Search Results (111)

TGR5 has emerged as a promising therapeutic target for obesity and metabolic disorders due to its regulatory roles in energy expenditure, glucose homeostasis, thermogenesis, and gut hormone secretion. This review summarizes the structural mechanisms of TGR5 activation, focusing on orthosteric and allosteric ligand interactions, toggle switch dynamics, and G protein coupling based on cryo-EM and docking-based models. A wide range of bioactive natural compounds including oleanolic acid, curcumin, betulinic acid, ursolic acid, quinovic acid, obacunone, nomilin, and 5β-scymnol are examined for their ability to modulate TGR5 signaling and elicit favorable metabolic effects. Molecular docking simulations using CB-Dock2 and PDB ID 7BW0 revealed key interactions within the orthosteric pocket, supporting their mechanistic potential as TGR5 agonists. Emerging strategies in TGR5-directed drug development are also discussed, including gut-restricted agonism to minimize gallbladder-related side effects, biased and allosteric modulation to fine-tune signaling specificity, and AI-guided optimization of natural product scaffolds. These integrated insights provide a structural and pharmacological framework for the rational design of safe and effective TGR5-targeted therapeutics. Full article

Rhodopsin, the prototypical Class A G protein-coupled receptor (GPCR) and visual pigment of rod photoreceptors, has long served as a structural and mechanistic model for GPCR biology. Mutations in rhodopsin are the leading cause of autosomal dominant retinitis pigmentosa (adRP), making this receptor a critical therapeutic target. In this review, we summarize the chemical, structural, and biophysical features of small-molecule modulators of this receptor, spanning both classical retinoid analogs and emerging non-retinoid scaffolds. These ligands reveal recurrent binding modes within the orthosteric chromophore pocket as well as peripheral allosteric and bitopic sites, where they mediate folding, rescue trafficking, photocycle modulation, and mutant stabilization. We organize ligand performance into a three-tier framework linking binding affinity, cellular rescue potency, and stability gains. Chemotypes in tier 2, which show sub-micromolar to low-micromolar activity with broad mutant coverage, emerge as promising candidates for optimization into next-generation scaffolds. Across scaffolds, a recurring minimal pharmacophore is evident by a contiguous hydrophobic π-surface anchored in the β-ionone region, coupled with a strategically oriented polar handle that modulates the Lys296/Glu113 microenvironment, offering tractable design vectors for non-retinoid chemotypes. Beyond the chromophore binding pocket, we highlight opportunities to exploit extracellular loop epitopes, cytoplasmic microswitch clefts, dimer/membrane interfaces, and ion co-binding sites to engineer safer, state-biased control with fewer photochemical liabilities. By integrating rhodopsin photobiophysics with environment-aware, multi-state medicinal chemistry, and by addressing current translational challenges in drug delivery, this review outlines a rational framework for advancing rhodopsin-targeted therapeutics toward clinically credible interventions for RP and related retinal degenerations. Full article

