Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β2-adrenergic receptor (β2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4-1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β2-adrenoceptor (β2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M-1 respectively, which were two orders of magnitude higher than those on the Halo-β2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.

Formyl peptide receptors (FPR), part of the G-protein coupled receptor superfamily, are pivotal in directing phagocyte migration towards chemotactic signals from bacteria and host tissues. Although their roles in acute bacterial infections are well-documented, their involvement in immunity against tuberculosis (TB) remains unexplored. This study investigates the functions of Fpr1 and Fpr2 in defense against Mycobacterium tuberculosis (Mtb), the causative agent of TB. Elevated levels of Fpr1 and Fpr2 were found in the lungs of mice, rabbits and peripheral blood of humans infected with Mtb, suggesting a crucial role in the immune response. The effects of Fpr1 and Fpr2 deletion on bacterial load, lung damage, and cellular inflammation were assessed using a TB model of hypervirulent strain of Mtb from the W-Beijing lineage. While Fpr2 deletion showed no impact on disease outcome, Fpr1-deficient mice demonstrated improved bacterial control, especially by macrophages. Bone marrow-derived macrophages from these Fpr1 -/- mice exhibited an enhanced ability to contain bacterial growth over time. Contrarily, treating genetically susceptible mice with Fpr1-specific inhibitors caused impaired early bacterial control, corresponding with increased bacterial persistence in necrotic neutrophils. Furthermore, ex vivo assays revealed that Fpr1 -/- neutrophils were unable to restrain Mtb growth, indicating a differential function of Fpr1 among myeloid cells. These findings highlight the distinct and complex roles of Fpr1 in myeloid cell-mediated immunity against Mtb infection, underscoring the need for further research into these mechanisms for a better understanding of TB immunity.

Amyotrophic lateral sclerosis can be caused by abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping and subsequent dissection of the neuromuscular circuit, we show that TDP-43 worms have profound defects in GABA neurons. Moreover, acetylcholine neurons appear functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of, among others, G-protein-coupled receptors restores neurotransmission, but inefficiently rescues locomotion. Rebalancing the excitatory-to-inhibitory ratio in the neuromuscular system by simultaneous stimulation of the affected GABA- and acetylcholine neurons, however, not only synergizes the effects of boosting individual neurotransmitter systems, but instantaneously improves movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the β-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with β-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for β-arrestin-2 binding and to investigate the role of β-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to β-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to β-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the β-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the β-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

ACE2 (angiotensin-converting enzyme 2), a multifunctional transmembrane protein, is well recognized as an important member of the (RAS) renin-angiotensin system with important roles in the regulation of cardiovascular function by opposing the harmful effects of Ang-II (angiotensin II) and AT1R (Ang-II type 1 receptor) activation. More recently, ACE2 was found to be the entry point for the SARS-CoV-2 virus into cells, causing COVID-19. This finding has led to an exponential rise in the number of publications focused on ACE2, albeit these studies often have opposite objectives to the preservation of ACE2 in cardiovascular regulation. However, notwithstanding accumulating data of the role of ACE2 in the generation of angiotensin-(1-7) and SARS-CoV-2 internalization, numerous other putative roles of this enzyme remain less investigated and not yet characterized. Currently, no drug modulating ACE2 function or expression is available in the clinic, and the development of new pharmacological tools should attempt targeting each step of the lifespan of the protein from synthesis to degradation. The present review expands on our presentation during the 2023 Lewis K. Dahl Memorial Lecture Sponsored by the American Heart Association Council on Hypertension. We provide a critical summary of the current knowledge of the mechanisms controlling ACE2 internalization and intracellular trafficking, the mutual regulation with GPCRs (G-protein-coupled receptors) and other proteins, and posttranslational modifications. A major focus is on ubiquitination which has become a critical step in the modulation of ACE2 cellular levels.

Melanocortin receptor accessory protein 2 (MRAP2) is a membrane protein that binds multiple G protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, including prokineticin receptors. These GPCRs are expressed both centrally and peripherally, and their endogenous ligands are prokineticin 1 (PK1) and prokineticin 2 (PK2). PKRs couple all G-protein subtypes, such as Gαq/11, Gαs, and Gαi, and recruit β-arrestins upon PK2 stimulation, although the interaction between PKR2 and β-arrestins does not trigger receptor internalisation. MRAP2 inhibits the anorexigenic effect of PK2 by binding PKR1 and PKR2. The aim of this work was to elucidate the role of MRAP2 in modulating PKR2-induced β-arrestin-2 recruitment and β-arrestin-mediated signalling. This study could allow the identification of new specific targets for potential new drugs useful for the treatment of the various pathologies correlated with prokineticin, in particular, obesity.

