Davide Calebiro

Alveolar macrophages are the primary lung immune cell. They play a crucial role in both maintenance of tissue homeostasis and the initiation of inflammation, secreting multiple immune mediators including the chemokine interleukin-8 (IL-8). Inflammatory settings are often characterised by tissue hypoxia, increased glycolytic rates and lactate secretion, yet how lactate influences alveolar macrophage function remains unclear. Here we investigated how lactate, once considered a waste-product, shapes alveolar macrophage function. To do so, primary human alveolar macrophages (hAMs) and a combination of flow cytometry, ELISA/Luminex, western blot, light microscopy, bioluminescence resonance energy transfer (BRET) and analysis of publicly available datasets were used to understand the role of lactate in lung pathological environments. We demonstrate that hAMs sense extracellular lactate via the expression of different lactate transporters (e.g. MCT1, MCT4) and receptors (i.e. GPR132). Lactate treatment of hAMs increased IL-8 secretion in a MCT1-dependent manner. Lipid metabolism and lipid droplet formation, as well as direct lactate-driven GPR132 signalling were required for lactate-dependent IL-8 release. Our findings uncover a previously unrecognised dual mechanism by which lactate orchestrates immune regulation in hAMs. Specifically, lactate-driven IL-8 production requires two distinct lactate-driven processes: uptake via MCT1, which reprograms lipid metabolism, and signaling through the lactate receptor GPR132. This functional integration of lactate transport and receptor-mediated signaling provides new mechanistic insight into lactate’s role in hAM biology and highlights potential targets for therapeutic intervention.

The ITAM receptors, GPVI and CLEC-2, are activated by a diversity of ligands and are targets for anti-platelet drugs. We used two-colour single-particle tracking to monitor GPVI and CLEC-2 diffusion and dimerisation in resting and stimulated CHO-K1 cells. GPVI and CLEC-2 were fluorescently labelled via SNAP and Halo tags. When expressed at ~10% of their level in platelets, GPVI and CLEC-2 are monomeric. Divalent and trivalent nanobody ligands induce GPVI and CLEC-2 homo-dimerisation, a reduction in free diffusion and an increase in immobile receptors proportionate to ligand valency. Dimers of CLEC-2 are longer-lived than those of GPVI despite a lower affinity of the monomeric nanobody that forms the backbone of the ligands. Recombinant monomeric CLEC-2 but not GPVI dimerises with an affinity (K_D) of 18.5 µM. Synergy between ligand-induced crosslinking and dimerisation underlies the longer lifetime of the CLEC-2 interactions. Targeting dimerisation may be an effective way to inhibit activation of CLEC-2 by all of its ligands.

G-protein-coupled receptors (GPCRs) can signal from intracellular compartments but the occurrence and relevance of this phenomenon for metabolite-sensing GPCRs is largely unknown. Here, we investigate free fatty acid receptor 4 (FFA4), a metabolite-sensing GPCR activated by medium-chain and long-chain fatty acids. Using live-cell imaging, bioluminescence resonance energy transfer, super-resolution microscopy and cell fractionation, we show that FFA4 localizes to intracellular membranes, particularly endoplasmic reticulum subdomains surrounding lipid droplets, in both immortalized adipocytes and mouse adipose tissue. Upon lipolysis, locally released fatty acids appear to rapidly activate this intracellular FFA4 pool, leading to G _{i /o} protein signaling and preferential reduction of cyclic adenosine monophosphate levels near lipid droplets. These mechanisms are required for efficient FFA4-mediated lipolysis regulation, as shown using tethered mini-Gα _i/o proteins to locally inhibit G _i/o signaling. These findings reveal an unexpected ‘intracrine’ GPCR signaling modality involved in the local regulation of cell metabolism, with biological and pharmacological implications.

The Wingless/Int-1 (WNT) signaling network is essential to orchestrate central physiological processes such as embryonic development and tissue homeostasis. In the currently held tenet, WNT/β-catenin signaling is initiated by WNT-induced recruitment of Frizzleds (FZDs) and LRP5/6 followed by the formation of a multiprotein signalosome complex. Here, we use bioluminescence resonance energy transfer (BRET) to show that different WNT paralogs dynamically trigger FZD-LRP6 association. While WNT-induced receptor interaction was independent of C-terminal LRP6 phosphorylation, it was allosterically modulated by binding of the phosphoprotein Dishevelled (DVL) to FZD. WNT-16B emerged as a ligand of particular interest, as it efficiently promoted FZD-LRP6 association but, unlike WNT-3A, did not lead to WNT/β-catenin signaling. Transcriptomic analysis further revealed distinct transcriptional fingerprints of WNT-3A and WNT-16B stimulation in HEK293 cells. Additionally, single-molecule tracking demonstrated that, despite increasing FZD₅ and LRP6 confinement, WNT-16B stimulation did not result in formation of higher-order receptor clusters, in contrast to WNT-3A. Our results suggest that FZD-WNT-LRP5/6 complex formation alone is not sufficient for the initiation of WNT/β-catenin signaling. Instead, we propose a two-step model, where initial ligand-induced FZD-LRP6 association must be followed by receptor clustering into higher-order complexes and subsequent phosphorylation of LRP6 for efficient activation of WNT/β-catenin signaling.

