Journal articles: 'Class A GPCR'

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Vidad, Ashley Ryan, Stephen Macaspac, and Ho Leung Ng. "Locating ligand binding sites in G-protein coupled receptors using combined information from docking and sequence conservation." PeerJ 9 (September 24, 2021): e12219. http://dx.doi.org/10.7717/peerj.12219.

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GPCRs (G-protein coupled receptors) are the largest family of drug targets and share a conserved structure. Binding sites are unknown for many important GPCR ligands due to the difficulties of GPCR recombinant expression, biochemistry, and crystallography. We describe our approach, ConDockSite, for predicting ligand binding sites in class A GPCRs using combined information from surface conservation and docking, starting from crystal structures or homology models. We demonstrate the effectiveness of ConDockSite on crystallized class A GPCRs such as the beta2 adrenergic and A2A adenosine receptors. We also demonstrate that ConDockSite successfully predicts ligand binding sites from high-quality homology models. Finally, we apply ConDockSite to predict the ligand binding sites on a structurally uncharacterized GPCR, GPER, the G-protein coupled estrogen receptor. Most of the sites predicted by ConDockSite match those found in other independent modeling studies. ConDockSite predicts that four ligands bind to a common location on GPER at a site deep in the receptor cleft. Incorporating sequence conservation information in ConDockSite overcomes errors introduced from physics-based scoring functions and homology modeling.

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Hou, Tianling, Yuemin Bian, Terence McGuire, and Xiang-Qun Xie. "Integrated Multi-Class Classification and Prediction of GPCR Allosteric Modulators by Machine Learning Intelligence." Biomolecules 11, no. 6 (June 11, 2021): 870. http://dx.doi.org/10.3390/biom11060870.

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G-protein-coupled receptors (GPCRs) are the largest and most diverse group of cell surface receptors that respond to various extracellular signals. The allosteric modulation of GPCRs has emerged in recent years as a promising approach for developing target-selective therapies. Moreover, the discovery of new GPCR allosteric modulators can greatly benefit the further understanding of GPCR cell signaling mechanisms. It is critical but also challenging to make an accurate distinction of modulators for different GPCR groups in an efficient and effective manner. In this study, we focus on an 11-class classification task with 10 GPCR subtype classes and a random compounds class. We used a dataset containing 34,434 compounds with allosteric modulators collected from classical GPCR families A, B, and C, as well as random drug-like compounds. Six types of machine learning models, including support vector machine, naïve Bayes, decision tree, random forest, logistic regression, and multilayer perceptron, were trained using different combinations of features including molecular descriptors, Atom-pair fingerprints, MACCS fingerprints, and ECFP6 fingerprints. The performances of trained machine learning models with different feature combinations were closely investigated and discussed. To the best of our knowledge, this is the first work on the multi-class classification of GPCR allosteric modulators. We believe that the classification models developed in this study can be used as simple and accurate tools for the discovery and development of GPCR allosteric modulators.

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Sato. "Conserved 2nd Residue of Helix 8 of GPCR May Confer the Subclass-Characteristic and Distinct Roles through a Rapid Initial Interaction with Specific G Proteins." International Journal of Molecular Sciences 20, no. 7 (April 9, 2019): 1752. http://dx.doi.org/10.3390/ijms20071752.

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To obtain a systematic view of the helix-8-second residue responsible for G protein-coupled receptor (GPCR)–G protein initial specific interactions, 786 human GPCRs were subclassified based on the pairs of agonist groups and target G proteins and compared with their conserved second residue of helix 8. Of 314 non-olfactory and deorphanized GPCRs, 273 (87%) conserved single amino acids in the subclasses, while 93 (58%) of the 160 subclasses possessed only a single GPCR member. Class B, C, Frizzled, and trace amine-associated GPCRs demonstrated 100% conservation, whereas class Ⅰ and Ⅱ olfactory and vomeronasal 1 receptors demonstrated much lower rates of conservation (20–47%). These conserved residues are characteristic of GPCR classes and G protein subtypes and confer their functionally-distinct roles.

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Vohra, Shabana, Bruck Taddese, Alex C. Conner, David R. Poyner, Debbie L. Hay, James Barwell, Philip J. Reeves, Graham J. G. Upton, and Christopher A. Reynolds. "Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies." Journal of The Royal Society Interface 10, no. 79 (February 6, 2013): 20120846. http://dx.doi.org/10.1098/rsif.2012.0846.

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Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A–class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg 2.39 , His 2.43 and Glu 3.46 , which makes a polar lock with T 6.37 . These alignments and models provide useful tools for understanding class B GPCR function.

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Pellissier, Lucie P., Gaël Barthet, Florence Gaven, Elisabeth Cassier, Eric Trinquet, Jean-Philippe Pin, Philippe Marin, et al. "G Protein Activation by Serotonin Type 4 Receptor Dimers." Journal of Biological Chemistry 286, no. 12 (January 19, 2011): 9985–97. http://dx.doi.org/10.1074/jbc.m110.201939.

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The discovery that class C G protein-coupled receptors (GPCRs) function as obligatory dimeric entities has generated major interest in GPCR oligomerization. Oligomerization now appears to be a common feature among all GPCR classes. However, the functional significance of this process remains unclear because, in vitro, some monomeric GPCRs, such as rhodopsin and β2-adrenergic receptors, activate G proteins. By using wild type and mutant serotonin type 4 receptors (5-HT4Rs) (including a 5-HT4-RASSL) expressed in COS-7 cells as models of class A GPCRs, we show that activation of one protomer in a dimer was sufficient to stimulate G proteins. However, coupling efficiency was 2 times higher when both protomers were activated. Expression of combinations of 5-HT4, in which both protomers were able to bind to agonists but only one could couple to G proteins, suggested that upon agonist occupancy, protomers did not independently couple to G proteins but rather that only one G protein was activated. Coupling of a single heterotrimeric Gs protein to a receptor dimer was further confirmed in vitro, using the purified recombinant WT RASSL 5-HT4R obligatory heterodimer. These results, together with previous findings, demonstrate that, differently from class C GPCR dimers, class A GPCR dimers have pleiotropic activation mechanisms.