Background/Objectives: G protein-coupled receptors (GPCRs) can promote ligand-biased signaling, yet the mechanisms that promote bias are not well understood. We have shown that C-C Chemokine Ligand 19 (CCL19) and CCL21 promote ligand-biased internalization and signaling of C-C Chemokine Receptor 7 (CCR7) in T cells. The roles of GPCR kinases (GRKs) in regulating biased CCR7 internalization and biased signaling in T cells are unclear. GRK2 is a serine/threonine kinase that phosphorylates GPCRs in response to ligand binding and is recruited to the plasma membrane via its C-terminal pleckstrin homology domain to phosphatidylinositol 4,5-bisphosphate (PIP₂). Methods: Human embryonic kidney cells (HEK293) transfected to express wild-type and mutant GRK2 and human CCR7, human T cell lines harboring heterozygous deletions of GRK2, and naïve primary T cells from GRK2 heterozygous (GRK2^+/−) or GRK2^f/f CD4-Cre mice were used to examine the effects of GRK2 on ligand-induced CCR7 signaling in T cells. We used flow cytometry to assay the effect of GRK2 on CCR7 internalization, Fluorescence Resonance Energy Transfer (FRET) to define the effect of GRK2 on CCR7 activation of Gα_i isoforms and transwell migration assays to examine the effect of GRK2 on chemotaxis. Since chemotaxis via CCR7 is mediated by phospholipase Cγ1 (PLCγ1), Western blot assays were used to measure the effect of GRK2 during downstream signaling via phosphorylation of PLCγ1. Results: We found that following CCL19 binding, GRK2 promoted kinase-dependent CCR7 recruitment of arrestin-3, rapid CCR7 internalization and Gα_i3 recruitment to CCR7. In contrast, following binding of CCL21 to CCR7, GRK2 slowed CCR7 internalization, induced recruitment of Gα_i2 to the activated receptor_, and promoted chemotaxis. Since we have shown that CCL21 promotes chemotaxis via PLCγ1, we examined the effect of GRK2 on PLCγ1 activation and found that GRK2 had no effect on CCL21-mediated PLCγ1 phosphorylation. Conclusions: GRK2 promotes differential signaling downstream of CCR7 activation by CCL19 and CCL21 and provides a model for biased signaling downstream of a GPCR driven by GRK2. Full article

MRGPRX2 (Mas-related G protein-coupled receptor member X2) is implicated in mast cell (MC)-driven disorders due to its ability to bind diverse ligands, which may be G-protein-biased or balanced, with the latter activating both G-proteins and the β-arrestin pathway. Icatibant, a peptide drug, produces injection-site reactions in most patients and is used experimentally to probe MRGPRX2 function in skin tests. While reported to be G-protein-biased, it is unknown how skin MCs respond to icatibant, although these are the primary target cells during therapy. We therefore compared responses to icatibant with those induced by the balanced agonist substance P (SP) in skin MCs. Degranulation and desensitization were assessed via β-hexosaminidase release, receptor internalization by flow cytometry, and downstream signaling by immunoblotting. Skin MCs degranulated in response to SP and icatibant, relying on Gi proteins and calcium channels; Gq and PI3K (Phosphoinositide 3-kinase) contributed more strongly to exocytosis following icatibant, while JNK (c-Jun n-terminal kinase) was more relevant for SP. Both agonists activated ERK, PI3K/AKT, and (weakly) p38. Surprisingly, and in contrast to the LAD2 (Laboratory of Allergic Diseases 2 mast cell line) MC line, icatibant was at least as potent as SP in eliciting MRGPRX2 internalization and (cross-)desensitization in skin MCs. These findings suggest that icatibant functions differently in primary versus transformed MCs, acting as a fully balanced ligand in the former by triggering not only degranulation but also receptor internalization and desensitization. Therefore, not only the ligand but also the MRGPRX2-expressing cell plays a decisive role in whether a ligand is balanced or biased. These findings are relevant to our understanding of icatibant’s clinical effects on edema and itch. Full article

Drought is the single largest abiotic threat to soybean yield, yet the lectin genes that mediate drought perception and signaling in this crop have never been systematically mapped. We reanalyzed the public RNA-seq dataset GSE237798 (Williams 82 leaves, 7-day water withdrawal) with an updated fastp–HISAT2–featureCounts–DESeq2 pipeline and a curated catalog of 359 soybean lectin loci. Of the 127 lectin transcripts showing any drought-dependent shift, only 15 were stringently differentially expressed with substantial fold changes: 7 were upregulated and 8 downregulated. These genes span four families, GNA, legume, LysM and Nictaba-related lectins, and are heavily biased toward lectin receptor-like kinases (11 of 15), pinpointing the plasma membrane as the main control node. Gene Ontology enrichment highlights protein autophosphorylation and signal-transduction terms, and the inspection of AlphaFold models together with established lectin knowledge indicates that G- and L-type lectin domains have largely lost canonical carbohydrate-binding residues, whereas LysM and Nictaba proteins retain conserved folds compatible with ligand binding. The data expose a focused, modular lectin program rather than the broad activation often assumed: most soybean lectins stay silent under drought conditions, and only a defined subset toggles their expression, albeit mildly. Full article

