Bilayer lipids modulate ligand binding to atypical chemokine receptor 3

Eberle, Stefanie Alexandra; Gustavsson, Martin

doi:10.1016/j.str.2024.04.018

“…Single receptor molecules of labeled CXCR4 or ACKR3 were reconstituted into phospholipid nanodiscs to mimic the native membrane bilayer environment. Nanodiscs have been used in a wide variety of structural and biophysical studies of integral membrane proteins, including ACKR3, and reconstitute protein-lipid interactions lost in detergent systems (43)(44)(45). The nanodisc-receptor complexes were then tethered to a quartz slide through biotinylation of the nanodisc membrane scaffolding protein (MSP) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Schafer,

Pauszek,

Gustavsson

et al. 2023

Preprint

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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.

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

Schafer,

Pauszek,

Gustavsson

et al. 2024

Preprint

0

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The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G protein- independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high- FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.

show abstract

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

Schafer,

Pauszek,

Gustavsson

et al. 2024

Preprint

0

View full text Add to dashboard Cite

The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G protein- independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high- FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.

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Bilayer lipids modulate ligand binding to atypical chemokine receptor 3

Cited by 3 publications

References 95 publications

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

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

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

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

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