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Chattopadhyay, Amitabha. "GPCRs: Lipid-Dependent Membrane Receptors That Act as Drug Targets." Advances in Biology 2014 (October 2, 2014): 1–12. http://dx.doi.org/10.1155/2014/143023.

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G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across cell membranes and represent major targets in the development of novel drug candidates in all clinical areas. Although there have been some recent leads, structural information on GPCRs is relatively rare due to the difficulty associated with crystallization. A specific reason for this is the intrinsic flexibility displayed by GPCRs, which is necessary for their functional diversity. Since GPCRs are integral membrane proteins, interaction of membrane lipids with them constitutes an important area of research in GPCR biology. In particular, membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. The role of membrane cholesterol in GPCR function is discussed with specific example of the serotonin1A receptor. Recent results show that GPCRs are characterized with structural motifs that preferentially associate with cholesterol. An emerging and important concept is oligomerization of GPCRs and its role in GPCR function and signaling. The role of membrane cholesterol in GPCR oligomerization is highlighted. Future research in GPCR biology would offer novel insight in basic biology and provide new avenues for drug discovery.

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Ellisdon, Andrew M., and Michelle L. Halls. "Compartmentalization of GPCR signalling controls unique cellular responses." Biochemical Society Transactions 44, no. 2 (April 11, 2016): 562–67. http://dx.doi.org/10.1042/bst20150236.

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With >800 members, G protein-coupled receptors (GPCRs) are the largest class of cell-surface signalling proteins, and their activation mediates diverse physiological processes. GPCRs are ubiquitously distributed across all cell types, involved in many diseases and are major drug targets. However, GPCR drug discovery is still characterized by very high attrition rates. New avenues for GPCR drug discovery may be provided by a recent shift away from the traditional view of signal transduction as a simple chain of events initiated from the plasma membrane. It is now apparent that GPCR signalling is restricted to highly organized compartments within the cell, and that GPCRs activate distinct signalling pathways once internalized. A high-resolution understanding of how compartmentalized signalling is controlled will probably provide unique opportunities to selectively and therapeutically target GPCRs.

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Guo, Yan-Zhi, Meng-Long Li, Ke-Long Wang, Zhi-Ning Wen, Min-Chun Lu, Li-Xia Liu, and Lin Jiang. "Fast Fourier Transform-based Support Vector Machine for Prediction of G-protein Coupled Receptor Subfamilies." Acta Biochimica et Biophysica Sinica 37, no. 11 (November 1, 2005): 759–66. http://dx.doi.org/10.1111/j.1745-7270.2005.00110.x.

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Abstract Although the sequence information on G-protein coupled receptors (GPCRs) continues to grow, many GPCRs remain orphaned (i.e. ligand specificity unknown) or poorly characterized with little structural information available, so an automated and reliable method is badly needed to facilitate the identification of novel receptors. In this study, a method of fast Fourier transform-based support vector machine has been developed for predicting GPCR subfamilies according to protein's hydrophobicity. In classifying Class B, C, D and F subfamilies, the method achieved an overall Matthew's correlation coefficient and accuracy of 0.95 and 93.3%, respectively, when evaluated using the jackknife test. The method achieved an accuracy of 100% on the Class B independent dataset. The results show that this method can classify GPCR subfamilies as well as their functional classification with high accuracy. A web server implementing the prediction is available at http://chem.scu.edu.cn/blast/Pred-GPCR.

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Fu, Zhe, Linjie Zhang, Sijin Hang, Shiyi Wang, Na Li, Xiaojing Sun, Zian Wang, et al. "Synthesis of Coumarin Derivatives: A New Class of Coumarin-Based G Protein-Coupled Receptor Activators and Inhibitors." Polymers 14, no. 10 (May 15, 2022): 2021. http://dx.doi.org/10.3390/polym14102021.

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To expand the range of daphnetin-based inhibitors/activators used for targeting G protein-coupled receptors (GPCRs) in disease treatment, twenty-five coumarin derivatives 1–25, including 7,8-dihydroxycoumarin and 7-hydroxycoumarin derivatives with various substitution patterns/groups at C3-/4- positions, were synthesized via mild Pechmann condensation and hydroxyl modification. The structures were characterized by 1H NMR, 13C NMR and ESI-MS. Their inhibition or activation activities relative to GPCRs were evaluated by double-antibody sandwich ELISA (DAS–ELISA) in vitro. The results showed that most of the coumarin derivatives possessed a moderate GPCR activation or inhibitory potency. Among them, derivatives 14, 17, 18, and 21 showed a remarkable GPCR activation potency, with EC50 values of 0.03, 0.03, 0.03, and 0.02 nM, respectively. Meanwhile, derivatives 4, 7, and 23 had significant GPCR inhibitory potencies against GPCRs with IC50 values of 0.15, 0.02, and 0.76 nM, respectively. Notably, the acylation of hydroxyl groups at the C-7 and C-8 positions of 7,8-dihydroxycoumarin skeleton or the etherification of the hydroxyl group at the C-7 position of the 7-hydroxycoumarin skeleton could successfully change GPCRs activators into inhibitors. This work demonstrated a simple and efficient approach to developing coumarin derivatives as remarkable GPCRs activators and inhibitors via molecular diversity-based synthesis.

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Wright, Shane C., Maria Consuelo Alonso Cañizal, Tobias Benkel, Katharina Simon, Christian Le Gouill, Pierre Matricon, Yoon Namkung, et al. "FZD5 is a Gαq-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs." Science Signaling 11, no. 559 (December 4, 2018): eaar5536. http://dx.doi.org/10.1126/scisignal.aar5536.

