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  3. Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

  • Proc Natl Acad Sci U S A. 2020 Nov 17;117(46):29144-29154. doi: 10.1073/pnas.2013319117.
Ali Işbilir 1 2,  Jan Möller 1 2,  Marta Arimont 3,  Vladimir Bobkov 3 4,  Cristina Perpiñá-Viciano 2 5,  Carsten Hoffmann 2 5,  Asuka Inoue 6,  Raimond Heukers 3 7,  Chris de Graaf 3 8,  Martine J Smit 3,  Paolo Annibale 9 2,  Martin J Lohse 9 2 10
Affiliations

Affiliations

  • 1 Receptor Signaling Group, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
  • 2 Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany.
  • 3 Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands.
  • 4 argenx BVBA, Zwijnaarde 9052, Belgium.
  • 5 Institute of Molecular Cell Biology, University Hospital Jena, University of Jena, 07745 Jena, Germany.
  • 6 Graduate School of Pharmaceutical Sciences, Tohoku University, 980-8578 Sendai, Japan.
  • 7 QVQ Holding B.V., 3584 CL Utrecht, The Netherlands.
  • 8 Sosei Heptares, CB21 6DG Cambridge, United Kingdom.
  • 9 Receptor Signaling Group, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; Paolo.Annibale@mdc-berlin.de m.lohse@mdc-berlin.de.
  • 10 ISAR Bioscience Institute, 82152 Munich, Germany.
PMID: 33148803 DOI: 10.1073/pnas.2013319117
Abstract

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC Chemokine Receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in Cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

Keywords

GPCR; basal activity; chemokine receptor; dimerization; microscopy.

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