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

Phys Chem Chem Phys. 2020 Oct 28;22(41):24086-24096. doi: 10.1039/d0cp01464h.

Abstract

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.

MeSH terms

  • Animals
  • Arthropod Proteins / metabolism
  • Biosensing Techniques
  • Immobilized Proteins / genetics
  • Immobilized Proteins / metabolism*
  • Lipid Bilayers / chemistry
  • Mutation
  • Phosphatidylcholines / chemistry
  • Protein Binding
  • Quartz Crystal Microbalance Techniques
  • Rhodopsin / metabolism*
  • Spiders / chemistry
  • Surface Plasmon Resonance
  • beta-Arrestin 2 / genetics
  • beta-Arrestin 2 / metabolism*

Substances

  • Arthropod Proteins
  • Immobilized Proteins
  • Lipid Bilayers
  • Phosphatidylcholines
  • beta-Arrestin 2
  • Rhodopsin
  • 1,2-oleoylphosphatidylcholine