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Frizzleds (FZDs) are a group of seven transmembrane–spanning (7TM) receptors that belong to class F of the G protein–coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the β-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)–based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.

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Lazim, Raudah, Donghyuk Suh, Jai Woo Lee, Thi Ngoc Lan Vu, Sanghee Yoon, and Sun Choi. "Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers." International Journal of Molecular Sciences 22, no. 6 (March 22, 2021): 3241. http://dx.doi.org/10.3390/ijms22063241.

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G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).

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Kruse, Andrew C., Aashish Manglik, Brian K. Kobilka, and William I. Weis. "Applications of molecular replacement to G protein-coupled receptors." Acta Crystallographica Section D Biological Crystallography 69, no. 11 (October 18, 2013): 2287–92. http://dx.doi.org/10.1107/s090744491301322x.

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G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

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Berger, Miles, David W. Scheel, Hector Macias, Takeshi Miyatsuka, Hail Kim, Phuong Hoang, Greg M. Ku, et al. "Gαi/o-coupled receptor signaling restricts pancreatic β-cell expansion." Proceedings of the National Academy of Sciences 112, no. 9 (February 18, 2015): 2888–93. http://dx.doi.org/10.1073/pnas.1319378112.

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Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs—including the α-2a adrenergic receptor, ADRA2A—increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.

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Kwon, Yong-Jun, Weontae Lee, Auguste Genovesio, and Neil Emans. "A High-Content Subtractive Screen for Selecting Small Molecules Affecting Internalization of GPCRs." Journal of Biomolecular Screening 17, no. 3 (November 15, 2011): 379–85. http://dx.doi.org/10.1177/1087057111427347.

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G-protein–coupled receptors (GPCRs) are pivotal in cellular responses to the environment and are common drug targets. Identification of selective small molecules acting on single GPCRs is complicated by the shared machinery coupling signal transduction to physiology. Here, we demonstrate a high-content screen using a panel of GPCR assays to identify receptor selective molecules acting within the kinase/phosphatase inhibitor family. A collection of 88 kinase and phosphatase inhibitors was screened against seven agonist-induced GPCR internalization cell models as well as transferrin uptake in human embryonic kidney cells. Molecules acting on a single receptor were identified through excluding pan-specific compounds affecting housekeeping endocytosis or disrupting internalization of multiple receptors. We identified compounds acting on a sole GPCR from activities in a broad range of chemical structures that could not be easily sorted by conventional means. Selective analysis can therefore rapidly select compounds selectively affecting GPCR activity with specificity to one receptor class through high-content screening.

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Kapolka, N. J., G. J. Taghon, J. B. Rowe, W. M. Morgan, J. F. Enten, N. A. Lambert, and D. G. Isom. "DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery." Proceedings of the National Academy of Sciences 117, no. 23 (May 20, 2020): 13117–26. http://dx.doi.org/10.1073/pnas.2000430117.

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More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR–Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR–metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs.

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Bemister-Buffington, Joseph, Alex J. Wolf, Sebastian Raschka, and Leslie A. Kuhn. "Machine Learning to Identify Flexibility Signatures of Class A GPCR Inhibition." Biomolecules 10, no. 3 (March 14, 2020): 454. http://dx.doi.org/10.3390/biom10030454.

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We show that machine learning can pinpoint features distinguishing inactive from active states in proteins, in particular identifying key ligand binding site flexibility transitions in GPCRs that are triggered by biologically active ligands. Our analysis was performed on the helical segments and loops in 18 inactive and 9 active class A G protein-coupled receptors (GPCRs). These three-dimensional (3D) structures were determined in complex with ligands. However, considering the flexible versus rigid state identified by graph-theoretic ProFlex rigidity analysis for each helix and loop segment with the ligand removed, followed by feature selection and k-nearest neighbor classification, was sufficient to identify four segments surrounding the ligand binding site whose flexibility/rigidity accurately predicts whether a GPCR is in an active or inactive state. GPCRs bound to inhibitors were similar in their pattern of flexible versus rigid regions, whereas agonist-bound GPCRs were more flexible and diverse. This new ligand-proximal flexibility signature of GPCR activity was identified without knowledge of the ligand binding mode or previously defined switch regions, while being adjacent to the known transmission switch. Following this proof of concept, the ProFlex flexibility analysis coupled with pattern recognition and activity classification may be useful for predicting whether newly designed ligands behave as activators or inhibitors in protein families in general, based on the pattern of flexibility they induce in the protein.

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Huang, Shuya Kate, and R. Scott Prosser. "Dynamics and mechanistic underpinnings to pharmacology of class A GPCRs: an NMR perspective." American Journal of Physiology-Cell Physiology 322, no. 4 (April 1, 2022): C739—C753. http://dx.doi.org/10.1152/ajpcell.00044.2022.

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One-third of current pharmaceuticals target G protein-coupled receptors (GPCRs), the largest receptor superfamily in humans and mediators of diverse physiological processes. This review summarizes the recent progress in GPCR structural dynamics, focusing on class A receptors and insights derived from nuclear magnetic resonance (NMR) and other spectroscopic techniques. We describe the structural aspects of GPCR activation and the various pharmacological models that capture aspects of receptor signaling behavior. Spectroscopic studies revealed that receptors and their signaling complexes are dynamic allosteric systems that sample multiple functional states under basal conditions. The distribution of states within the conformational ensemble and the kinetics of transitions between states are regulated through the binding of ligands, allosteric modulators, and the membrane environment. This ensemble view of GPCRs provides a mechanistic framework for understanding many of the pharmacological phenomena associated with receptor signaling, such as basal activity, efficacy, and functional bias.

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Sakmar, Thomas P. "Clicking class B GPCR ligands." Nature Chemical Biology 7, no. 8 (July 18, 2011): 500–501. http://dx.doi.org/10.1038/nchembio.621.

