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    Immobilization of arrestin-3 on different biosensor platforms for evaluating GPCR binding

    Year: 2020

    Journal: Phys. Chem. Chem. Phys., Volume 22, NOV 7, page 24086–24096

    Authors: Avsar, Saziye Yorulmaz; Kapinos, Larisa E.; Schoenenberger, Cora-Ann; Schertler, Gebhard F. X.; Muhle, Jonas; Meger, Benoit; Lim, Roderick Y. H.; Ostermaier, Martin K.; Lesca, Elena; Palivan, Cornelia G.

    G protein-coupled receptors (GPCRs) are a large and ubiquitous family of membrane receptors of great pharmacological interest. Cell-based assays are the primary tool for assessing GPCR interactions and activation but their design and intrinsic complexity limit their application. Biosensor-based assays that directly and specifically report GPCR-protein binding (e.g. arrestin or G protein) could provide a good alternative. We present an approach based on the stable immobilization of different arrestin-3 proteins (wild type, and two mutants, mutant X (arrestin-3 I386A) and mutant Y (arrestin-3 R393E)) via histidine tags on NTA(Ni2+)-coated sensors in a defined orientation. Using biolayer interferometry (BLI), surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D), we were able to follow the interaction between the different arrestin-3 proteins and a representative GPCR, jumping spider rhodopsin-1 (JSR1), in a label-free manner in real-time. The interactions were quantified as binding affinity, association and dissociation rate constants. The combination of surface-based biosensing methods indicated that JSR1 showed the strongest binding to arrestin mutant Y. Taken together, this work introduces direct label-free, biosensor-based screening approaches that can be easily adapted for testing interactions of proteins and other compounds with different GPCRs.
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