Biology subjects

Handel, T. M.

Publications and source records attributed to Handel, T. M..

11 recordsLinked to original sources

bioRxiv2026

Elevated conformational dynamics makes ACKR3 activation-prone and G protein-incompetent

The atypical receptor ACKR3 works together with the canonical chemokine receptor CXCR4 to drive cell migration along gradients of their shared agonist CXCL12. CXCR4 promotes chemotaxis by activating canonical G protein pathways and recruiting {beta}-arrestins. ACKR3 indirectly regulates CXCR4-mediated chemotaxis by scavenging CXCL12. Unlike canonical chemokine receptors, ACKR3 does not couple to G proteins and instead is 100% biased towards {beta}-arrestins. CXCR4 activation by CXCL12 is exquisitely sensitive to subtle changes in both receptor and ligand. By contrast, ACKR3 is activation-prone: it recruits {beta}-arrestins in response to many ligands and is much less sensitive to mutations, suggesting distinct activation mechanisms compared to CXCR4. To explore the basis of these differences, we compared the dynamics of ACKR3 and CXCR4 complexes with chemokines using molecular dynamic (MD) simulations. Ten-microsecond atomistic MD simulations revealed that CXCR4 adopts a stable active state when bound to WT CXCL12 but transitions to an inactive state when in complex with the antagonist variant, [P2G]CXCL12. By comparison, ACKR3 exhibits a variable transmembrane (TM) 6 state distribution and persistently "active" TM7 when complexed with either WT CXCL12 or [P2G]CXCL12, the latter retaining substantial agonistic activity at ACKR3. We further identified ligand-mediated residue interaction networks in the TM core that regulate TM6 and TM7 activation in CXCR4 but are absent or disrupted in ACKR3, resulting in less constrained receptor dynamics. These findings were validated by BRET-based assays with CXCL12 and ACKR3 mutants. Together, the data suggests that the unique conformational dynamics of ACKR3 govern its activation propensity, its ligand promiscuity, and its atypical effector coupling.

pharmacology and toxicology↗

bioRxiv2026

Monocyte Migration Emerges from a Divergent Chemokine Signaling Network

Migration of leukocytes in the context of immune homeostasis or inflammatory diseases is regulated by activation of chemokine receptors by chemokine ligands. To elucidate how these interactions give rise to cell migration, we mapped the chemokine-stimulated signal transduction network in monocytic THP-1 cells. Global phosphoproteomics revealed 630 time-resolved changes in phosphorylated proteins downstream of the chemokine receptor CCR2. We used the "PHONEMeS" network modeling algorithm to generate the most parsimonious signal transduction network consistent with the observed protein phosphorylation data. The CCR2 signaling network is highly divergent, acting via multiple branches to regulate proteins required for cell migration. We validated this model using kinase inhibitors targeting different branches of the network and successfully blocked chemokine-stimulated cell migration. Thus, chemotaxis is an emergent property resulting from an integrated cellular response to divergent signaling pathways. This paradigm suggests that physiological regulation or pharmacological blockade of chemokine-driven inflammation could potentially be achieved by inhibiting any of the divergent pathways within the network.

systems biology↗

bioRxiv2025

Exploiting the CXCR3/CXCL10 pathway to override tumor immune suppression by enhancing immune trafficking and effector cell priming in head and neck squamous cell carcinoma

The immune-suppressive nature of the tumor microenvironment (TME) has limited the impact of immune checkpoint blockade in many cancers, often by restricting the infiltration and activation of anti-tumoral CD8+ T, CD4+ T, and NK cells. Here, we utilized murine models of head and neck squamous cell carcinoma and demonstrated that intratumoral (IT) delivery of CXCL10 drives tumor elimination and inhibits recurrence. CD8+ T cells recruited to tumors display enhanced activation, increased tumor antigen specificity, and decreased markers of T cell exhaustion, indicating that CXCL10 not only directs cell migration, but also enhances T cell effector functions. Despite delivery of CXCL10 into tumors, CD8+ and CD4+ T cells also show enhanced presence and proliferation in tumor-draining lymph nodes (TdLNs), consistent with antigen presentation and trafficking of these cells between tumors and TdLNs. CXCL10 also stunts angiogenesis and lymphangiogenesis within the TME, which likely contributes to its antitumoral effects. Finally, enhanced tumor clearance was observed by combining IT CXCL10 and anti-PD-1. Together, these findings provide the rationale for the clinical evaluation of CXCL10 as a strategy to enhance the efficacy of immunotherapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/650529v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@4d0f3borg.highwire.dtl.DTLVardef@122f4e6org.highwire.dtl.DTLVardef@1fc50a0org.highwire.dtl.DTLVardef@117a05_HPS_FORMAT_FIGEXP M_FIG C_FIG SUMMARYCXCL10 suppresses tumor growth and promotes immune memory by recruiting T and NK cells into the tumor microenvironment, promoting tumor-specific antigen recognition and effector functions, slowing T cell exhaustion, and inhibiting angiogenesis. CXCL10 directly and indirectly mobilizes an immune cell network that together supports an anti-tumoral microenvironment.