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Saikia, Surovi, Manobjyoti Bordoloi, and Rajeev Sarmah. "Established and In-trial GPCR Families in Clinical Trials: A Review for Target Selection." Current Drug Targets 20, no. 5 (March 5, 2019): 522–39. http://dx.doi.org/10.2174/1389450120666181105152439.

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The largest family of drug targets in clinical trials constitute of GPCRs (G-protein coupled receptors) which accounts for about 34% of FDA (Food and Drug Administration) approved drugs acting on 108 unique GPCRs. Factors such as readily identifiable conserved motif in structures, 127 orphan GPCRs despite various de-orphaning techniques, directed functional antibodies for validation as drug targets, etc. has widened their therapeutic windows. The availability of 44 crystal structures of unique receptors, unexplored non-olfactory GPCRs (encoded by 50% of the human genome) and 205 ligand receptor complexes now present a strong foundation for structure-based drug discovery and design. The growing impact of polypharmacology for complex diseases like schizophrenia, cancer etc. warrants the need for novel targets and considering the undiscriminating and selectivity of GPCRs, they can fulfill this purpose. Again, natural genetic variations within the human genome sometimes delude the therapeutic expectations of some drugs, resulting in medication response differences and ADRs (adverse drug reactions). Around ~30 billion US dollars are dumped annually for poor accounting of ADRs in the US alone. To curb such undesirable reactions, the knowledge of established and currently in clinical trials GPCRs families can offer huge understanding towards the drug designing prospects including “off-target” effects reducing economical resource and time. The druggability of GPCR protein families and critical roles played by them in complex diseases are explained. Class A, class B1, class C and class F are generally established family and GPCRs in phase I (19%), phase II(29%), phase III(52%) studies are also reviewed. From the phase I studies, frizzled receptors accounted for the highest in trial targets, neuropeptides in phase II and melanocortin in phase III studies. Also, the bioapplications for nanoparticles along with future prospects for both nanomedicine and GPCR drug industry are discussed. Further, the use of computational techniques and methods employed for different target validations are also reviewed along with their future potential for the GPCR based drug discovery.

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Dijkman, Patricia M., Juan C. Muñoz-García, Steven R. Lavington, Patricia Suemy Kumagai, Rosana I. dos Reis, Daniel Yin, Phillip J. Stansfeld, Antonio José Costa-Filho, and Anthony Watts. "Conformational dynamics of a G protein–coupled receptor helix 8 in lipid membranes." Science Advances 6, no. 33 (August 2020): eaav8207. http://dx.doi.org/10.1126/sciadv.aav8207.

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G protein–coupled receptors (GPCRs) are the largest and pharmaceutically most important class of membrane proteins encoded in the human genome, characterized by a seven-transmembrane helix architecture and a C-terminal amphipathic helix 8 (H8). In a minority of GPCR structures solved to date, H8 either is absent or adopts an unusual conformation. The controversial existence of H8 of the class A GPCR neurotensin receptor 1 (NTS1) has been examined here for the nonthermostabilized receptor in a functionally supporting membrane environment using electron paramagnetic resonance, molecular dynamics simulations, and circular dichroism. Lipid-protein interactions with phosphatidylserine and phosphatidylethanolamine lipids, in particular, stabilize the residues 374 to 390 of NTS1 into forming a helix. Furthermore, introduction of a helix-breaking proline residue in H8 elicited an increase in ß-arrestin–NTS1 interactions observed in pull-down assays, suggesting that the structure and/or dynamics of H8 might play an important role in GPCR signaling.

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Wheatley, Mark, Jack Charlton, Mohammed Jamshad, Sarah J. Routledge, Sian Bailey, Penelope J. La-Borde, Maria T. Azam, et al. "GPCR–styrene maleic acid lipid particles (GPCR–SMALPs): their nature and potential." Biochemical Society Transactions 44, no. 2 (April 11, 2016): 619–23. http://dx.doi.org/10.1042/bst20150284.

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G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a ‘molecular pastry cutter’ to ‘solubilize’ GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR–SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

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Akondi, Kalyana Bharati, Marianne Paolini-Bertrand, and Oliver Hartley. "Precision-engineered Peptide and Protein Analogs: Establishing a New Discovery Platform for Potent GPCR Modulators." CHIMIA International Journal for Chemistry 75, no. 6 (June 30, 2021): 489–94. http://dx.doi.org/10.2533/chimia.2021.489.

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Numerous members of the human G protein-coupled receptor (GPCR) superfamily are receptors of therapeutic interest. GPCRs are considered to be highly tractable for drug discovery, representing the targets of approximately one-third of currently licensed drugs. These successful drug discovery outcomes cover only a relatively small subset of the superfamily, however, and many other attractive receptors have proven to present significant challenges. Among these difficult GPCRs are those whose natural ligands are peptides and proteins. In this review we explain the obstacles faced by GPCR drug discovery campaigns, with particular focus on those related to peptide and protein GPCRs. We describe a novel and promising approach for these targets based on engineering of their natural ligands and describe an integrated discovery platform that allows potent ligand analogs to be discovered rapidly and efficiently. Finally, we present a case study involving the chemokine receptor CCR5 to show that this approach can be used to generate new drugs for peptide and protein GPCR targets combining best-in-class potency with tunable signaling activity.

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Torrens-Fontanals, Mariona, Tomasz Maciej Stepniewski, David Aranda-García, Adrián Morales-Pastor, Brian Medel-Lacruz, and Jana Selent. "How Do Molecular Dynamics Data Complement Static Structural Data of GPCRs." International Journal of Molecular Sciences 21, no. 16 (August 18, 2020): 5933. http://dx.doi.org/10.3390/ijms21165933.

