Schafer Christopher T, Pauszek Raymond F, Gustavsson Martin, Handel Tracy M, Millar David P
Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology, University of California San Diego, La Jolla, United States.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States.
Elife. 2025 Apr 15;13:RP100098. doi: 10.7554/eLife.100098.
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.
典型趋化因子受体CXCR4和非典型受体ACKR3均对CXCL12作出反应，但诱导不同的效应反应来调节细胞迁移。CXCR4与G蛋白偶联并直接促进细胞迁移，而ACKR3不依赖G蛋白，它清除CXCL12以调节细胞外趋化因子水平并维持CXCR4的反应性，从而间接影响迁移。这些受体也有不同的激活要求。CXCR4仅对野生型CXCL12作出反应，并且对趋化因子的突变敏感。相比之下，ACKR3募集GPCR激酶（GRK）和β-抑制蛋白，并对CXCL12、CXCL12变体、其他肽和蛋白质有广泛反应，并且对突变相对不敏感。为了研究构象动力学在CXCR4和ACKR3不同药理行为中的作用，我们采用单分子荧光共振能量转移实时追踪受体的离散构象状态。数据显示，无配体的CXCR4优先处于高荧光共振能量转移非活性状态，而无配体的ACKR3在多个非活性、中间和活性构象之间几乎没有构象偏好且具有高转变概率，这与其激活倾向一致。与单一的CXCR4活性状态相比，多种类似活性的ACKR3构象在激动剂作用下出现。受体这种明显不同的构象格局表明，与CXCR4相比，ACKR3的激活可能通过更广泛分布的构象状态来实现。ACKR3的许多构象异质性与ACKR3和CXCR4之间不同的单个残基有关。ACKR3的动态特性可能是其无法与驱动典型GPCR信号传导的G蛋白形成有效相互作用的原因。
