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

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.

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

“…We have championed the use of SpIDA to monitor GPCR quaternary organization for a number of years ( 21 , 25 , 26 , 34 , 35 ), and it is therefore gratifying to note that Işbilir et al . ( 36 ) have now also adopted this approach and in so doing have generated results strongly in concordance with our own observations.…”

“…More clearly, whilst the current article was under review, Işbilir et al . ( 36 ) reported, as we do, that AMD3100 is unable to alter the dimeric proportion of CXCR4. Equally, as we do, they noted that IT1t was highly effective at causing monomerization of CXCR4 and did so rapidly ( 36 ).…”

“…( 36 ) reported, as we do, that AMD3100 is unable to alter the dimeric proportion of CXCR4. Equally, as we do, they noted that IT1t was highly effective at causing monomerization of CXCR4 and did so rapidly ( 36 ). We have championed the use of SpIDA to monitor GPCR quaternary organization for a number of years ( 21 , 25 , 26 , 34 , 35 ), and it is therefore gratifying to note that Işbilir et al .…”

“…Oligomerization became more complex with increased expression, suggesting an effect of mass action. However, significant levels of higher-order organization above dimers were largely restricted to receptor levels beyond those found in either normal or pathophysiological conditions and at receptor expression level higher than studied by others ( 19 , 36 ). To reinforce such conclusions, we employed blue native-PAGE as a completely different approach ( 37 ).…”

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

“…The proposed dimerization of family A GPCRs, such as rhodopsin or the beta-adrenergic receptors, has sparked intense debate based on the wide range of results that are seen across techniques and preparations (Ferré et al, 2014, Milligan et al, 2019, Sleno and Hébert, 2019). However, a reasonable interpretation of the litany of studies is that dimerization can indeed occur and contribute to various modes of regulation of family A GPCR function, but is likely transient, based on single molecule imaging studies (Hern et al, 2010, Kasai et al, 2011, Meral et al, 2018, Işbilir et al, 2020, Möller et al, 2020, Felce et al, 2017), and is not required for G protein activation, based on reconstitution studies (Bayburt et al, 2007, Whorton et al, 2008, Whorton et al, 2007, Kuszak et al, 2009). Importantly, a structural understanding of GPCR dimerization has remained elusive with a wide range of dimer interfaces proposed for different GPCRs based on cross-linking, mutagenesis and x-ray crystallography data.…”