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12 pages, 4783 KB

Open AccessArticle

Functional Coupling of Calcium-Sensing Receptor and Polycystin-2 in Renal Epithelial Cells: Physiological Role and Potential Therapeutic Target in Polycystic Kidney Disease

by Annarita Di Mise, Angela Ferrulli, Mariangela Centrone, Maria Venneri, Marianna Ranieri, Grazia Tamma, Rosa Caroppo and Giovanna Valenti

Int. J. Mol. Sci. 2025, 26(24), 12004; https://doi.org/10.3390/ijms262412004 - 13 Dec 2025

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Abstract

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutations in PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively, characterized by excessive cell proliferation and fluid secretion, resulting in renal cyst formation and growth. PC1 and PC2 form a complex [...] Read more.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutations in PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively, characterized by excessive cell proliferation and fluid secretion, resulting in renal cyst formation and growth. PC1 and PC2 form a complex localized on the plasma membrane, endoplasmic reticulum, and primary cilia. PC2 is a non-selective cation channel which, in renal epithelial cells, contributes to calcium transport and signaling. It has been previously shown in renal cells that high external calcium increases whole-cell currents likely mediated by PC2. In this study, we explored the possibility that the Calcium Sensing Receptor (CaSR) is involved in the functional regulation of PC2. To test this hypothesis, human conditionally immortalized Proximal Tubular Epithelial cells, isolated from urine sediments, wt or with stably downregulated PKD1 (PC1KD) or PKD2 (PC2KD) were used. Interestingly, CaSR and PC2 co-immunoprecipitated and Proximity Ligation Assay demonstrated a direct physical interaction at endogenous protein levels. Membrane potential measurements demonstrated that selective CaSR activation, elicited by the calcimimetic R568, caused plasma membrane depolarization, consistent with the modulation of PC2-mediated cation currents, which was significantly lower in PC2KD with respect to wt and PC1KD cells. To conclude, this study provides evidence for a functional coupling of CaSR and PC2, which might be relevant for therapeutic strategies to correct dysregulations occurring in ADPKD. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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36 pages, 7997 KB

Open AccessArticle

The Cannabinoid CB₁ Receptor Inverse Agonist/Antagonist SR141716A Activates the Adenylate Cyclase/PKA Signaling Pathway Among Other Intracellular Emetic Signals to Evoke Vomiting in Least Shrews (Cryptotis parva)

by Yina Sun, Louiza Belkacemi, Weixia Zhong, Zollie Daily and Nissar A. Darmani

Int. J. Mol. Sci. 2025, 26(20), 9884; https://doi.org/10.3390/ijms26209884 - 11 Oct 2025

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Abstract

Intracellular emetic signals involved in the cannabinoid CB₁ receptor inverse agonist/antagonist SR141716A were investigated. SR141716A (20 mg/kg, i.p.)-evoked vomiting occurred via both the central and peripheral mechanisms. This was accompanied by robust emesis-associated increases in the following: (i) c-fos- and [...] Read more.

Intracellular emetic signals involved in the cannabinoid CB₁ receptor inverse agonist/antagonist SR141716A were investigated. SR141716A (20 mg/kg, i.p.)-evoked vomiting occurred via both the central and peripheral mechanisms. This was accompanied by robust emesis-associated increases in the following: (i) c-fos- and phospho-glycogen synthase kinase-3α/β (p-GSK-3αβ)-expression in the shrew’s dorsal vagal complex (DVC), (ii) phospho-extracellular signal-regulated kinase1/2 (p-ERK_1/2) expression in both the DVC and jejunal enteric nervous system, and (iii) time-dependent upregulation of cAMP levels and phosphorylation of protein kinase A (PKA), protein kinase B (Akt), GSK-3α/β, ERK_1/2, and protein kinase C αβII (PKCαβII) in the brainstem. SR141716A-evoked emetic parameters were attenuated by diverse inhibitors of the following: PKA, ERK_1/2, GSK-3, phosphatidylinositol 3-kinase (PI3K)-Akt pathway, phospholipase C (PLC), PKC, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), L-type Ca²⁺ channel (LTCC), store-operated Ca²⁺ entry (SOCE), inositol trisphosphate receptor (IP3R), ryanodine receptor (RyRs), both 5-HT₃-, and D_2/3-receptor antagonists, and the transient receptor potential vanilloid 1 receptor (TRPV1R) agonist. SR141716A appears to evoke vomiting via inverse agonist activity involving emesis-associated kinases, including cAMP/PKA, ERK_1/2, PI3K/Akt/GSK-3, PLC/PKCαβII, and CaMKII, which depend upon Ca²⁺ mobilization linking extracellular Ca²⁺ entry via plasma membrane Ca²⁺ channels (LTCC, SOCE, TRIPV1R) and intracellular Ca²⁺ release via IP3Rs and RyRs. The 5-HT₃, NK₁, and D_2/3 receptors also contribute to SR141716A-mediated vomiting. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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12 pages, 3251 KB