G protein-coupled receptors (GPCRs) transmit through G proteins upon agonist activation, followed by phosphorylation by GPCR kinases (GRKs) to initiate β-arrestin signaling. However, the molecular mechanisms underlying GPCR signaling regulation by distinct agonists, GRK subtypes, and phosphorylation patterns remain poorly understood. The angiotensin II (AngII) type 1 receptor (AT1R), a prototypical GPCR, serves as an ideal model for studying biased ligands and signaling. Here, we investigated the wild-type (WT) AT1R and mutants of three potential phosphorylation motifs at its C-terminus (Motif I: S326/S328/S331, Motif II: T332/S335/T336/S338, and Motif III: S346/S347/S348/T349) using unbiased agonist AngII, β-arrestin-biased agonist TRV026, and G protein-biased agonist TRV056, along with GRK2/3/5/6 subtypes. We employed phosphorylation assays, β-arrestin pull-down experiments, molecular dynamics simulations, and AlphaFold3 predictions to dissect these mechanisms. Our results reveal that GRK2-mediated AT1R phosphorylation is abolished by mutations in Motifs I and II, with Motif II exhibiting a more pronounced effect. This phosphorylation was enhanced by Gβγ subunits. In contrast, GRK3-mediated phosphorylation remained unaffected by any mutations. GRK5 specifically phosphorylated Motif II, while GRK6 phosphorylated Motif II with the unbiased agonist AngII and both Motifs I and II with biased agonists TRV026 and TRV056. Notably, Motif II mutations reduced β-arrestin1/2 recruitment by GRK5/6 but not GRK2/3. Molecular dynamics simulations demonstrated that Motif II phosphorylation minimized steric hindrance, facilitating stable β-arrestin interactions, whereas Motif I phosphorylation increased intramolecular contacts that potentially impede recruitment. AlphaFold3 models provided detailed insights into the interactions between Motif II and β-arrestin1/2. Collectively, our findings elucidate diverse AT1R phosphorylation patterns driven by different agonists and GRK subtypes, offering a framework for developing signaling-biased AT1R therapeutics by decoding GRK-specific phosphorylation barcodes. Full article

The tomato leafminer (Tuta absoluta), a globally invasive pest, poses a major economic threat to tomato production. Although chemical control remains the primary management method, sustainable alternatives are urgently needed. Sex pheromone communication is critical for moth courtship and mating, with pheromone-binding proteins (PBPs) playing a key role in this process. In this study, we identified a PBP gene, TabsPBP2, from the T. absoluta transcriptome. Real-time quantitative PCR (RT-qPCR) revealed that TabsPBP2 is highly expressed in the antennae, with a strong male-biased expression pattern. Ligand-binding assays demonstrated that TabsPBP2 has the highest affinity for the sex pheromones (3E, 8Z, 11Z)-tetradecatrienyl acetate (TDTA) and (3E, 8Z)-tetradecadienyl acetate (TDDA). It also demonstrated a moderate-to-strong binding affinity to several tomato volatiles, including 2-carene, myrcene, α-pinene, cis-3-hexen-l-ol, methyl salicylate, sabinene, and α-terpinene. Molecular docking suggested that hydrophobic interactions predominantly stabilize the TabsPBP2–ligand complexes, with PHE118, PHE12, LEU90, LEU68, and ALA73 identified as key interacting residues. Electroantennogram (EAG) and Y-tube olfactometer assays confirmed that TDTA and TDDA act as strong attractants for male T. absoluta. This study enhances our understanding of the pheromone recognition in T. absoluta and provides a foundation for developing novel, pheromone-based pest control strategies. Full article

