Structural studies interaction between CRF G-protein coupled receptors & ligands

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Corticotropin releasing factor (CRF) and related ligands play key not only roles in the modulation of adaptive responses of the endocrine, central nervous and immune system to stress but also within the cardiovascular and gastrointestinal systems. The ligand's effects are mediated by binding to CRF G-protein coupled receptors (GPCR's). The aim of this application is the structural elucidation of the interaction between peptides of the CRF family and their receptors. The proposed studies will exploit the specificities of agonist/antagonist-receptor interactions, will elucidate the molecular mechanism of the signaling pathway through the membrane and will facilitate the improvement of drugs targeting stress-related and other diseases. The important observation towards the aims described above is the determination of a major binding site of the CRF family of peptides, which is located in the N-terminal extracellular domain (ECD1) of CRFR's. We determined the three-dimensional NMR structure of the extracellular domain of CRFR2beta and identified its ligand binding site. Here, we propose to elucidate the dynamics and the three-dimensional structures of the extracellular CRF receptors both free and in complex with agonists and antagonists. It is furthermore proposed to determine the three dimensional structure of a hormone analogue when bound to full-length receptor through transferred NOEs and "transferred" residual dipolar couplings, and in parallel, it is aimed to obtain structural insights of the full-length receptor using our established expression protocol of functional CRFR1 in E. coli and TROSY-NMR. We believe that these studies will provide molecular insights of the ligand-receptor specificities and set a structural base for the improvement of drugs targeting stress-related diseases. The structures and dynamics will be determined using standard protocols.