G protein–coupled receptors (GPCRs) are transmembrane detectors of extracellular signals that activate heterotrimeric G proteins to regulate intracellular responses. Because there are only 16 Gα proteins that can couple to GPCRs, variation in a single Gα can affect the function of numerous receptors. Here, we investigated two mutant forms of Gα _s (L388R and E392K) that are associated with pseudohypoparathyroidism type Ic (PHPIc), a maternally inherited rare disease. Gα _s is encoded by an imprinted gene, resulting in the mutant form of Gα _s being the only version of the protein present in certain tissues, which leads to tissue-specific disease manifestations. By integrating data from three-dimensional structures, GPCR–G protein coupling specificity, transcriptomics, biophysics, and molecular dynamics with systems pharmacology modeling, we identified GPCRs whose signaling could be altered by Gα _s mutations in the kidney, a tissue involved in the pathophysiology of PHPIc. Analysis of G protein activation by the parathyroid hormone receptor 1 (PTH1R) revealed that L388R impaired Gα _s interaction with the receptor, whereas E392K reduced the receptor-induced activation of heterotrimeric G _s . This indicates that different signal transduction steps can be altered by specific Gα _s mutants associated with the same disease. These findings highlight the importance of investigating mutation-specific perturbations in GPCR signaling to suggest patient-specific treatment strategies. Furthermore, our methods provide a blueprint for interrogating GPCR signaling diversity in different physiological and pathophysiological contexts.

The Wingless/Int-1 (WNT) signaling network is essential to orchestrate central physiological processes such as embryonic development and tissue homeostasis. In the currently held tenet, WNT/β-catenin signaling is initiated by WNT-induced recruitment of Frizzleds (FZDs) and LRP5/6 followed by the formation of a multiprotein signalosome complex. Here, we use bioluminescence resonance energy transfer (BRET) to show that different WNT paralogs dynamically trigger FZD-LRP6 association. While WNT-induced receptor interaction was independent of C-terminal LRP6 phosphorylation, it was allosterically modulated by binding of the phosphoprotein Dishevelled (DVL) to FZD. WNT-16B emerged as a ligand of particular interest, as it efficiently promoted FZD-LRP6 association but, unlike WNT-3A, did not lead to WNT/β-catenin signaling. Transcriptomic analysis further revealed distinct transcriptional fingerprints of WNT-3A and WNT-16B stimulation in HEK293 cells. Additionally, single-molecule imaging demonstrated that, despite increasing FZD₅ and LRP6 confinement, WNT-16B stimulation did not result in formation of large receptor clusters, in contrast to WNT-3A. Our results suggest that FZD-WNT-LRP5/6 complex formation alone is not sufficient for the initiation of WNT/β-catenin signaling. Instead, we propose a two-step model, where initial ligand-induced FZD-LRP6 association must be followed by LRP6 phosphorylation and receptor clustering into higher-order complexes for efficient activation of WNT/β-catenin signaling.

The delta opioid receptor (δOR or DOR) is a G protein-coupled receptor (GPCR) showing a promising profile as a drug target for nociception and analgesia. Herein, we design and synthesize new fluorescent antagonist probes with high δOR selectivity that are ideally suited for single-molecule microscopy (SMM) applications in unmodified, untagged receptors. Using our new probes, we investigated wild-type δOR localization and mobility at low physiological receptor densities for the first time. Furthermore, we investigate the potential formation of δOR homodimers, as such a receptor organization might exhibit distinct pharmacological activity, potentially paving the way for innovative pharmacological therapies. Our findings indicate that the majority of δORs labeled with these probes exist as freely diffusing monomers on the cell surface in a simple cell model. This discovery advances our understanding of OR behavior and offers potential implications for future therapeutic research.

G protein-coupled receptors (GPCRs) are transmembrane proteins capable of detecting signals as diverse as odours, neurotransmitters, and hormones. Upon activation, receptor signalling converges onto four Gα protein subtypes to regulate intracellular responses. Therefore, variation in a single Gα protein gene can potentially impact the function of numerous receptors. In this work, we have performed a multidimensional study of rare disease mutations in Gαs, a prototypical Gα protein. By integrating data from 3D structures, GPCR / G protein functional pairings, transcriptomics, biophysics, and molecular dynamics with systems pharmacology modelling, our results reveal why mutations impairing receptor / Gαs coupling result in highly specific context-based signalling defects. Furthermore, we show that mutations leading to the same rare disease can alter different signal transduction steps, highlighting the importance of patient-specific treatment strategies. By closely dissecting G protein coupling, our study provides a blueprint to interrogate GPCR pathway signalling diversity in different (patho)physiological contexts.