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G protein-coupled receptors (GPCRs) are implicated in nearly every physiological process in the human body and therefore represent an important drug targeting class. Advances in X-ray crystallography and cryo-electron microscopy (cryo-EM) have provided multiple static structures of GPCRs in complex with various signaling partners. However, GPCR functionality is largely determined by their flexibility and ability to transition between distinct structural conformations. Due to this dynamic nature, a static snapshot does not fully explain the complexity of GPCR signal transduction. Molecular dynamics (MD) simulations offer the opportunity to simulate the structural motions of biological processes at atomic resolution. Thus, this technique can incorporate the missing information on protein flexibility into experimentally solved structures. Here, we review the contribution of MD simulations to complement static structural data and to improve our understanding of GPCR physiology and pharmacology, as well as the challenges that still need to be overcome to reach the full potential of this technique.

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Kozielewicz, Paweł, Konrad Owczarek, and Joanna J. Sajkowska-Kozielewicz. "Class F GPCR – activation mechanism and pharmacology." Farmacja Polska 75, no. 8 (August 31, 2019): 451–56. http://dx.doi.org/10.32383/farmpol/116110.

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Erlandson, Sarah C., Conor McMahon, and Andrew C. Kruse. "Structural Basis for G Protein–Coupled Receptor Signaling." Annual Review of Biophysics 47, no. 1 (May 20, 2018): 1–18. http://dx.doi.org/10.1146/annurev-biophys-070317-032931.

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G protein–coupled receptors (GPCRs), which mediate processes as diverse as olfaction and maintenance of metabolic homeostasis, have become the single most effective class of therapeutic drug targets. As a result, understanding the molecular basis for their activity is of paramount importance. Recent technological advances have made GPCR structural biology increasingly tractable, offering views of these receptors in unprecedented atomic detail. Structural and biophysical data have shown that GPCRs function as complex allosteric machines, communicating ligand-binding events through conformational change. Changes in receptor conformation lead to activation of effector proteins, such as G proteins and arrestins, which are themselves conformational switches. Here, we review how structural biology has illuminated the agonist-induced cascade of conformational changes that culminate in a cellular response to GPCR activation.

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Velazhahan, Vaithish, Ning Ma, Nagarajan Vaidehi, and Christopher G. Tate. "Activation mechanism of the class D fungal GPCR dimer Ste2." Nature 603, no. 7902 (March 16, 2022): 743–48. http://dx.doi.org/10.1038/s41586-022-04498-3.

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AbstractThe fungal class D1 G-protein-coupled receptor (GPCR) Ste2 has a different arrangement of transmembrane helices compared with mammalian GPCRs and a distinct mode of coupling to the heterotrimeric G protein Gpa1–Ste2–Ste181. In addition, Ste2 lacks conserved sequence motifs such as DRY, PIF and NPXXY, which are associated with the activation of class A GPCRs2. This suggested that the activation mechanism of Ste2 may also differ. Here we determined structures of Saccharomyces cerevisiae Ste2 in the absence of G protein in two different conformations bound to the native agonist α-factor, bound to an antagonist and without ligand. These structures revealed that Ste2 is indeed activated differently from other GPCRs. In the inactive state, the cytoplasmic end of transmembrane helix H7 is unstructured and packs between helices H1–H6, blocking the G protein coupling site. Agonist binding results in the outward movement of the extracellular ends of H6 and H7 by 6 Å. On the intracellular surface, the G protein coupling site is formed by a 20 Å outward movement of the unstructured region in H7 that unblocks the site, and a 12 Å inward movement of H6. This is a distinct mechanism in GPCRs, in which the movement of H6 and H7 upon agonist binding facilitates G protein coupling.

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Plante, Ambrose, Derek M. Shore, Giulia Morra, George Khelashvili, and Harel Weinstein. "A Machine Learning Approach for the Discovery of Ligand-Specific Functional Mechanisms of GPCRs." Molecules 24, no. 11 (June 2, 2019): 2097. http://dx.doi.org/10.3390/molecules24112097.

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G protein-coupled receptors (GPCRs) play a key role in many cellular signaling mechanisms, and must select among multiple coupling possibilities in a ligand-specific manner in order to carry out a myriad of functions in diverse cellular contexts. Much has been learned about the molecular mechanisms of ligand-GPCR complexes from Molecular Dynamics (MD) simulations. However, to explore ligand-specific differences in the response of a GPCR to diverse ligands, as is required to understand ligand bias and functional selectivity, necessitates creating very large amounts of data from the needed large-scale simulations. This becomes a Big Data problem for the high dimensionality analysis of the accumulated trajectories. Here we describe a new machine learning (ML) approach to the problem that is based on transforming the analysis of GPCR function-related, ligand-specific differences encoded in the MD simulation trajectories into a representation recognizable by state-of-the-art deep learning object recognition technology. We illustrate this method by applying it to recognize the pharmacological classification of ligands bound to the 5-HT2A and D2 subtypes of class-A GPCRs from the serotonin and dopamine families. The ML-based approach is shown to perform the classification task with high accuracy, and we identify the molecular determinants of the classifications in the context of GPCR structure and function. This study builds a framework for the efficient computational analysis of MD Big Data collected for the purpose of understanding ligand-specific GPCR activity.

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Toneatti, Rudy, Jong M. Shin, Urjita H. Shah, Carl R. Mayer, Justin M. Saunders, Miguel Fribourg, Paul T. Arsenovic, et al. "Interclass GPCR heteromerization affects localization and trafficking." Science Signaling 13, no. 654 (October 20, 2020): eaaw3122. http://dx.doi.org/10.1126/scisignal.aaw3122.