Open AccessArticle

Neuromedin U Receptor NMUR3 Regulates Autophagy, Thereby Enhancing Thermal Tolerance in C. elegans

by Limei He, Jianqi Yang, Shufang Wang, Yicheng Ma and Chenggang Zou

Int. J. Mol. Sci. 2025, 26(17), 8471; https://doi.org/10.3390/ijms26178471 - 31 Aug 2025

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Abstract

Neuromedin U receptors (NMURs) represent a class of evolutionarily conserved G-protein-coupled receptors that play a pivotal role in a variety of physiological processes. However, the role of NMURs in the heat shock response has yet to be elucidated. Using the nematode Caenorhabditis elegans [...] Read more.

Neuromedin U receptors (NMURs) represent a class of evolutionarily conserved G-protein-coupled receptors that play a pivotal role in a variety of physiological processes. However, the role of NMURs in the heat shock response has yet to be elucidated. Using the nematode Caenorhabditis elegans as a model system, we demonstrate herein that functional loss of NMUR-3 results in reduced survival upon heat shock. The regulation of thermal tolerance by NMUR-3 is dependent on AMPK. Furthermore, our data demonstrate that NMUR-3 activates autophagy via AMPK, thereby contributing to protection against heat shock. The results of this study suggest that NMUR-3 is crucial for thermal tolerance in C. elegans. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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8 pages, 1035 KB

Open AccessCommunication

The G-Protein-Coupled Estrogen Receptor Selective Agonist G-1 Attenuates Cell Viability and Migration in High-Grade Serous Ovarian Cancer Cell Lines

by Donia Hanafi, Rob U. Onyenwoke and K. Sean Kimbro

Int. J. Mol. Sci. 2024, 25(12), 6499; https://doi.org/10.3390/ijms25126499 - 13 Jun 2024

Cited by 4 | Viewed by 2290

Abstract

The G-protein-coupled estrogen receptor (GPER; G-protein-coupled estrogen receptor 30, also known as GPR30) is a novel estrogen receptor and has emerged as a promising target for ovarian cancer. GPER, a seven-transmembrane receptor, suppresses cellular viability and migration in studied ovarian cancer cells. However, [...] Read more.

The G-protein-coupled estrogen receptor (GPER; G-protein-coupled estrogen receptor 30, also known as GPR30) is a novel estrogen receptor and has emerged as a promising target for ovarian cancer. GPER, a seven-transmembrane receptor, suppresses cellular viability and migration in studied ovarian cancer cells. However, its impact on the fallopian tube, which is the potential origin of high-grade serous (HGSC) ovarian cancer, has not been addressed. This study was conducted to evaluate the relationship of GPER, ovarian cancer subtypes, i.e., high-grade serous cell lines (OV90 and OVCAR420), as well as the cell type that is the potential origin of HGSC ovarian cancer (i.e., the fallopian tube cell line FT190). The selective ligand assessed here is the agonist G-1, which was utilized in an in vitro study to characterize its effects on cellular viability and migration. As a result, this study has addressed the effect of a specific GPER agonist on cell viability, providing a better understanding of the effects of this compound on our diverse group of studied cell lines. Strikingly, attenuated cell proliferation and migration behaviors were observed in the presence of G-1. Thus, our in vitro study reveals the impact of the origin of HGSC ovarian cancers and highlights the GPER agonist G-1 as a potential therapy for ovarian cancer. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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23 pages, 7622 KB

Open AccessArticle

Keras/TensorFlow in Drug Design for Immunity Disorders

by Paulina Dragan, Kavita Joshi, Alessandro Atzei and Dorota Latek

Int. J. Mol. Sci. 2023, 24(19), 15009; https://doi.org/10.3390/ijms241915009 - 9 Oct 2023

Cited by 10 | Viewed by 4339

Abstract

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue [...] Read more.

