Determining topology of beta-arrestin binding to non-visual G-protein coupled receptors in live cells via genetic incorporation of chemical tools

• Applicant: Professorin Dr. Irene Coin
• Subject Area: Biochemistry
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 316443431

Beta-arrestins are direct signalling regulators of the about 800 G protein-coupled receptors (GPCRs) encoded in the human genome. Arrestin-GPCR complexes are highly dynamic and represent challenging objects for crystallography and cryo-EM. The few structures that have been solved so far all include receptors that feature a highly phosphorylated C-tail and carry often heavy artificial modifications. General aim of this project is to identify topological features of beta-arrestin binding to fully-glycosylated and minimally modified GPCRs directly in the natural context of the live mammalian cell. We apply genetically encoded non-canonical amino acids for extensive photo- and chemical crosslinking. We map proximity points within arrestin-GPCR complexes and pin-point intermolecular pairs of arrestin-receptor amino acids that lie in reciprocal proximity. We apply this information to molecular modelling experiments and build detailed models for the GPCR-arrestin interaction. In the first funding period, we used this strategy to characterize the stable interaction between beta-arrestin-1 and the parathyroid hormone receptor (PTH1R). We have built a detailed model for the PTH1R-beta-arr-1 complex that is based on more than three hundred experimental constraints. The model reveals a number of details in flexible receptor parts like loops and C-terminal region that are missing in all published GPCR-arrestin structures and reveals a hereto unknown role of beta-arrestin-1 regions, especially the distal region of the N-domain (N-edge). Moreover, we demonstrate that the approach is applicable to transient GPCR-arrestin interactions, which are not accessible to classical structure determination methods. Now, we want to clarify whether the observed role of the N-edge is a conserved feature for different GPCRs and possibly also for beta-arrestin-2. We want also to characterize alternative arrangements of the PTH1R-arrestin complex that we have observed, and that are not compatible either with the predominant state represented in the model or with any of the published structures. In the second place, we want to characterize arrestin binding to GPCRs that do not have a highly phosphorylated C-tail, for which 3D data are completely missing. Third, we want to investigate the effect of phosphorylation patterns generated by distinct GPCR kinases (GRKs) to conformational features of arrestin binding, as well as characterize transient GPCR-arrestin complexes. We expect our data will contribute to complete the understanding of the GPCR-arrestin interaction and in particular the connection between GPCR phosphorylation and structural features of GPCR-bound arrestin.

• DFG Programme: Research Grants