The glucagon-like peptide-1 receptor (GLP-1R), which belongs to the class B1 G protein-coupled receptor (GPCR) family, is an important target for treatment of metabolic disorders, including type 2 diabetes and obesity. The growing interest in GLP-1R-based therapies is driven by the development of various functional agonists as well as the huge commercial market. Thus, understanding the structural details of ligand-induced signaling are important for developing improved GLP-1R drugs. Here, we investigated the conformational dynamics of the receptor in complex with a selection of prototypical functional agonists, including CHU-128 (small molecule-biased), danuglipron (small molecule balanced), and Peptide 19 (peptide balanced), which exhibit unique, distinct binding modes and induced helix packing. Furthermore, our all-atom molecular dynamics (MD) simulations revealed atomic feature how different those ligands led to signaling pathway preference. Our findings offer valuable insights into the mechanistic principle of GLP-1R activation, which are helpful for the rational design of next-generation GLP-1R drug molecules. Full article

Spurred by the enormous therapeutic success of glucagon-like peptide-1 receptor (GLP-1R) agonists (GLP1-RAs) against diabetes and obesity, glucagon family receptor pharmacology has garnered a tremendous amount of interest. Glucagon family receptors, e.g., the glucagon receptor itself (GCGR), the GLP-1R, and the glucose-dependent insulinotropic peptide receptor (GIPR), belong to the incretin receptor superfamily, i.e., receptors that increase blood glucose-dependent insulin secretion. All incretin receptors are class B1 G protein-coupled receptors (GPCRs), coupling to the G_s type of heterotrimeric G proteins which activates adenylyl cyclase (AC) to produce cyclic adenosine monophosphate (cAMP). Most GPCRs undergo desensitization, i.e., uncouple from G proteins and internalize, thanks to interactions with the βarrestins (arrestin-2 and -3). Since the βarrestins can also mediate their own G protein-independent signaling, any given GPCR can theoretically signal (predominantly) either via G proteins or βarrestins, i.e., be a G protein- or βarrestin-“biased” receptor, depending on the bound ligand. A plethora of experimental evidence suggests that the GLP-1R does not undergo desensitization in physiologically relevant tissues in vivo, but rather, it produces robust and prolonged cAMP signals. A particular property of constant cycling between the cell membrane and caveolae/lipid rafts of the GLP-1R may underlie its lack of desensitization. In contrast, GIPR signaling is extensively mediated by βarrestins and the GIPR undergoes significant desensitization, internalization, and downregulation, which may explain why both agonists and antagonists of the GIPR exert the same physiological effects. Here, we discuss this evidence and make a case for the GLP-1R being a phenotypically or functionally G_s-selective receptor. We also discuss the implications of this for the development of GLP-1R poly-ligands, which are increasingly pursued for the treatment of obesity and other diseases. Full article

Comparative studies using lysophosphatidic acid (LPA) and the synthetic agonist, oleoyl-methoxy glycerophosphothionate (OMPT), in cells expressing the LPA₃ receptor revealed differences in the action of these agents. The possibility that more than one recognition cavity might exist for these ligands in the LPA₃ receptor was considered. We performed agonist docking studies exploring the whole protein to obtain tridimensional details of the ligand–receptor interaction. Functional in cellulo experiments using mutants were also executed. Our work includes blind docking using the unrefined and refined proteins subjected to hot spot predictions. Distinct ligand protonation (charge −1 and −2) states were evaluated. One LPA recognition cavity is located near the lower surface of the receptor close to the cytoplasm (Lower Cavity). OMPT displayed an affinity for an additional identification cavity detected in the transmembrane and extracellular regions (Upper Cavity). Docking targeted to Trp102 favored binding of both ligands in the transmembrane domain near the extracellular areas (Upper Cavity), but the associating amino acids were not identical due to close sub-cavities. A receptor model was generated using AlphaFold3, which properly identified the transmembrane regions of the sequence and co-modeled the lipid environment accordingly. These two models independently generated (with and without the membrane) and adopted essentially the same conformation, validating the data obtained. A DeepSite analysis of the model predicted two main binding pockets, providing additional confidence in the predicted ligand-binding regions and support for the relevance of the docking-based interaction models. In addition, mutagenesis was performed of the amino acids of the two detected cavities. In the in cellulo studies, LPA action was much less affected by the distinct mutations than that of OMPT (which was almost abolished). Therefore, docking and functional data indicate the presence of distinct agonist binding cavities in the LPA₃ receptor. Full article