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue or pathogens. Dysregulation of the immune system leading to disorders such as allergies, autoimmune diseases, or cancer requires efficient, fast-acting drugs to minimize the long-term effects of chronic inflammation. Here, we performed structure-based virtual screening (SBVS) assisted by the Keras/TensorFlow neural network (NN) to find novel compound scaffolds acting on three chemokine receptors: CCR2, CCR3, and one CXC receptor, CXCR3. Keras/TensorFlow NN was used here not as a typically used binary classifier but as an efficient multi-class classifier that can discard not only inactive compounds but also low- or medium-activity compounds. Several compounds proposed by SBVS and NN were tested in 100 ns all-atom molecular dynamics simulations to confirm their binding affinity. To improve the basic binding affinity of the compounds, new chemical modifications were proposed. The modified compounds were compared with known antagonists of these three chemokine receptors. Known CXCR3 compounds were among the top predicted compounds; thus, the benefits of using Keras/TensorFlow in drug discovery have been shown in addition to structure-based approaches. Furthermore, we showed that Keras/TensorFlow NN can accurately predict the receptor subtype selectivity of compounds, for which SBVS often fails. We cross-tested chemokine receptor datasets retrieved from ChEMBL and curated datasets for cannabinoid receptors. The NN model trained on the cannabinoid receptor datasets retrieved from ChEMBL was the most accurate in the receptor subtype selectivity prediction. Among NN models trained on the chemokine receptor datasets, the CXCR3 model showed the highest accuracy in differentiating the receptor subtype for a given compound dataset. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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18 pages, 4098 KB

Open AccessArticle

Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and β-Arrestin through BRET Assays and Molecular Docking

by Dongxue Wang, Yunjin Yao, Shiqi Wang, Yifei Hou, Lanxue Zhao, Hao Wang, Hongzhuan Chen and Jianrong Xu

Int. J. Mol. Sci. 2023, 24(8), 7356; https://doi.org/10.3390/ijms24087356 - 16 Apr 2023

Cited by 11 | Viewed by 6957

Abstract

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently [...] Read more.

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing Gαq and β-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and β-arrestin2. Pilocarpine preferentially promoted the recruitment of β-arrestin2 (∆∆RAi = −0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to β-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards Gαq and β-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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14 pages, 1579 KB

Open AccessArticle

Starvation Induces Extracellular Accumulation of Polyphosphate in Dictyostelium discoideum to Inhibit Macropinocytosis, Phagocytosis, and Exocytosis

by Ramesh Rijal, Issam Ismail, Shiyu Jing and Richard H. Gomer

Int. J. Mol. Sci. 2023, 24(6), 5923; https://doi.org/10.3390/ijms24065923 - 21 Mar 2023

Cited by 1 | Viewed by 3590

Abstract

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit [...] Read more.

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity; this effect requires GrlD and Ppk1, but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, and to both decrease energy expenditures and retain nutrients. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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13 pages, 2454 KB

Open AccessArticle

GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

by Yunkyung Heo, Eunhee Yang, Yechan Lee, Yohan Seo, Kunhi Ryu, Hyejin Jeon and Wan Namkung

Int. J. Mol. Sci. 2022, 23(18), 10631; https://doi.org/10.3390/ijms231810631 - 13 Sep 2022

Cited by 5 | Viewed by 4157

Abstract

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor (GPCR) activated by proteolytic cleavage of its N-terminal domain. Once activated, PAR2 is rapidly desensitized and internalized by phosphorylation and β-arrestin recruitment. Due to its irreversible activation mechanism, some agonists that rapidly desensitized PAR2 have [...] Read more.

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor (GPCR) activated by proteolytic cleavage of its N-terminal domain. Once activated, PAR2 is rapidly desensitized and internalized by phosphorylation and β-arrestin recruitment. Due to its irreversible activation mechanism, some agonists that rapidly desensitized PAR2 have been misconceived as antagonists, and this has impeded a better understanding of the pathophysiological role of PAR2. In the present study, we found that GB83, initially identified as a PAR2 antagonist, is a bona fide agonist of PAR2 that induces unique cellular signaling, distinct from trypsin and PAR2-activating peptide (AP). Activation of PAR2 by GB83 markedly elicited an increase in intracellular calcium levels and phosphorylation of MAPKs, but in a delayed and sustained manner compared to the rapid and transient signals induced by trypsin and PAR2-AP. Interestingly, unlike PAR2-AP, GB83 and trypsin induced sustained receptor endocytosis and PAR2 colocalization with β-arrestin. Moreover, the recovery of the localization and function of PAR2 was significantly delayed after stimulation by GB83, which may be the reason why GB83 is recognized as an antagonist of PAR2. Our results revealed that GB83 is a bona fide agonist of PAR2 that uniquely modulates PAR2-mediated cellular signaling and is a useful pharmacological tool for studying the pathophysiological role of PAR2. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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Review