Doxorubicin (Doxo)-associated cardio-and vasotoxicity has been recognised as a serious complication of cancer chemotherapy. The purpose of this novel paper was to determine the effect of Doxo on G-protein coupled receptor (GPCR)-mediated vasocontraction located on vascular smooth muscle cells. Rat left anterior descending artery segments were incubated for 24 h with 0.5 µM Doxo. The vasocontractile responses by activation of endothelin receptor type A (ETA) and type B (ETB), serotonin receptor 1B (5-HT1B) and thromboxane A2 prostanoid receptor (TP) were investigated by a sensitive myography using specific agonists, while the specificity of the GPCR agonists was verified by applying selective antagonists (i.e. ETA and ETB agonist = 10- 14-10- 7.5 M endothelin-1 (ET-1); ETA antagonist = 10 µM BQ123; ETB agonists = 10- 14-10- 7.5 M sarafotoxin 6c (S6c) and ET-1; ETB antagonist = 0.1 µM BQ788; 5-HT1B agonist = 10- 12-10- 5.5 M 5-carboxamidotryptamine (5-CT); 5-HT1B antagonist = 1 µM GR55562; TP agonist = 10- 12-10- 6.5 M U46619; TP antagonist = 1 µM Seratrodast). Our results show that 0.5 µM Doxo incubation of LAD segments leads to an increased VSMC vasocontraction through the ETB, 5-HT1B and TP GPCRs, with a 2.2-fold increase in ETB-mediated vasocontraction at 10- 10.5 M S6c, a 2.0-fold increase in 5-HT1B-mediated vasocontraction at 10- 5.5 M 5-CT, and a 1.3-fold increase in TP-mediated vasocontraction at 10- 6.5 M U46619. Further studies unravelling the involvement of intracellular GPCR signalling pathways will broaden our understanding of the Doxo-induced vasotoxicity, and thus pave the way to mitigate the adverse effects by potential implementation of adjunct therapy options.

This review explores the evolving landscape of G-protein-coupled receptor (GPCR)-based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand-binding properties, structural insights, and spectral diversity.

Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes. Fluorescence-based techniques are routinely employed for accurate and robust measurements of in-vitro activity of molecular targets and for discovering novel chemical molecules that modulate the activity of molecular targets. In the current review, the authors focus on the applications of fluorescence-based high throughput screening (HTS) and fragment-based ligand discovery (FBLD) techniques such as fluorescence polarization (FP), Förster resonance energy transfer (FRET), fluorescence thermal shift assay (FTSA) and microscale thermophoresis (MST) for the discovery of chemical probe to exploring target\'s role in disease biology and ultimately, serve as a foundation for drug discovery. Some recent advancements in these techniques for compound library screening against important classes of drug targets, such as G-protein-coupled receptors (GPCRs) and GTPases, as well as phosphorylation- and acetylation-mediated protein-protein interactions, are discussed. Overall, this review presents a landscape of how these techniques paved the way for the discovery of small-molecule modulators and biologics against these targets for therapeutic benefits.

Colorectal cancer (CRC) is one of the most common neoplasms worldwide. Among the risk factors of CRC, inflammatory bowel disease (IBD) is one of the most important ones leading to the development of colitis-associated CRC (CAC). G-protein coupled receptors (GPCR) are transmembrane receptors that orchestrate a multitude of signaling cascades in response to external stimuli. Because of their functionality, they are promising targets in research on new strategies for CRC diagnostics and treatment. Recently, regulators of G-proteins (RGS) have been attracting attention in the field of oncology. Typically, they serve as negative regulators of GPCR responses to both physiological stimuli and medications. RGS activity can lead to both beneficial and harmful effects depending on the nature of the stimulus. However, the atypical RGS-AXIN uses its RGS domain to antagonize key signaling pathways in CRC development through the stabilization of the β-catenin destruction complex. Since AXIN does not limit the efficiency of medications, it seems to be an even more promising pharmacological target in CRC treatment. In this review, we discuss the current state of knowledge on RGS significance in sporadic CRC and CAC with particular emphasis on the regulation of GPCR involved in IBD-related inflammation comprising opioid, cannabinoid and serotonin receptors.