Recent studies have revealed marked sex differences in pathophysiological roles of spinal microglia in neuropathic pain, with microglia contributing to pain exacerbation exclusively in males. However, the characteristics of pain-enhancing microglia, which are more prominent in males, remain poorly understood. Here, we reanalyzed a previously published single-cell RNA sequencing dataset and identified a microglial subpopulation that significantly increases in the spinal dorsal horn (SDH) of male mice following peripheral nerve injury. CC-chemokine ligand 4 (CCL4) was highly expressed in this subpopulation and its mRNA levels were increased in the SDH after partial sciatic nerve ligation (PSL) only in male mice. Notably, CCL4 expression was reduced in male mice following microglial depletion, indicating that microglia are the primary source of CCL4. Intrathecal administration of maraviroc, an inhibitor of the CCL4–CC-chemokine receptor 5 (CCR5) signaling pathway, after PSL, significantly suppressed mechanical allodynia only in male mice. Furthermore, intrathecal administration of CCL4 induced mechanical allodynia in both sexes, accompanied by increased expression of c-fos, a neuronal excitation marker, in the SDH. These findings highlight a sex-biased difference in the gene expression profile of spinal microglia following peripheral nerve injury, with elevated CCL4 expression in male mice potentially contributing to pain exacerbation. Full article

G-protein coupled receptors (GPCRs) are the largest family of membrane proteins engaged in transducing signals from the extracellular environment into the cell. GPCR-biased signaling occurs when two different ligands, sharing the same binding site, induce distinct signaling pathways. This selective signaling offers significant potential for the design of safer and more effective drugs. Although its molecular mechanism remains elusive, big efforts are made to try to explain this mechanism using a wide range of methods. Recent advances in computational techniques and AI technology have introduced a variety of simulations and machine learning tools that facilitate the modeling of GPCR signal transmission and the analysis of ligand-induced biased signaling. In this review, we present the current state of in silico approaches to elucidate the structural mechanism of GPCR-biased signaling. This includes molecular dynamics simulations that capture the main interactions causing the bias. We also highlight the major contributions and impacts of transmembrane domains, loops, and mutations in mediating biased signaling. Moreover, we discuss the impact of machine learning models on bias prediction and diffusion-based generative AI to design biased ligands. Ultimately, this review addresses the future directions for studying the biased signaling problem through AI approaches. Full article

The Chinese tongue sole (Cynoglossus semilaevis) is a marine flatfish of significant economic value, characterized by pronounced female-biased sexual size dimorphism (SSD). Sexual differences of cell number and gene expression within the PIT-1 lineage of the pituitary gland may be crucial for interpreting the female-biased SSD of C. semilaevis. Among hormones secreted by PIT-1 cell lineage, growth hormone (gh), prolactin (prl), prolactin 2 (prl2), and somatolactin (sl) comprise a gene family within the extensive superfamily of class-1 helical cytokines. To better understand the function of the gh/prl/sl in teleost SSD, we firstly identified five genes of the gh/prl/sl family (gh, sl, prl, prl2a, and prl2b) and their receptors (ghra, ghrb, prlra, prlrb, and prlr-like) from C. semilaevis at the genome-wide level. Phylogenetic analyses revealed that the gh/prl/sl family and their receptors were each clustered into five distinct groups. More microsatellites were revealed in the intron 2 of gh gene of female rather than the male and pseudo-male individuals, which is positively correlated with its sexual expression pattern. Interaction network prediction indicated that gh, prl, and sl may collectively contribute to individual growth and development. A FRET experiment showed that ghra can act as a receptor for sl. Additionally, the transcripts of the gh/prl/sl family and their receptors exhibited varying abundances in the pituitary, brain, gonad, and liver of both female and male C. semilaevis, with most ligands showing the highest abundance in the female pituitary. Furthermore, gh and sl were found to be maternally expressed. The knock-down of gh, prl, and sl in the pituitary cells could lead to the expression change of igf1, c-fos, and sos2. This study provided a foundation for further functional characterization of the gh/prl/sl gene family, contributing to a deeper understanding of the growth and reproductive mechanisms in C. semilaevis. Full article

