small GTPases, Rap[1] and Rap[21, 2]
Several recent studies have reported the effective development of exchange protein directly activated by cAMP (EPAC)-specific antagonists using high throughput screening (HTS) biochemical assays[27,28,29]
Activation of EPAC1 or EPAC2 in these cell lines by cAMP elevating agents leads to the accumulation of Flag-Rap1-GTP, which can be captured by RalGDSRBD immobilized in a nickel-coated 96-well microplate

Summary

Introduction

Exchange proteins directly activated by cAMP (EAPC1 and EPAC2) mediate the intracellular functions of cAMP by acting as guanine nucleotide exchange factors for the Ras-like small GTPases, Rap[1] and Rap[21, 2]. Developing small molecule EPAC-specific modulators has evolved into an active area of research within the field for the last few years[24,25,26]. A robust cell-based assay equipped to measure the activity of EPAC proteins in a medium to high throughput setup is lacking. The design and execution of an isoform specific cell-based assay capable of measuring cellular activity of EPAC proteins in a microplate format are described

Methods

Results

Conclusion