class-A GPCRs Research Articles

Ligand-Dependent Sodium Ion Dynamics within the A2A Adenosine Receptor: A Molecular Dynamics Study.

Sodium ions have long been known to reduce the binding of agonists in many class-A GPCRs while having little effect on antagonist binding. Here, using long-time scale classical all-atom molecular dynamics simulations, we explore, in atomic detail, the motion of sodium ions within the ligand-binding pocket of the A2A adenosine receptor (A2A-AR) both in the presence and absence of ligands and in the active and inactive state. We identify novel secondary ion binding sites within the pocket and find that the types of ion motions within the pocket are highly dependent on the presence and type of ligand within the pocket. Our results provide a first step toward developing a molecular understanding of the impact of sodium ions on class-A GPCRs.

Structural basis for the interaction of diapause hormone with its receptor in the silkworm, Bombyx mori.

Diapause hormone (DH) is a 24-aa amidated neuropeptide that elicits the embryonic diapause of the silkworm, Bombyx mori ( Bommo), via sensitive and selective interaction with its receptor, Bommo DH receptor ( Bommo-DHR). Previous studies of the structure-activity relationship of Bommo-DH were all based on an in vivo diapause-induction bioassay, which has provided little information on the structure of Bommo-DHR or its iteration with DH. Here, to unveil the interaction of Bommo-DH with its receptor, N-terminally truncated analogs and alanine-scanning mutants of Bommo-DH were chemically synthesized and functionally evaluated by using a Cy5.5-labeled Bommo-DH competitive binding assay and Bommo-DHR-based functional assays, including cAMP assay and Ca2+ mobilization assay. Our study demonstrates that the C-terminal residues of Arg23 and Leu24 of Bommo-DH are essential for the binding and activation of Bommo-DHR, and that Trp19 and Phe20 also contribute to the functional activity of Bommo-DH. In contrast, when Gly21 or Pro22 were replaced with alanine, both mutants exhibited binding and signaling activities that were indistinguishable from the wild-type peptide. Furthermore, our homology modeling and molecular dynamics simulations, together with experimental validations, have identified the residues of Glu89, Phe172, Phe194, and Tyr299 in Bommo-DHR that are critically involved in the interaction with Bommo-DH. These results may deepen our understanding of the interactions of class-A GPCRs with their peptidic ligands, particularly those between pheromone biosynthesis-activating neuropeptide/DH family neuropeptides and their cognate receptors.-Shen, Z., Jiang, X., Yan, L., Chen, Y., Wang, W., Shi, Y., Shi, L., Liu, D., Zhou, N. Structural basis for the interaction of diapause hormone with its receptor in the silkworm, Bombyx mori.

A promiscuous recognition mechanism between GPR17 and SDF-1: Molecular insights

Recent data and publications suggest a promiscuous behaviour for GPR17, a class-A GPCR operated by different classes of ligands, such as uracil nucleotides, cysteinyl-leukotrienes and oxysterols. This observation, together with the ability of several class-A GPCRs to form homo- and hetero-dimers, is likely to unveil new pathophysiological roles and novel emerging pharmacological properties for some of these GPCRs, including GPR17. This receptor shares structural, phylogenetic and functional properties with some chemokine receptors, CXCRs. Both GPR17 and CXCR2 are operated by oxysterols, and both GPR17 and CXCR ligands have been demonstrated to have a role in orchestrating inflammatory responses and oligodendrocyte precursor cell differentiation to myelinating cells in acute and chronic diseases of the central nervous system. Here, by combining in silico modelling data with in vitro validation in (i) a classical reference pharmacological assay for GPCR activity and (ii) a model of maturation of primary oligodendrocyte precursor cells, we demonstrate that GPR17 can be activated by SDF-1, a ligand of chemokine receptors CXCR4 and CXCR7, and investigate the underlying molecular recognition mechanism. We also demonstrate that cangrelor, a GPR17 orthosteric antagonist, can block the SDF-1-mediated activation of GPR17 in a concentration-dependent manner. The ability of GPR17 to respond to different classes of GPCR ligands suggests that this receptor modifies its function depending on the extracellular mileu changes occurring under specific pathophysiological conditions and advocates it as a strategic target for neurodegenerative diseases with an inflammatory/immune component.

