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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. Editor's summary: The specific patterns of phosphorylation in the intracellular tail of a G protein–coupled receptor (GPCR) are thought to act as a barcode that determines whether G proteins are stimulated or β-arrestins are recruited. Gareri et al. investigated the role of phosphorylation barcodes in signaling by the angiotensin II type 1 receptor (AT1R). AT1R elicits both G protein activation and β-arrestin recruitment in response to the endogenous agonist AngII but stimulates only β-arrestin recruitment in response to a synthetic biased agonist. The authors identified patterns of serine and threonine phosphorylation that stabilized specific conformations of β-arrestin and were necessary for β-arrestin function. These findings demonstrate the importance of phosphorylation barcodes in determining the consequences of AT1R activation. —Annalisa M. VanHook [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the role of CB1 palmitoylation in modulating the functional interaction with G proteins both in the absence and presence of agonist binding. Our data show that the nonpalmitoylated CB1 receptor significantly reduced its association with Gαi2. The agonist stimulation induced a partial dissociation of Gαi2 proteins from the wild‐type receptor, while on the C415A mutant the agonist binding was not able to induce a significant dissociation of Gαi2 from the receptor. The lack of palmitoyl chain seems to hamper the ability of the receptor to functionally interact with the Gαi2 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 CB1 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 CB1 receptor may offer a coordinated process of regulation and could open new therapeutic approaches. [ABSTRACT FROM AUTHOR]

The forward (kon) and reverse (koff) 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 kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon 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, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Kds derived from the koff and kon estimates correlated well with EC50 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 D2R agonist candidate drugs. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusions and Implications 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.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.The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs 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 Gαs 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.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. [ABSTRACT FROM AUTHOR]

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.

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.

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.