| Rap proteins are Ras-like small G-proteins, which functions as molecular switches by cycling between a GDP-bound inactive and a GTP-bound active state. They control a wide variety of cellular process, most notably cell adhesion and cell junction formation, which play a crucial role in cell migration and tumour formation. Guanine nucleotide exchange factors (GEFs) are activating G-proteins by catalysing the exchange of GDP to GTP. A number of GEFs for Rap proteins have been identified. These multi-domain proteins are highly regulated and responsible for the temporal and spatial activation of Rap. The Rap-GEFs Epac1 and Epac2 are directly regulated by cAMP and particular Epac2 modulates insulin secretion from pancreatic -cells. The applicant determined the crystal structure of full length Epac2 in the inactive conformation, showing the mechanism of auto-inhibition and suggesting a model of activation1. In continuation the following is proposed: 1. Structure determination of the active complex, which include Epac, cAMP and Rap. 2. Biochemical and biophysical analysis of Epac2, including the mechanism of super-activation by certain cAMP-analogues. 3. Extension of these studies to other Rap-GEFs, most notably CalDAG-GEF which is directly or indirectly regulated by Ca2+ and DAG, and PDZ-GEF, for which it is currently unclear how it is regulated and whether second messengers are involved. The applicant will determine the crystal structure of these GEFs and study their biochemical and biophysical properties in detail. 4. As a further ambition, the applicant will aim to determine the structure of protein complexes including Rap-GEFs and associated proteins. The proposed studies are complementary to the research line of the host laboratory, which studies the biological aspects of Rap-GEFs, allowing a fruitful exchange between in vitro and in vivo approaches. Furthermore, several Rap-GEFs have been proposed as drug targets and detailed analysis of the proteins is vital for developing such drugs. |
