A method for identifying G protein-coupled receptor dimers and their interfaces

Biochim Biophys Acta Mol Cell Res. 2021 Jan;1868(1):118887. doi: 10.1016/j.bbamcr.2020.118887. Epub 2020 Oct 16.

Abstract

The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers.

Keywords: Bioluminescence resonance energy transfer (BRET); Dimer; G protein-coupled receptor; Interface; Mass spectrometry; Transmembrane domain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs / genetics
  • Cell Membrane / chemistry
  • Dimerization*
  • GTP-Binding Proteins / chemistry
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Protein Conformation*
  • Protein Domains / genetics
  • Protein Multimerization / genetics
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / ultrastructure*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Survivin / chemistry
  • Survivin / genetics*
  • tat Gene Products, Human Immunodeficiency Virus / chemistry
  • tat Gene Products, Human Immunodeficiency Virus / genetics*

Substances

  • HIV-TAT-survivin (T34A) protein
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Survivin
  • tat Gene Products, Human Immunodeficiency Virus
  • GTP-Binding Proteins