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21 pages, 2014 KB

Open AccessReview

GPR75: Advances, Challenges in Deorphanization, and Potential as a Novel Drug Target for Disease Treatment

by Jingyi Han, Jiaojiao Li, Sirui Yao, Zao Wei, Hui Jiang, Tao Xu, Junwei Zeng, Lin Xu and Yong Han

Int. J. Mol. Sci. 2025, 26(9), 4084; https://doi.org/10.3390/ijms26094084 - 25 Apr 2025

Cited by 4 | Viewed by 8208

Abstract

G protein-coupled receptor 75 (GPR75), a novel member of the rhodopsin-like G protein-coupled receptor (GPCR) family, has been identified across various tissues and organs, where it contributes to biological regulation and disease progression. Recent studies suggest potential interactions between GPR75 and ligands such [...] Read more.

G protein-coupled receptor 75 (GPR75), a novel member of the rhodopsin-like G protein-coupled receptor (GPCR) family, has been identified across various tissues and organs, where it contributes to biological regulation and disease progression. Recent studies suggest potential interactions between GPR75 and ligands such as 20-hydroxyeicosatetraenoic acid (20-HETE) and C-C motif chemokine ligand 5 (CCL5/RANTES); however, its definitive endogenous ligand remains unidentified, and GPR75 is currently classified as an orphan receptor by International Union of Basic and Clinical Pharmacology (IUPHAR). Research on GPR75 deorphanization has underscored its critical roles in disease models, particularly in metabolic health, glucose regulation, and stability of the nervous and cardiovascular systems. However, the signaling pathways of GPR75 across different pathological conditions require further investigation. Importantly, ongoing studies are targeting GPR75 for drug development, exploring small molecule inhibitors, antibodies, and gene silencing techniques, positioning GPR75 as a promising GPCR target for treating related diseases. This review summarizes the recent advancements in GPR75 deorphanization research, examines its functions across tissues and systems, and highlights its links to metabolic, cardiovascular, and neurological disorders, thereby providing a resource for researchers to better understand the biological functions of this receptor. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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11 pages, 2147 KB

Open AccessTechnical Note

GPCRVS - AI-driven Decision Support System for GPCR Virtual Screening

by Dorota Latek, Khushil Prajapati, Paulina Dragan, Matthew Merski and Przemysław Osial

Int. J. Mol. Sci. 2025, 26(5), 2160; https://doi.org/10.3390/ijms26052160 - 27 Feb 2025

Cited by 6 | Viewed by 3200

Abstract

G protein-coupled receptors (GPCRs) constitute the largest and most frequently used family of molecular drug targets. The simplicity of GPCR drug design results from their common seven-transmembrane-helix topology and well-understood signaling pathways. GPCRs are extremely sensitive to slight changes in the chemical structure [...] Read more.

G protein-coupled receptors (GPCRs) constitute the largest and most frequently used family of molecular drug targets. The simplicity of GPCR drug design results from their common seven-transmembrane-helix topology and well-understood signaling pathways. GPCRs are extremely sensitive to slight changes in the chemical structure of compounds, which allows for the reliable design of highly selective and specific drugs. Only recently has the number of GPCR structures, both in their active and inactive conformations, together with their active ligands, become sufficient to comprehensively apply machine learning in decision support systems to predict compound activity in drug design. Here, we describe GPCRVS, an efficient machine learning system for the online assessment of the compound activity against several GPCR targets, including peptide- and protein-binding GPCRs, which are the most difficult for virtual screening tasks. As a decision support system, GPCRVS evaluates compounds in terms of their activity range, the pharmacological effect they exert on the receptor, and the binding mode they could demonstrate for different types and subtypes of GPCRs. GPCRVS allows for the evaluation of compounds ranging from small molecules to short peptides provided in common chemical file formats. The results of the activity class assignment and the binding affinity prediction are provided in comparison with predictions for known active ligands of each included GPCR. Multiclass classification in GPCRVS, handling incomplete and fuzzy biological data, was validated on ChEMBL and Google Patents-retrieved data sets for class B GPCRs and chemokine CC and CXC receptors. Full article

(This article belongs to the Special Issue G Protein-Coupled Receptors)

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