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The Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced via an unusual mechanism: upon agonist-induced phosphorylation, the noncanonical G protein-coupled receptor SMOOTHENED (SMO) binds the catalytic subunit of protein kinase A (PKA-C) and physically blocks its enzymatic activity. By combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary structural homology. An intrinsically disordered region of SMO binds the PKA-C active site, resembling the PKA regulatory subunit (PKA-R) / PKA-C holoenzyme, while the SMO transmembrane domain binds a conserved PKA-C interaction hub, similar to other GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key structural elements within the SMO cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly., Competing Interests: Competing interest statement Authors declare that they have no competing interests.

Different ligands stabilize specific conformations of the angiotensin II type 1 receptor (AT1R) that direct distinct signaling cascades mediated by heterotrimeric G proteins or β-arrestin. These different active conformations are thought to engage distinct intracellular transducers because of differential phosphorylation patterns in the receptor C-terminal tail (the "barcode" hypothesis). Here, we identified the AT1R barcodes for the endogenous agonist AngII, which stimulates both G protein activation and β-arrestin recruitment, and for a synthetic biased agonist that only stimulates β-arrestin recruitment. The endogenous and β-arrestin-biased agonists induced two different ensembles of phosphorylation sites along the C-terminal tail. The phosphorylation of eight serine and threonine residues in the proximal and middle portions of the tail was required for full β-arrestin functionality, whereas phosphorylation of the serine and threonine residues in the distal portion of the tail had little influence on β-arrestin function. Similarly, molecular dynamics simulations showed that the proximal and middle clusters of phosphorylated residues were critical for stable β-arrestin-receptor interactions. These findings demonstrate that ligands that stabilize different receptor conformations induce different phosphorylation clusters in the C-terminal tail as barcodes to evoke distinct receptor-transducer engagement, receptor trafficking, and signaling.

G protein-coupled receptors (GPCRs) are one of the major drug targets. In recent years, computational drug design for GPCRs has mainly focused on static structures obtained through X-ray crystallography, cryogenic electron microscopy (cryo-EM) or in silico modelling as a starting point for virtual screening campaigns. However, GPCRs are highly flexible entities with the ability to adopt different conformational states that elicit different physiological responses. Including this knowledge in the drug discovery pipeline can help to tailor novel conformation-specific drugs with an improved therapeutic profile. In this review, we outline our current knowledge about GPCR dynamics that is relevant for receptor activation, signalling bias and allosteric modulation. Ultimately, we highlight new technological implementations such as time-resolved X-ray crystallography and cryo-EM as well as computational algorithms that can contribute to a more comprehensive understanding of receptor dynamics and its relevance for GPCR functionality., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus., Experimental Approach: The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel., Key Results: The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for G αs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their G αs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail., Conclusions and Implications: Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

G protein-coupled receptors (GPCRs) are sophisticated signaling machines able to simultaneously elicit multiple intracellular signaling pathways upon activation. Complete (in)activation of all pathways can be counterproductive for specific therapeutic applications. This is the case for the serotonin 2 A receptor (5-HT 2A R), a prominent target for the treatment of schizophrenia. In this study, we elucidate the complex 5-HT 2A R coupling signature in response to different signaling probes, and its physiological consequences by combining computational modeling, in vitro and in vivo experiments with human postmortem brain studies. We show how chemical modification of the endogenous agonist serotonin dramatically impacts the G protein coupling profile of the 5-HT 2A R and the associated behavioral responses. Importantly, among these responses, we demonstrate that memory deficits are regulated by G αq protein activation, whereas psychosis-related behavior is modulated through G αi1 stimulation. These findings emphasize the complexity of GPCR pharmacology and physiology and open the path to designing improved therapeutics for the treatment of stchizophrenia., (© 2024. The Author(s).)

G protein-coupled receptor (GPCR)-biased agonism, selective activation of certain signaling pathways relative to others, is thought to be directed by differential GPCR phosphorylation "barcodes." At chemokine receptors, endogenous chemokines can act as "biased agonists", which may contribute to the limited success when pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomics studies. Mutation of CXCR3 phosphosites altered β-arrestin 2 conformation in cellular assays and was consistent with conformational changes observed in molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes, leading to distinct physiological processes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

G protein-coupled receptor (GPCR) biased agonism, the activation of some signaling pathways over others, is thought to largely be due to differential receptor phosphorylation, or "phosphorylation barcodes." At chemokine receptors, ligands act as "biased agonists" with complex signaling profiles, which contributes to the limited success in pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of CXCR3 phosphosites altered β-arrestin conformation in cellular assays and was confirmed by molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes and lead to distinct physiological processes., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V 2 R), agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V 2 R (i.e., V 2 R T360A ) results in near-complete loss of βarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated βarr1, efficiently rescues the endosomal trafficking of βarr1 and ERK1/2 activation for V 2 R T360A . Molecular dynamics simulations reveal that Ib30 enriches active-like βarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances βarr1-β 2 -adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery., (© 2022. The Author(s).)

Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a potential antiviral drug.

The function of several G protein-coupled receptors (GPCRs) exhibits cholesterol sensitivity. Cholesterol sensitivity of GPCRs could be attributed to specific sequence and structural features, such as the cholesterol recognition/interaction amino acid consensus (CRAC) motif, that facilitate their cholesterol-receptor interaction. In this work, we explored the molecular basis of cholesterol sensitivity exhibited by the serotonin 1A receptor, the most studied GPCR in the context of cholesterol sensitivity, by generating mutants of key residues in CRAC motifs in transmembrane helix 2 (TM2) and TM5 of the receptor. Our results show that a lysine residue (K101) in one of the CRAC motifs is crucial for sensing altered membrane cholesterol levels. Insights from all-atom molecular dynamics simulations showed that cholesterol-sensitive functional states of the serotonin 1A receptor are associated with reduced conformational dynamics of extracellular loops of the receptor. These results constitute one of the first reports on the molecular mechanism underlying cholesterol sensitivity of GPCRs., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Brain functions rely on neurotransmitters that mediate communication between billions of neurons. Disruption of this communication can result in a plethora of psychiatric and neurological disorders. In this work, we combine molecular dynamics simulations, live-cell biosensor and electrophysiological assays to investigate the action of the neurotransmitter dopamine at the dopaminergic D 2 receptor (D 2 R). The study of dopamine and closely related chemical probes reveals how neurotransmitter binding translates into the activation of distinct subsets of D 2 R effectors ( i.e. : G i2 , G oB , G z and β-arrestin 2). Ligand interactions with key residues in TM5 (S5.42) and TM6 (H6.55) in the D 2 R binding pocket yield a dopamine-like coupling signature, whereas exclusive TM5 interaction is typically linked to preferential G protein coupling (in particular G oB ) over β-arrestin. Further experiments for serotonin receptors indicate that the reported molecular mechanism is shared by other monoaminergic neurotransmitter receptors. Ultimately, our study highlights how sequence variation in position 6.55 is used by nature to fine-tune β-arrestin recruitment and in turn receptor signaling and internalization of neurotransmitter receptors., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

The forward (k on ) and reverse (k off ) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the k on s and k off s of four dopamine D 2 receptor (D 2 R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S193 5.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar k off s for the two 5-OH-DPAT enantiomers at wild-type (WT) D 2 R, both being slower than the k off s of DA and p-tyramine. Conversely, the k on of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S193 5.42 A mutation lowered the k on of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic K d s derived from the k off and k on estimates correlated well with EC 50 values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing D 2 R agonist candidate drugs.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V 2 R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V 2 R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

β-arrestins (βarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of βarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of βarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B 2 R), analogous to that present in the vasopressin receptor (V 2 R), reverses the contribution of βarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of βarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys 294 in the lariat loop of βarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of βarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics., (© 2020 The Authors.)

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.

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.

The δ-opioid receptor (δ-OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C-terminal region of δ-OR using its N-terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull-down experiments we delimit this interaction to 12 helix 8 residues of δ-ΟR and to the first 17 N-terminal residues (NT) of RGS4. Monitoring the complex arrangement and stabilization between RGS4 and δ-OR by molecular dynamics simulations combined with mutagenesis studies, we defined that two critical interactions are formed: one between Phe329 of helix8 of δ-ΟR and Pro9 of the NT of RGS4 and the other a salt bridge between Glu323 of δ-ΟR and Lys17 of RGS4. Our observations allow drafting for the first time a structural model of a ternary complex including the δ-opioid receptor, a G protein and a RGS protein. Furthermore, the high degree of conservation among opioid receptors of the RGS4-binding region, points to a conserved interaction mode between opioid receptors and this important regulatory protein., (© 2019 Federation of European Biochemical Societies.)