A mechanistic role of Helix 8 in GPCRs: Computational modeling of the dopamine D2 receptor interaction with the GIPC1–PDZ-domain

Helix-8 (Hx8) is a structurally conserved amphipathic helical motif in class-A GPCRs, adjacent to the C-terminal sequence that is responsible for PDZ-domain-recognition. The Hx8 segment in the dopamine D2 receptor (D2R) constitutes the C-terminal segment and we investigate its role in the function of D2R by studying the interaction with the PDZ-containing GIPC1 using homology models based on the X-ray structures of very closely related analogs: the D3R for the D2R model, and the PDZ domain of GIPC2 for GIPC1–PDZ. The mechanism of this interaction was investigated with all-atom unbiased molecular dynamics (MD) simulations that reveal the role of the membrane in maintaining the helical fold of Hx8, and with biased MD simulations to elucidate the energy drive for the interaction with the GIPC1–PDZ. We found that it becomes more favorable energetically for Hx8 to adopt the extended conformation observed in all PDZ–ligand complexes when it moves away from the membrane, and that C-terminus palmitoylation of D2R enhanced membrane penetration by the Hx8 backbone. De-palmitoylation enables Hx8 to move out into the aqueous environment for interaction with the PDZ domain. All-atom unbiased MD simulations of the full D2R–GIPC1-PDZ complex in sphingolipid/cholesterol membranes show that the D2R carboxyl C-terminus samples the region of the conserved GFGL motif located on the carboxylate-binding loop of the GIPC1–PDZ, and the entire complex distances itself from the membrane interface. Together, these results outline a likely mechanism of Hx8 involvement in the interaction of the GPCR with PDZ-domains in the course of signaling.

Oxysterols act as promiscuous ligands of class-A GPCRs: In silico molecular modeling and in vitro validation

According to classical pharmacology, each neurotransmitter/hormonal receptor, including GPCRs, is exclusively activated by highly specific ligands. However, recent evidence challenges this dogma. Oxysterols are produced at inflammatory sites and can surprisingly potently activate the Epstein Barr virus induced gene receptor-2 (EBI2), a GPCR involved in adaptive immune responses. Similarly, oxysterols promiscuously operate CXCR2, a chemokine receptor participating to immune reactions and cancer development. Both EBI2 and CXCR2 are phylogenetically related to GPR17, another GPCR implicated in inflammatory/immune neurodegenerative events. Here, we used an integrated approach combining comparative modeling, molecular docking and in vitro experiments to investigate their potential interactions with oxysterols. All three receptors share the binding site to allocate oxysterols with different local arrangements, higher sensitivity to specific oxysterols and different activation thresholds. Such differences may dictate the diverse biological effects induced by oxysterols, depending on production site, concentration, specific spatiotemporal features and receptor expression on targeted cells. Thus, EBI2, CXCR2 and GPR17 are promiscuously operated by oxysterols making this class of ligands a ‘fil rouge’ linking oxidative stress, inflammation and neurodegeneration. Such a transversal role may represent a conserved, “unspecific” (but selective) signaling mode, by which emergency molecules activate multiple receptors involved in inflammatory/immune responses.

Simple Models Characterize the Activation of G Protein-Coupled Receptors

G Protein-Coupled Receptors, the largest family of proteins in the human genome, are integral membrane proteins responsible for transducing signals across the cell membrane. They play vital roles in vision, olfaction, taste and many other processes. They are major drug targets, so understanding their activation mechanism may lead to better drug design.In this investigation, we studied the activation and deactivation of two class-A GPCRs, rhodopsin and β2 Adrenergic Receptor (B2AR), using computational methods. Specifically, we used structure-based potentials (Gō-models) to rapidly simulate the transition from the inactive state to the active state, as well as the reverse process. We monitored the transition using a biologically motivated quantity, as well as a novel tool based on principal component analysis of inter-residue contacts. The latter approach proved to be more informative, and helped us develop novel insights into the difference in activation pathway for structurally similar proteins.

