FLIM-FRET-Based Structural Characterization of a Class-A GPCR Dimer in the Cell Membrane

J Mol Biol. 2020 Jul 24;432(16):4596-4611. doi: 10.1016/j.jmb.2020.06.009. Epub 2020 Jun 15.

Abstract

Class-A G protein-coupled receptors (GPCRs) are known to homo-dimerize in the membrane. Yet, methods to characterize the structure of GPCR dimer in the native environment are lacking. Accordingly, the molecular basis and functional relevance of the class-A GPCR dimerization remain unclear. Here, we present the dimeric structural model of GPR17 in the cell membrane. The dimer mainly involves transmembrane helix 5 (TM5) at the interface, with F229 in TM5, a critical residue. An F229A mutation makes GPR17 monomeric regardless of the expression level of the receptor. Monomeric mutants of GPR17 display impaired ERK1/2 activation and cannot be properly internalized upon agonist treatment. Conversely, the F229C mutant is cross-linked as a dimer and behaves like wild-type. Importantly, the GPR17 dimer structure has been modeled using sparse inter-protomer FRET distance restraints obtained from fluorescence lifetime imaging microscopy. The same approach can be applied to characterizing the interactions of other important membrane proteins in the cell.

Keywords: FLIM FRET; GPCR; GPR17; MD simulation; rigid-body refinement.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / metabolism*
  • Fluorescence Resonance Energy Transfer
  • HEK293 Cells
  • Humans
  • Mice
  • Microscopy, Fluorescence
  • Models, Molecular
  • Mutation*
  • Nerve Tissue Proteins / chemistry*
  • Nerve Tissue Proteins / genetics
  • Protein Multimerization
  • Protein Structure, Secondary
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / genetics

Substances

  • GPR17 protein, human
  • GPR17 protein, mouse
  • Nerve Tissue Proteins
  • Receptors, G-Protein-Coupled