Browsing by Author "Chen, Qiuyan"

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    ACKR3–arrestin2/3 complexes reveal molecular consequences of GRK-dependent barcoding
    (bioRxiv, 2023-07-19) Chen, Qiuyan; Schafer, Christopher T.; Mukherjee, Somnath; Gustavsson, Martin; Agrawal, Parth; Yao, Xin-Qiu; Kossiakoff, Anthony A.; Handel, Tracy M.; Tesmer, John J. G.; Biochemistry and Molecular Biology, School of Medicine
    Atypical chemokine receptor 3 (ACKR3, also known as CXCR7) is a scavenger receptor that regulates extracellular levels of the chemokine CXCL12 to maintain responsiveness of its partner, the G protein-coupled receptor (GPCR), CXCR4. ACKR3 is notable because it does not couple to G proteins and instead is completely biased towards arrestins. Our previous studies revealed that GRK2 and GRK5 install distinct distributions of phosphates (or "barcodes") on the ACKR3 carboxy terminal tail, but how these unique barcodes drive different cellular outcomes is not understood. It is also not known if arrestin2 (Arr2) and 3 (Arr3) bind to these barcodes in distinct ways. Here we report cryo-electron microscopy structures of Arr2 and Arr3 in complex with ACKR3 phosphorylated by either GRK2 or GRK5. Unexpectedly, the finger loops of Arr2 and 3 directly insert into the detergent/membrane instead of the transmembrane core of ACKR3, in contrast to previously reported "core" GPCR-arrestin complexes. The distance between the phosphorylation barcode and the receptor transmembrane core regulates the interaction mode of arrestin, alternating between a tighter complex for GRK5 sites and heterogenous primarily "tail only" complexes for GRK2 sites. Arr2 and 3 bind at different angles relative to the core of ACKR3, likely due to differences in membrane/micelle anchoring at their C-edge loops. Our structural investigations were facilitated by Fab7, a novel Fab that binds both Arr2 and 3 in their activated states irrespective of receptor or phosphorylation status, rendering it a potentially useful tool to aid structure determination of any native GPCR-arrestin complex. The structures provide unprecedented insight into how different phosphorylation barcodes and arrestin isoforms can globally affect the configuration of receptor-arrestin complexes. These differences may promote unique downstream intracellular interactions and cellular responses. Our structures also suggest that the 100% bias of ACKR3 for arrestins is driven by the ability of arrestins, but not G proteins, to bind GRK-phosphorylated ACKR3 even when excluded from the receptor cytoplasmic binding pocket.
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    Atypical Chemokine Receptor 3 "Senses" CXC Chemokine Receptor 4 Activation Through GPCR Kinase Phosphorylation
    (Aspet, 2023) Schafer, Christopher T.; Chen, Qiuyan; Tesmer, John J. G.; Handel, Tracy M.; Biology, School of Science
    Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging process restricts the availability of the chemokine agonist CXCL12 for the G protein-coupled receptor (GPCR) CXCR4 and requires phosphorylation of the ACKR3 C-terminus by GPCR kinases (GRKs). ACKR3 is phosphorylated by GRK2 and GRK5, but the mechanisms by which these kinases regulate the receptor are unresolved. Here we determined that GRK5 phosphorylation of ACKR3 results in more efficient chemokine scavenging and β-arrestin recruitment than phosphorylation by GRK2 in HEK293 cells. However, co-activation of CXCR4-enhanced ACKR3 phosphorylation by GRK2 through the liberation of Gβγ, an accessory protein required for efficient GRK2 activity. The results suggest that ACKR3 "senses" CXCR4 activation through a GRK2-dependent crosstalk mechanism, which enables CXCR4 to influence the efficiency of CXCL12 scavenging and β-arrestin recruitment to ACKR3. Surprisingly, we also found that despite the requirement for phosphorylation and the fact that most ligands promote β-arrestin recruitment, β-arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet-to-be-determined function for these adapter proteins. Since ACKR3 is also a receptor for CXCL11 and opioid peptides, these data suggest that such crosstalk may also be operative in cells with CXCR3 and opioid receptor co-expression. Additionally, kinase-mediated receptor cross-regulation may be relevant to other atypical and G protein-coupled receptors that share common ligands. SIGNIFICANCE STATEMENT: The atypical receptor ACKR3 indirectly regulates CXCR4-mediated cell migration by scavenging their shared agonist CXCL12. Here, we show that scavenging and β-arrestin recruitment by ACKR3 are primarily dependent on phosphorylation by GRK5. However, we also show that CXCR4 co-activation enhances the contribution of GRK2 by liberating Gβγ. This phosphorylation crosstalk may represent a common feedback mechanism between atypical and G protein-coupled receptors with shared ligands for regulating the efficiency of scavenging or other atypical receptor functions.
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    Structural Basis of Arrestin Binding to Cell Membranes
    (2024-04) Miller, Kyle Warren; Chen, Qiuyan; Takagi, Yuichiro; Georgiadis, Millie M.; Hurley, Thomas D.
    Two non-visual arrestins, arrestin2 (Arr2) and arrestin3 (Arr3), selectively interact with activated and phosphorylated G protein-coupled receptors (GPCRs) and play crucial roles in regulating many important physiological processes. Arrestins also engage the lipid bilayer surrounding activated GPCRs, which further potentiates arrestin activation and regulates GPCR trafficking in cells. Because of this, structural and functional understanding of arrestins would provide insight in enhancing arrestin’s GPCR desensitization for various diseases where constitutively active GPCR mutants play a role including congenital endocrine disorders and familial gestational hyperthyroidism. To better understand the membrane binding role of arrestins, we performed in vitro binding assays and demonstrated that Arr2 selectively binds to nanodiscs containing Phosphatidylinositol 4,5-bisphosphate (PIP2) even in the absence of different binding sites. Our cryo-electron microscopy (Cryo-EM) structure of Arr2 in complex with PIP2 nanodisc reveals that multiple structural elements of Arr2, including the finger loop, C domain and C-edge loop, contribute to membrane binding. Eliminating one individual site does not significantly impact Arr2 binding to the nanodisc. Moreover, a preactivated variant of Arr2 shows increased binding to the nanodisc than wildtype. We also labeled four potential membrane binding sites with monobromobimane (mBrB) and detected different levels of fluorescence increase in the presence of nanodisc containing various types of phospholipids. Overall, our study provides detailed structural evidence on how arrestins engage the membrane via multiple contact points and how this can impact arrestin-mediated signaling.