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Membrane trafficking processes regulate G protein–coupled receptor (GPCR) activity. Although class A GPCRs are capable of activating G proteins in a monomeric form, they can also potentially assemble into functional GPCR heteromers. Here, we showed that the class A serotonin 5-HT2A receptors (5-HT2ARs) affected the localization and trafficking of class C metabotropic glutamate receptor 2 (mGluR2) through a mechanism that required their assembly as heteromers in mammalian cells. In the absence of agonists, 5-HT2AR was primarily localized within intracellular compartments, and coexpression of 5-HT2AR with mGluR2 increased the intracellular distribution of the otherwise plasma membrane–localized mGluR2. Agonists for either 5-HT2AR or mGluR2 differentially affected trafficking through Rab5-positive endosomes in cells expressing each component of the 5-HT2AR–mGluR2 heterocomplex alone, or together. In addition, overnight pharmacological 5-HT2AR blockade with clozapine, but not with M100907, decreased mGluR2 density through a mechanism that involved heteromerization between 5-HT2AR and mGluR2. Using TAT-tagged peptides and chimeric constructs that are unable to form the interclass 5-HT2AR–mGluR2 complex, we demonstrated that heteromerization was necessary for the 5-HT2AR–dependent effects on mGluR2 subcellular distribution. The expression of 5-HT2AR also augmented intracellular localization of mGluR2 in mouse frontal cortex pyramidal neurons. Together, our data suggest that GPCR heteromerization may itself represent a mechanism of receptor trafficking and sorting.

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Wang, Jialu, Clarice Gareri, and Howard A. Rockman. "G-Protein–Coupled Receptors in Heart Disease." Circulation Research 123, no. 6 (August 31, 2018): 716–35. http://dx.doi.org/10.1161/circresaha.118.311403.

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GPCRs (G-protein [guanine nucleotide-binding protein]–coupled receptors) play a central physiological role in the regulation of cardiac function in both health and disease and thus represent one of the largest class of surface receptors targeted by drugs. Several antagonists of GPCRs, such as βARs (β-adrenergic receptors) and Ang II (angiotensin II) receptors, are now considered standard of therapy for a wide range of cardiovascular disease, such as hypertension, coronary artery disease, and heart failure. Although the mechanism of action for GPCRs was thought to be largely worked out in the 80s and 90s, recent discoveries have brought to the fore new and previously unappreciated mechanisms for GPCR activation and subsequent downstream signaling. In this review, we focus on GPCRs most relevant to the cardiovascular system and discuss traditional components of GPCR signaling and highlight evolving concepts in the field, such as ligand bias, β-arrestin–mediated signaling, and conformational heterogeneity.

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Patel, Jyoti, Keith M. Channon, and Eileen McNeill. "The Downstream Regulation of Chemokine Receptor Signalling: Implications for Atherosclerosis." Mediators of Inflammation 2013 (2013): 1–12. http://dx.doi.org/10.1155/2013/459520.

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Heterotrimeric G-protein-coupled receptors (GPCRs) are key mediators of intracellular signalling, control numerous physiological processes, and are one of the largest class of proteins to be pharmacologically targeted. Chemokine-induced macrophage recruitment into the vascular wall is an early pathological event in the progression of atherosclerosis. Leukocyte activation and chemotaxis during cell recruitment are mediated by chemokine ligation of multiple GPCRs. Regulation of GPCR signalling is critical in limiting vascular inflammation and involves interaction with downstream proteins such as GPCR kinases (GRKs), arrestin proteins and regulator of G-protein signalling (RGS) proteins. These have emerged as new mediators of atherogenesis by functioning in internalisation, desensitisation, and signal termination of chemokine receptors. Targeting chemokine signalling through these proteins may provide new strategies to alter atherosclerotic plaque formation and plaque biology.

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Mattedi, Giulio, Silvia Acosta-Gutiérrez, Timothy Clark, and Francesco Luigi Gervasio. "A combined activation mechanism for the glucagon receptor." Proceedings of the National Academy of Sciences 117, no. 27 (June 22, 2020): 15414–22. http://dx.doi.org/10.1073/pnas.1921851117.

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We report on a combined activation mechanism for a class B G-protein–coupled receptor (GPCR), the glucagon receptor. By computing the conformational free-energy landscape associated with the activation of the receptor–agonist complex and comparing it with that obtained with the ternary complex (receptor–agonist–G protein) we show that the agonist stabilizes the receptor in a preactivated complex, which is then fully activated upon binding of the G protein. The proposed mechanism contrasts with the generally assumed GPCR activation mechanism, which proceeds through an opening of the intracellular region allosterically elicited by the binding of the agonist. The mechanism found here is consistent with electron cryo-microscopy structural data and might be general for class B GPCRs. It also helps us to understand the mode of action of the numerous allosteric antagonists of this important drug target.

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Toh, H. "Molecular Phylogenetic Analysis of Class A GPCR." Seibutsu Butsuri 41, supplement (2001): S20. http://dx.doi.org/10.2142/biophys.41.s20_1.

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Harini, K., S. Jayashree, Vikas Tiwari, Sneha Vishwanath, and Ramanathan Sowdhamini. "Ligand Docking Methods to Recognize Allosteric Inhibitors for G-Protein-Coupled Receptors." Bioinformatics and Biology Insights 15 (January 2021): 117793222110377. http://dx.doi.org/10.1177/11779322211037769.

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G-protein-coupled receptors (GPCRs) are membrane proteins which play an important role in many cellular processes and are excellent drug targets. Despite the existence of several US Food and Drug Administration (FDA)-approved GPCR-targeting drugs, there is a continuing challenge of side effects owing to the nonspecific nature of drug binding. We have investigated the diversity of the ligand binding site for this class of proteins against their cognate ligands using computational docking, even if their structures are known already in the ligand-complexed form. The cognate ligand of some of these receptors dock at allosteric binding site with better score than the binding at the conservative site. Interestingly, amino acid residues at such allosteric binding site are not conserved across GPCR subfamilies. Such a computational approach can assist in the prediction of specific allosteric binders for GPCRs.

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Zaidman, Nathan A., Viktor N. Tomilin, Naghmeh Hassanzadeh Khayyat, Mahendra Damarla, Josephine Tidmore, Diane E. Capen, Dennis Brown, Oleh M. Pochynyuk, and Jennifer L. Pluznick. "Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion." Proceedings of the National Academy of Sciences 117, no. 42 (October 1, 2020): 26470–81. http://dx.doi.org/10.1073/pnas.2007620117.