The oxoeicosanoid receptor 1 (OXER1) is a member of the G-protein coupled receptors (GPCR) family, and is involved in inflammatory processes and oncogenesis. As such it is an attractive target for pharmacological intervention. The present study aimed to shed light on the molecular fundaments of OXER1 modulation using chemical probes structurally related to the natural agonist 5-oxo-ETE. In a first step, 5-oxo-ETE and its closely related derivatives (5-oxo-EPE and 4-oxo-DHA) were obtained by conducting concise and high-yielding syntheses. The biological activity of obtained compounds was assessed in terms of potency (EC 50 ) and efficacy (E max ) for arrestin recruitment. Finally, molecular modelling and simulation were used to explore binding characteristics of 5-oxo-ETE and derivatives with the aim to rationalize biological activity. Our data suggest that the tested 5-oxo-ETE derivatives (i) insert quickly into the membrane, (ii) access the receptor via transmembrane helices (TMs) 5 and 6 from the membrane side and (iii) drive potency and efficacy by differential interaction with TM5 and 7. Most importantly, we found that the methyl ester of 5-oxo-ETE (1a) showed even a higher maximum response than the natural agonist (1). In contrast, shifting the 5-oxo group into position 4 results in inactive compounds (4-oxo DHA compounds (3) and (3a)). All in all, our study provides relevant structural data that help understanding better OXER1 functionality and its modulation. The structural information presented herein will be useful for designing new lead compounds with desired signalling profiles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The observation of biased agonism in G protein-coupled receptors (GPCRs) has provided new approaches for the development of more efficacious and safer drugs. However, in order to rationally design biased drugs, one must understand the molecular basis of this phenomenon. Computational approaches can help in exploring the conformational universe of GPCRs and detecting conformational states with relevance for distinct functional outcomes. This information is extremely valuable for the development of new therapeutic agents that promote desired conformational receptor states and responses while avoiding the ones leading to undesired side-effects.This book chapter intends to introduce the reader to powerful computational approaches for sampling the conformational space of these receptors, focusing first on molecular dynamics and the analysis of the produced data through methods such as dimensionality reduction, Markov State Models and adaptive sampling. Then, we show how to seek for compounds that target distinct conformational states via docking and virtual screening. In addition, we describe how to detect receptor-ligand interactions that drive signaling bias and comment current challenges and opportunities of presented methods.

In this study, we investigated the role of CB 1 palmitoylation in modulating the functional interaction with G proteins both in the absence and presence of agonist binding. Our data show that the nonpalmitoylated CB 1 receptor significantly reduced its association with Gα i2 . The agonist stimulation induced a partial dissociation of Gα i2 proteins from the wild-type receptor, while on the C415A mutant the agonist binding was not able to induce a significant dissociation of Gα i2 from the receptor. The lack of palmitoyl chain seems to hamper the ability of the receptor to functionally interact with the Gα i2 and indicate that the palmitoyl chain is responsible for the functional transmission of the agonist-induced conformational change in the receptor of the G protein. These data were further corroborated by molecular dynamics simulations. Overall these results suggest that palmitoylation of the CB 1 receptor finely tunes its interaction with G proteins and serves as a targeting signal for its functional regulation. Of note, the possibility to reversibly modulate the palmitoylation of CB 1 receptor may offer a coordinated process of regulation and could open new therapeutic approaches., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)

We previously demonstrated that CB 1 receptor is palmitoylated at cysteine 415, and that such a post-translational modification affects its biological activity. To assess the molecular mechanisms responsible for modulation of CB 1 receptor function by S-palmitoylation, in this study biochemical and morphological approaches were paralleled with computational analyses. Molecular dynamics simulations suggested that this acyl chain stabilizes helix 8 as well as the interaction of CB 1 receptor with membrane cholesterol. In keeping with these in silico data, experimental results showed that the non-palmitoylated CB 1 receptor was unable to interact efficaciously with caveolin 1, independently of its activation state. Moreover, in contrast with the wild-type receptor, the lack of S-palmitoylation in the helix 8 made the mutant CB 1 receptor completely irresponsive to agonist-induced effects in terms of both lipid raft partitioning and receptor internalization. Overall, our results support the notion that palmitoylation of cysteine 415 modulates the conformational state of helix 8 and influences the interactions of CB 1 receptor with cholesterol and caveolin 1, suggesting that the palmitoyl chain may serve as a functional interface for CB 1 receptor localization and function., (Copyright © 2017 Elsevier B.V. All rights reserved.)