Mechanism of Arrestin Activation and GPCR Binding

• Applicant: Dr. Martin Heck, since 8/2021
• Subject Area: Biophysics
• Term: from 2009 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 156861807

Arrestin proteins regulate the function of G protein-coupled receptors (GPCRs), the largest and most pharmacologically important class of cell membrane receptors. Four structurally conserved arrestin isoforms bind hundreds of different GPCRs thereby influencing a wide array of physiological processes. It is still not understood at the molecular level how arrestin-receptor interactions affect signal transduction within the cell and the eventual cellular response. Our previous work on rod cell arrestin and the photoreceptor rhodopsin has shown that complex formation involves a sequential interaction of key sites of the proteins with each other and with the membrane. Initial contact with the receptor induces a partial activation of arrestin, leading to the formation of a ‘pre-complex’, which then transitions to a ‘high-affinity complex’ that entails full arrestin activation. In the proposed project we will characterize the molecular mechanism of this multi-step process by investigating the roles of the individual interaction sites of arrestin and receptor as well as the sequence of their interactions. A further goal is to understand within a physiological context the significant influence of the receptor activation state (as controlled by the type of ligand) and receptor phosphorylation (number and position of phosphate groups) on arrestin binding. In addition to native rhodopsin we will employ site-specifically labelled mutant proteins and synthetic peptides derived from the key interaction sites of arrestin and receptor. The experimental system will be mainly based on in vitro reconstitution of the well-established visual system (arrestin-1 and rhodopsin) and will further extend to arrestin-2/-3 and other GPCRs. Site-directed fluorescence spectroscopy will be our primary method to observe conformational changes and intermolecular interactions, complemented by other biochemical and biophysical methods (e.g. UV/Vis- and FTIR spectroscopy). The different binding modes and the role of conformational flexibility in complex formation will be explored with the help of computational modelling and molecular dynamics simulations. The long term goal of the project is to integrate the interactions between ligand, receptor, membrane and arrestin into an all-encompassing model, which will help answer the ‘big questions’ in the GPCR field: how do small changes in protein structure control the signalling pathway and the resulting physiological response, and what possibilities are there to pharmacologically influence these pathways.

• DFG Programme: Research Grants
• Major Instrumentation: Fluorescence spectrometer
• Instrumentation Group: 1850 Spektralfluorometer, Lumineszenz-Spektrometer (außer Filterfluorometer
• Ehemalige Antragstellerin: Dr. Martha E. Sommer, until 8/2021