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The diversity and near universal expression of G protein-coupled receptors (GPCR) reflects their involvement in most physiological processes. The GPCR superfamily is the largest in the human genome, and GPCRs are common pharmaceutical targets. Therefore, uncovering the function of understudied GPCRs provides a wealth of untapped therapeutic potential. We previously identified an adhesion-class GPCR, Gpr116, as one of the most abundant GPCRs in the kidney. Here, we show that Gpr116 is highly expressed in specialized acid-secreting A-intercalated cells (A-ICs) in the kidney using both imaging and functional studies, and we demonstrate in situ receptor activation using a synthetic agonist peptide unique to Gpr116. Kidney-specific knockout (KO) of Gpr116 caused a significant reduction in urine pH (i.e., acidification) accompanied by an increase in blood pH and a decrease in pCO2compared to WT littermates. Additionally, immunogold electron microscopy shows a greater accumulation of V-ATPase proton pumps at the apical surface of A-ICs in KO mice compared to controls. Furthermore, pretreatment of split-open collecting ducts with the synthetic agonist peptide significantly inhibits proton flux in ICs. These data suggest a tonic inhibitory role for Gpr116 in the regulation of V-ATPase trafficking and urinary acidification. Thus, the absence of Gpr116 results in a primary excretion of acid in KO mouse urine, leading to mild metabolic alkalosis (“renal tubular alkalosis”). In conclusion, we have uncovered a significant role for Gpr116 in kidney physiology, which may further inform studies in other organ systems that express this GPCR, such as the lung, testes, and small intestine.

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Isu, Ugochi H., Shadi A. Badiee, Ehsaneh Khodadadi, and Mahmoud Moradi. "Cholesterol in Class C GPCRs: Role, Relevance, and Localization." Membranes 13, no. 3 (March 3, 2023): 301. http://dx.doi.org/10.3390/membranes13030301.

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G-protein coupled receptors (GPCRs), one of the largest superfamilies of cell-surface receptors, are heptahelical integral membrane proteins that play critical roles in virtually every organ system. G-protein-coupled receptors operate in membranes rich in cholesterol, with an imbalance in cholesterol level within the vicinity of GPCR transmembrane domains affecting the structure and/or function of many GPCRs, a phenomenon that has been linked to several diseases. These effects of cholesterol could result in indirect changes by altering the mechanical properties of the lipid environment or direct changes by binding to specific sites on the protein. There are a number of studies and reviews on how cholesterol modulates class A GPCRs; however, this area of study is yet to be explored for class C GPCRs, which are characterized by a large extracellular region and often form constitutive dimers. This review highlights specific sites of interaction, functions, and structural dynamics involved in the cholesterol recognition of the class C GPCRs. We summarize recent data from some typical family members to explain the effects of membrane cholesterol on the structural features and functions of class C GPCRs and speculate on their corresponding therapeutic potential.

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Cong, Zhaotong, Yi-Lynn Liang, Qingtong Zhou, Sanaz Darbalaei, Fenghui Zhao, Wenbo Feng, Lihua Zhao, H. Eric Xu, Dehua Yang, and Ming-Wei Wang. "Structural perspective of class B1 GPCR signaling." Trends in Pharmacological Sciences 43, no. 4 (April 2022): 321–34. http://dx.doi.org/10.1016/j.tips.2022.01.002.

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Tse, Man Tsuey. "Two landmark class B GPCR structures unveiled." Nature Reviews Drug Discovery 12, no. 8 (August 2013): 579. http://dx.doi.org/10.1038/nrd4082.

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Katritch, Vsevolod, Gustavo Fenalti, Enrique E. Abola, Bryan L. Roth, Vadim Cherezov, and Raymond C. Stevens. "Allosteric sodium in class A GPCR signaling." Trends in Biochemical Sciences 39, no. 5 (May 2014): 233–44. http://dx.doi.org/10.1016/j.tibs.2014.03.002.

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Xu, Yueming, Yuxia Wang, Yang Wang, Kaiwen Liu, Yao Peng, Deqiang Yao, Houchao Tao, Haiguang Liu, and Gaojie Song. "Mutagenesis facilitated crystallization of GLP-1R." IUCrJ 6, no. 6 (October 17, 2019): 996–1006. http://dx.doi.org/10.1107/s2052252519013496.

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The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins.

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Guillien, Myriam, Assia Mouhand, Aurélie Fournet, Amandine Gontier, Aleix Martí Navia, Tiago N. Cordeiro, Frédéric Allemand, et al. "Structural Insights into the Intrinsically Disordered GPCR C-Terminal Region, Major Actor in Arrestin-GPCR Interaction." Biomolecules 12, no. 5 (April 21, 2022): 617. http://dx.doi.org/10.3390/biom12050617.

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Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the β2-adernergic receptor (β2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.

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Civciristov, Srgjan, Andrew M. Ellisdon, Ryan Suderman, Cindy K. Pon, Bronwyn A. Evans, Oded Kleifeld, Steven J. Charlton, William S. Hlavacek, Meritxell Canals, and Michelle L. Halls. "Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations." Science Signaling 11, no. 551 (October 9, 2018): eaan1188. http://dx.doi.org/10.1126/scisignal.aan1188.

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G protein–coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β2-adrenergic receptor (β2AR) and muscarinic acetylcholine M3 receptor (M3R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal–regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations.

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König, Caroline, Renè Alqézar, Alfredo Vellido, and Jesús Giraldo. "Reducing the n-gram feature space of class C GPCRs to subtype-discriminating patterns." Journal of Integrative Bioinformatics 11, no. 3 (December 1, 2014): 99–115. http://dx.doi.org/10.1515/jib-2014-254.