cancer biology↗

bioRxiv2024

Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer

The constitutive (ligand-independent) signaling of G protein-coupled receptors (GPCRs) is being increasingly appreciated as an integral aspect of their function; however, it can be technically hard to detect for poorly characterized, e.g. orphan, receptors of the cAMP-inhibitory Gi-coupled (GiPCR) family. In this study, we delineate the optimal strategies for the detection of such activity across several GiPCRs in two cell lines. As our study examples, we chose two canonical GiPCRs - the constitutively active Smoothened and the ligand-activated CXCR4,-and one atypical GPCRs, the chemokine receptor ACKR3. We verified the applicability of three Bioluminescence Resonance Energy Transfer (BRET)-based assays - one measuring changes in intracellular cAMP, another in G{beta}{gamma}/GRK3ct association and third in Gi-G{beta}{gamma} dissociation, - for assessing both constitutive and ligand-modulated activity of these receptors. We also revealed the possible caveats and sources of false positives, and proposed optimization strategies. All three types of assays confirmed the ligand-dependent activity of CXCR4, the controversial G protein incompetence of ACKR3, the constitutive Gi-directed activity of SMO, and its modulation by PTCH1. We also demonstrated that PTCH1 promotes SMO localization to the cell surface, thus enhancing its responsiveness not only to agonists but also to antagonists, which is a novel mechanism of regulation of a Class F GiPCR Smoothened.

pharmacology and toxicology↗

bioRxiv2024

Noncanonical roles of chemokine regions in CCR9 activation revealed by structural modeling and mutational mapping

The G protein-coupled CC chemokine receptor 9 (CCR9) plays a major role in inflammatory bowel disease and has been implicated in cancer. Despite its importance as a drug target, there is limited mechanistic understanding of how CCR9 engages and is activated by its endogenous chemokine agonist CCL25. Here, by combining structural modeling with multimodal pharmacological assessment of receptor mutants, we generated a functional map of the CCR9-CCL25 interaction interface and delineated key determinants of binding, agonism, constitutive activity, and G protein vs arrestin signaling. In contrast to all complexes studied to date, where chemokines drive receptor activation through their N-termini, we determined that CCL25 activates CCR9 via a distinct region, its 30s loop. In support of this non-canonical mechanism, CCR9 signaling is tolerant to alanine mutations in the N-terminus of CCL25 but strongly affected by modifications to the 30s loop. Through molecular evolution of the CCL25 N-terminus, we identified chemokine analogs with enhanced binding properties. However, in contrast to other receptor-chemokine systems, these analogs remained full agonists, consistent with the localization of CCL25 signaling determinants outside of the N-terminus. The non-canonical signature of CCR9 activation provides new insights to aid CCR9 drug discovery and may also inform structure-based design of drugs targeting other chemokine receptors.

molecular biology↗

bioRxiv2023

Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

pharmacology and toxicology↗

bioRxiv2023

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

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 {beta}-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.

biophysics↗

bioRxiv2023

Atypical Chemokine Receptor 3 'Senses' CXC Chemokine Receptor 4 Activation Through GPCR Kinase Phosphorylation

Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging action mediates the availability of the chemokine 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 mapped the phosphorylation patterns and determined that GRK5 phosphorylation of ACKR3 dominates {beta}-arrestin recruitment and chemokine scavenging over GRK2. Co-activation of CXCR4 significantly enhanced phosphorylation by GRK2 through the liberation of G{beta}{gamma}. These results suggest that ACKR3 senses CXCR4 activation through a GRK2-dependent crosstalk mechanism. Surprisingly, we also found that despite the requirement for phosphorylation, and the fact that most ligands promote {beta}-arrestin recruitment, {beta}-arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet to be determined function for these adapter proteins.

biochemistry↗

bioRxiv2022

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate wide-spread non-receptor mediated immune cell recruitment

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system, but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation, due to a lack of mechanistic understanding. Specifically, CXCL4 (PF4) has no established receptor that explains its function. Here we use biophysical, in vitro and in vivo techniques to determine the mechanism underlying CXCL4 mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars within the endothelial extracellular matrix resulting in increased vascular permeability and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulphation confers selectivity onto chemokine localisation. These findings represent a new understanding of chemokine biology, providing novel mechanisms for future therapeutic targeting. One sentence summaryCXCL4 binds to extracellular matrix proteoglycans resulting in increased vascular permeability and recruitment of a wide range of different leukocytes via a non-canonical mechanism.

immunology↗

bioRxiv2020

Functional anatomy of the full length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis

Due to their prominent role in development and infamy in cancer and HIV, the chemokine receptor CXCR4 and its ligand, CXCL12, have been the subject of numerous structural and functional studies. Nevertheless, a high resolution structure of the CXCR4-CXCL12 complex has not been reported. Even with several alternative computational models of the complex at hand, the relative contributions of different interaction epitopes to ligand binding, ligand selectivity and signaling are not readily apparent. Here, building upon our latest structural model, we employed a systematic mutagenesis strategy to dissect the functional anatomy of the of CXCR4-CXCL12 complex. Key charge swap mutagenesis experiments supported pairwise interactions between oppositely charged residues in the receptor and chemokine, confirming the accuracy of the predicted orientation of the chemokine relative to the receptor, while also providing insight into ligand selectivity. Progressive deletion of N-terminal residues revealed an unexpected contribution of the receptor N-terminus to chemokine signaling; this finding challenges a longstanding "two-site" hypothesis about the essential features of the receptor-chemokine interaction where the N-terminus is purported to only contribute to binding affinity. The results suggest that while the interaction of the chemokine N-terminus with the receptor binding pocket is the key driver of signaling, the signaling amplitude depends on the extent to which the receptor N-terminus binds the chemokine. Along with systematic characterization of other epitopes, the current data allow us to propose a comprehensive experimentally-consistent structural model for how the chemokine binds CXCR4 and initiates signal transmission through the receptor TM domain. One sentence summaryA systematic structure-guided mutagenesis study of chemokine receptor CXCR4 reveals novel insights into epitopes regulating ligand recognition, ligand specificity and CXCL12-mediated signaling.

pharmacology and toxicology↗

bioRxiv2020

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide crosslinking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 {beta}1-strand, while also recapitulating earlier findings from NMR, modeling and crystallography of homologous receptors. A crosslinking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N-terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor-targeting small molecules and biologics with novel pharmacology.

biochemistry↗