Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes

Abstract

Canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different intracellular effector responses to regulate cell migration: CXCR4 couples to G proteins and arrestins, while ACKR3 is arrestin-biased. CXCR4 also signals only in response to CXCL12, whereas ACKR3 recruits β-arrestin in response to CXCL12, CXCL12 variants, and other peptides and proteins. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we utilized single-molecule FRET. The data revealed that apo CXCR4 preferentially populates a high-FRET inactive state while apo ACKR3 shows little conformational preference, consistent with its promiscuous ligand recognition and propensity for activation. Markedly different conformational landscapes of the receptors in response to ligands suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. The dynamic properties of ACKR3 may also underly its inability to couple to G proteins, making it arrestin-biased.

Conformational dynamics underlying atypical chemokine receptor 3 activation

“…The high basal activity of ACKR3 is reflected by the less pronounced absolute ΔHDX values upon agonist-binding compared to inverse agonist-binding. This also correlates to a recent single-molecule Förster resonance energy transfer (FRET) study ( 52 ) demonstrating substantial populations of active and intermediate states of apo ACKR3. ICL2 and the intracellular tip of TM4 also exhibited significant deprotection upon agonist binding, in both the MD simulations and the HDX-MS experiments ( Fig.…”