Biology subjects

Handel, T.

Publications and source records attributed to Handel, T..

3 recordsLinked to original sources

bioRxiv2025

Rules of engagement: determinants of chemokine receptor activation and selectivity by CCL27 and CCL28

The distinct functional roles of chemokines CCL27 and CCL28 in epithelial immunity of skin and mucosal tissues, respectively, are coordinated by their shared receptor, CCR10 and the CCL28-specific receptor, CCR3. In this study, we conducted structure-function studies focused on the N-termini of these two chemokines to identify determinants of receptor activation, internalization and binding specificity. Deletion of two N-terminal residues of CCL27 resulted in a CCR10 antagonist, highlighting the critical roles of these residues in driving receptor pharmacology. Extension with a Phe produced a superagonist by occupying an available subpocket in the receptor binding site. Swapping the N-terminus of CCL28 onto the CCL27 globular domain (NT28-CCL27) also resulted in a superagonist of CCR10, but the opposite swap (NT27-CCL28) showed equivalent or reduced activity compared to WT CCL28, indicating that the CCL28 N-terminus is a stronger driver of CCR10 signaling. The effect of these and other mutations were rationalized by AlphaFold models of the CCR10 complexes. AlphaFold modeling also revealed that the reduced size of the binding pocket, and more basic nature of the N-terminus and extracellular loops of CCR3 compared to CCR10, contribute to its specificity for CCL28 while CCR10 accommodates both ligands. The overall basic nature of CCL28 also contributes to its high affinity for glycosaminoglycans, which is likely important for its retention in mucosal tissues. These data illustrate the modular compositions of these chemokines that has evolved to achieve overlapping but non-redundant functions, and the exploitation of this modular nature to produce engineered chemokines for probing or targeting CCR10 in disease contexts.

pharmacology and toxicology↗

bioRxiv2023

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics

CC chemokine receptor 5 (CCR5) promotes inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A CCR5 inhibitor has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of other diseases has had limited success, likely because of the complexity of CCR5 pharmacology and biology. CCR5 is activated by natural and engineered chemokines that elicit distinct signaling and trafficking responses, including receptor sequestration inside the cell. Intracellular sequestration may be therapeutically exploitable as a strategy for receptor inhibition, but the mechanisms by which different ligands promote receptor retention in the cell versus presence on the cell membrane are poorly understood. We employed live cell ascorbic acid peroxidase (APEX2) proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveal distinct ligand-dependent CCR5 trafficking patterns with temporal and spatial resolution. All three chemokines internalize CCR5 via {beta}-arrestin-dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the cellular mechanisms behind CCR5 intracellular sequestration and suggest how trafficking can be exploited for the development of functional antagonists of CCR5. Significance StatementCCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and transmission of HIV. It responds to different ligands with distinct signaling and trafficking behaviors; notably some ligands induce retention of the receptor inside the cell. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the cellular basis for receptor sequestration that can be exploited as a therapeutic strategy for inhibiting CCR5 function.

cell biology↗

bioRxiv2023

ACKR3-arrestin2/3 complexes reveal molecular consequences of GRK-dependent barcoding

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.

biochemistry↗