The beet webworm, Loxostege sticticalis, is a typical migratory pest. Although miRNAs participate in many physiological functions, little is known about the functions of miRNAs in olfactory regulation. In this study, 1120 (869 known and 251 novel) miRNAs were identified in the antennae of L. sticticalis by using high-throughput sequencing technology. Among the known miRNAs, 189 from 49 families were insect-specific, indicating that these miRNAs might play unique roles in insects. Furthermore, based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we found that 3647 and 1393 miRNAs were associated with localization and the regulation of localization, respectively, and 80 miRNAs were enriched in the neuroactive ligand–receptor interaction pathway. These miRNAs might be involved in the olfactory system of L. sticticalis. Notably, qRT-PCR showed that most of the tested miRNAs presented similar expression patterns compared with the RNA-seq data and that miR-87-3, novel-miR-78, and novel-miR-142 were significantly differentially expressed in the antennae of males and females. In addition, 21 miRNAs were predicted to target 23 olfactory genes, including 10 odorant-binding proteins (OBPs), 3 chemosensory proteins (CSPs), 4 odorant receptors (ORs), 1 ionotropic receptor (IR), and 5 gustatory receptors (GRs). The olfactory-related miRNAs exhibited low-abundance transcripts, except undef-miR-55 and undef-miR-523, and gender-biased expression was not observed for olfactory-related miRNAs. Our findings provide an overview of the potential miRNAs involved in olfactory regulation, which may provide important information on the function of miRNAs in the insect olfactory system. Full article

Chemosensory protein (CSP) genes significantly enriched in the female antennae are potential molecular candidates for mediating female oviposition behaviors. In this study, we presented the interaction mechanisms of a female-antenna-biased PxutCSP19 in Papilio xuthus to 47 host volatiles, four biopesticides and 24 synthetic insecticides. Using a bioinformatics-based homology search, 22 genes orthologous to PxutCSP19 were identified from 22 other Papilio butterflies with high sequence identities to each other (73.20~98.72%). Multiple alignment analyses revealed a particularly extended N-terminus of Papilio CSP19s (an average of 154 residues) compared to insects’ typical CSPs (approximately 120 residues). The expression profiles indicated that PxutCSP19 was significantly enriched in the female antennae, with a 31.81-fold difference relative to the male antennae. In ligand-binding assays, PxutCSP19 could strongly bind six host odorants with high affinities, ranging from dissociation constant (K_i) values of 20.44 ± 0.64 μM to 22.71 ± 0.73 μM. Notably, this protein was tuned to a monoterpenoid alcohol, linalool, which generally existed in the Rutaceae plants and elicited electrophysiological and behavioral activities of the swallowtail butterfly. On the other hand, PxutCSP19 was also capable of binding eight insecticides with stronger binding abilities (K_i < 12 μM) compared to host odorants. When an extended N-terminal region of PxutCSP19 was truncated into two different proteins, they did not significantly affect the binding of PxutCSP19 to ligands with high affinities, suggesting that this extended N-terminal sequences were not involved in the specificity of ligand recognition. Altogether, our study sheds light on the putative roles of PxutCSP19 enriched in the female antennae of P. xuthus in the perception of host volatiles and the sequestering of insecticides, and it complements the knowledge of butterfly CSPs in olfaction and insecticide resistance. Full article