Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is a ctivated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP) -binding domain. We investigated the equilibrium and dynamics of the intera ction of cAMP and Epac1 using a newly designed fluorescence analogue of cAM P, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by compe tition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (K-d) of 4 muM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparen tly not affected when the catalytic domain is present, despite the fact tha t binding of cAMP results in activation of Epac1. This indicates that for t he activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent K-d of Epac to cAMP was about fivefold lo wer, suggesting that substrate interaction stabilizes cAMP binding. Since t he fluorescent analogues used here were either less able or unable to induc e activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of CEF activity are uncoupled processes and that thus ap propriate cAMP analogues can be used as inhibitors of the Epac1-mediated si gnal transduction pathway of Rap. (C) 2001 Academic Press.