| Project Detail |
Signaling by G-protein coupled receptors (GPCRs) is a research topic of high interest in the fields of pharmaceutical sciences and fundamental biology. In the ongoing SNF project in our laboratory, we were able to establish techniques which allow us to study highly challenging GPCRs (i.e. ß1AR, ß2AR and Rhodopsin) and their interaction partners at atomic resolution in solution by nuclear magnetic resonance (NMR). This lays the basis for the current proposal, where, in the realm of fundamental biological research, we will focus on GPCR desensitization. In particular, we will focus on the interplay of GPCRs with G-protein coupled receptor kinases (GRKs), an area, which compared to other processes in the GPCR field is still only poorly understood. We will study the different activation states that GRKs can attain and explore how interactions with GPCRs and other binding partners shift equilibria between active and inactive states. For such studies of molecular interactions and dynamics at the atomic level, NMR is the ideal method. However, these proteins represent highly challenging systems for NMR (GRKs have molecular weights of 60-80 kDa and can to date only be expressed in insect cells), such that we will develop methods to overcome these challenges, which will be for the benefit of the entire NMR field. In the realm of fundamental biology, the aims of this project are the following:1: Biophysical and functional characterization of the effects of substrates and effector molecules on GRK1 and GRK2. Determination of affinities of binding partners and characterization of the activation states induced by the effectors.2: Detailed structural and dynamic studies of the conformational equilibrium of the N-terminal a helix of GRK1 and GRK2 and correlation with kinase activity.3: Dissection of the allosteric network that translates interactions with GPCRs and other effectors to GRK activation.4: Finding of ligands of GRK2 with novel binding modes and characterization of the induced activation state. Inhibitors will serve as starting points for novel drugs and activators as valuable chemical biology probes.The fact that we have established human ß1AR as a system amenable to NMR studies is of particularly high value to pharmaceutical research. Our goal is to find novel starting points for more specific drugs to ß1- and ß2AR and to provide tools for chemical biology approaches. To this end we will establish fragment-based screening (FBS) for membrane proteins. To reduce protein consumption by an order of magnitude, we will exploit dynamic nuclear polarization techniques.The specific aims addressing pharmaceutical questions are the following:5: Establishment of a fluorinated fragment library optimized for screening of membrane proteins and development of suitable hyperpolarization techniques.6: Finding and characterization of ligands that specifically bind to ß1AR and ß2AR in active and inactive states, potentially at unexplored binding sites. |
