Regulation of Epac by cAMP

Abstract

The role of cAMP as a second messenger in cellular signalling is well established. In the eukaryotic cell the targets of cAMP are PKA, cyclic nucleotide gated ion channels and Epac. All these proteins have a cAMP-binding domain of same architecture in common. Its function is to bind cAMP and thereby sense the presence of cAMP. Binding of cAMP to this sensor domain is transformed into an altered property of the whole protein. In spite of the important role of cAMP in regulating cellular events, the knowledge on a molecular level of how cAMP binding is translated into protein action was limited so far. For a long time structural information was available only for cAMP-bound (i.e. “active”) cAMP-binding domains, but information of cAMP free (i.e. “inactive”) cAMP-binding domains was missing. We determined the cAMP free crystal structure of the regulatory region of Epac2 containing two cAMP-binding domains. The high conservation of both sequence and structure between Epac and PKA allows to compare these structures as representatives of the ligand bound and the ligand free state of cAMP-binding domains in general. From these analyses a general model was developed of how cAMP-binding is sensed by cAMP-binding domains. A relative small rearrangement of residues directly involved in cAMP binding is transduced to a reorientation of the C-terminal helical part of the cAMP-binding domain. The helices of the different classes of cAMP-regulated proteins have common features related to the rearrangements induced by cAMP and unique features to allow communication to the respective remote parts of the protein. By adaptation of the C-terminal helix to the individual requirements in the context of the regulated protein class, the cAMP-binding domain can be used as an universal switch unit. The critical importance of this helix for the regulation of PKA, CAP and cyclic nucleotide gated ion channels is described in the literature and was shown for Epac by various approaches in this thesis. In Epac the helix forms an interface interacting with the catalytic region. This interaction blocks the catalytic centre in the absence of cAMP. This binding and therefore the blockage is terminated after binding of cAMP. To complete the structural investigation, extensive biochemical analysis was undertaken. This confirmed already mentioned the importance of the C-terminal helix and corroborated the mechanism suggested for cAMP sensing. In addition, the requirements for productive cAMP-binding and activation were characterised. Thus, the action of Epac specific activators such as 8-pCPT-2’-O-Me-cAMP can be explained. This analogue is able to activate Epac with higher efficiency than normal cAMP, but can neither activate nor inhibit PKA. These studies showed also that it is in principle possible to inhibit Epac with certain cAMP analogues. Based on these findings it should be possible to develop Epac specific inhibitors. This will allow the specific investigation of Epac mediated effects.