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Summary G protein-coupled receptors (GPCRs) are a large and heterogeneous superfamily of receptors that are key cell players for their role as extracellular signal transmitters. Class C GPCRs, in particular, are of great interest in pharmacology. The lack of knowledge about their full 3-D structure prompts the use of their primary amino acid sequences for the construction of robust classifiers, capable of discriminating their different subtypes. In this paper, we investigate the use of feature selection techniques to build Support Vector Machine (SVM)-based classification models from selected receptor subsequences described as n-grams. We show that this approach to classification is useful for finding class C GPCR subtype-specific motifs.

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Shen, Cangsong, Chunyou Mao, Chanjuan Xu, Nan Jin, Huibing Zhang, Dan-Dan Shen, Qingya Shen, et al. "Structural basis of GABAB receptor–Gi protein coupling." Nature 594, no. 7864 (April 28, 2021): 594–98. http://dx.doi.org/10.1038/s41586-021-03507-1.

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AbstractG-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

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Grundmann, Manuel, Eckhard Bender, Jens Schamberger, and Frank Eitner. "Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators." International Journal of Molecular Sciences 22, no. 4 (February 10, 2021): 1763. http://dx.doi.org/10.3390/ijms22041763.

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The physiological function of free fatty acids (FFAs) has long been regarded as indirect in terms of their activities as educts and products in metabolic pathways. The observation that FFAs can also act as signaling molecules at FFA receptors (FFARs), a family of G protein-coupled receptors (GPCRs), has changed the understanding of the interplay of metabolites and host responses. Free fatty acids of different chain lengths and saturation statuses activate FFARs as endogenous agonists via binding at the orthosteric receptor site. After FFAR deorphanization, researchers from the pharmaceutical industry as well as academia have identified several ligands targeting allosteric sites of FFARs with the aim of developing drugs to treat various diseases such as metabolic, (auto)inflammatory, infectious, endocrinological, cardiovascular, and renal disorders. GPCRs are the largest group of transmembrane proteins and constitute the most successful drug targets in medical history. To leverage the rich biology of this target class, the drug industry seeks alternative approaches to address GPCR signaling. Allosteric GPCR ligands are recognized as attractive modalities because of their auspicious pharmacological profiles compared to orthosteric ligands. While the majority of marketed GPCR drugs interact exclusively with the orthosteric binding site, allosteric mechanisms in GPCR biology stay medically underexploited, with only several allosteric ligands currently approved. This review summarizes the current knowledge on the biology of FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120), and GPR84, including structural aspects of FFAR1, and discusses the molecular pharmacology of FFAR allosteric ligands as well as the opportunities and challenges in research from the perspective of drug discovery.

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Zwier, Jurriaan M., Thomas Roux, Martin Cottet, Thierry Durroux, Stephanie Douzon, Sara Bdioui, Nathalie Gregor, et al. "A Fluorescent Ligand-Binding Alternative Using Tag-lite® Technology." Journal of Biomolecular Screening 15, no. 10 (October 25, 2010): 1248–59. http://dx.doi.org/10.1177/1087057110384611.

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G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite®. Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (δ, µ, and κ), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications.

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Fasciani, Irene, Marco Carli, Francesco Petragnano, Francesco Colaianni, Gabriella Aloisi, Roberto Maggio, Marco Scarselli, and Mario Rossi. "GPCRs in Intracellular Compartments: New Targets for Drug Discovery." Biomolecules 12, no. 10 (September 22, 2022): 1343. http://dx.doi.org/10.3390/biom12101343.

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The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches.

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Birdsall, Nigel J. M. "Class A GPCR heterodimers: evidence from binding studies." Trends in Pharmacological Sciences 31, no. 11 (November 2010): 499–508. http://dx.doi.org/10.1016/j.tips.2010.08.003.

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Rowe, Jacob B., Geoffrey J. Taghon, Nicholas J. Kapolka, William M. Morgan, and Daniel G. Isom. "CRISPR-addressable yeast strains with applications in human G protein–coupled receptor profiling and synthetic biology." Journal of Biological Chemistry 295, no. 24 (May 1, 2020): 8262–71. http://dx.doi.org/10.1074/jbc.ra120.013066.

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Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein–coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.

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Dankwah, Kwabena Owusu, Jonathon E. Mohl, Khodeza Begum, and Ming-Ying Leung. "What Makes GPCRs from Different Families Bind to the Same Ligand?" Biomolecules 12, no. 7 (June 21, 2022): 863. http://dx.doi.org/10.3390/biom12070863.

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G protein-coupled receptors (GPCRs) are the largest class of cell-surface receptor proteins with important functions in signal transduction and often serve as therapeutic drug targets. With the rapidly growing public data on three dimensional (3D) structures of GPCRs and GPCR-ligand interactions, computational prediction of GPCR ligand binding becomes a convincing option to high throughput screening and other experimental approaches during the beginning phases of ligand discovery. In this work, we set out to computationally uncover and understand the binding of a single ligand to GPCRs from several different families. Three-dimensional structural comparisons of the GPCRs that bind to the same ligand revealed local 3D structural similarities and often these regions overlap with locations of binding pockets. These pockets were found to be similar (based on backbone geometry and side-chain orientation using APoc), and they correlate positively with electrostatic properties of the pockets. Moreover, the more similar the pockets, the more likely a ligand binding to the pockets will interact with similar residues, have similar conformations, and produce similar binding affinities across the pockets. These findings can be exploited to improve protein function inference, drug repurposing and drug toxicity prediction, and accelerate the development of new drugs.

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Fisher, Gregory W., Margaret H. Fuhrman, Sally A. Adler, Christopher Szent-Gyorgyi, Alan S. Waggoner, and Jonathan W. Jarvik. "Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization." Journal of Biomolecular Screening 19, no. 8 (May 12, 2014): 1220–26. http://dx.doi.org/10.1177/1087057114534299.

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G protein–coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology—receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.

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Journal articles on the topic 'Class A GPCR'

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