Structure and Dynamics of Helix-8 in GPCR-PDZ-Domain Interactions

The interaction of GPCRs with PDZ-domain-containing proteins is essential for signaling. Helix-8 (H8) is a structurally conserved amphipathic helical motif in class-A GPCRs, adjacent to the C-terminal sequence that is responsible for PDZ-domain-recognition. To understand the role of H8 in molecular level mechanisms of GPCR/PDZ interactions we investigated the interaction of the PDZ-containing GIPC1 protein with the dopamine D2 receptor (D2R) in homology models of the systems based on the X-ray structures of very closely related analogs: the D3R, and the PDZ domain of GIPC2, respectively. The 5 C-terminal residues of D2 receptor's relatively short C-terminal stretch next to H8 was docked into the GIPC1-PDZ model using a simulated-annealing-based PDZ docking scheme. On the resulting GIPC1-D2R complex we carried out several regular MD simulations in sphingolipid/cholesterol membranes to determine the stability of the complex. The results show that the free carboxyl group of C-terminus of the D2R preferred to sample the space around the main-chain atoms of the conserved GFGL motif located on the carboxylate-binding loop of the GIPC1-PDZ. We investigated the relation between distance of H8 from the membrane and its secondary structure by metadynamics MD simulations and found that as the H8 moved away from the membrane, it became energetically easier for the helix to unravel into an extended conformation that is the canonical conformation for interaction with PDZ-domain. Because the C-termini of GPCRs are palmitoylated, we also investigated effect of palmitoylation on the structure and dynamics of H8 in the context of its role in repositioning H8 for the interaction with the PDZ domain. We find that in the palmitoylated C-terminus the H8 backbone penetrates deeper into the membrane, whereas de-palmitoylation renders it more accessible to the cytoplasm where it can interact with the PDZ domain-containing proteins.

Dynamic Monomer-Dimer Equilibrium of a Prototypical GPCR, Beta2 Adrenergic Receptor: A Single Molecule Imaging Study

Recently, by developing special single-molecule techniques and analysis theory, we first time ever succeeded in obtaining the two-dimensional association equilibrium constant in the membrane and fully characterizing the equilibrium by also obtaining both dissociation and association rate constants (Kasai et al., 2011). As an important paradigm for this work, we examined the monomer-dimer equilibrium of a GPCR, formyl-peptide receptor (FPR). Here, we applied this method to a prototypical class-A GPCR, beta2-adrenergic receptor (B-AR), to clarify whether it forms dimers. Although B-AR has been studied extensively, whether it (and other class-A GPCRs in addition to FPR) exists as dimers has been controversial. We found that B-AR does form dimers with rate and equilibrium constants similar to those for FPR (FPR values in parentheses): the monomer-dimer equilibrium constant = 1.6 (3.6) copies/square microns; the dimer dissociation rate constant of 12.6 (11.0) /s [dimer lifetime of 80 (90) ms]; and the monomer association rate constant of 7.9 (3.1)/[copies/square microns]/s. However, under physiological conditions, the behaviors of B-AR and FPR were quite different due to the difference in the expression levels, ∼260 and ∼2.1 copies/square microns [740,000 (6,000) copies/cell] in dog heart cells and neutrophils, respectively. The majority (95%) of B-AR would exist as dimers, whereas about half (42%) of FPR would do so. Interestingly, their dimer lifetimes are similar, ∼80 and ∼90 ms, respectively, but their monomer lifetimes would be vastly different, i.e., 0.49 ms and 154 ms, respectively. These results suggest that the dynamic monomer-dimer equilibrium might play a role in signal transduction and/or its regulation.

In silico identification of new ligands for GPR17: a promising therapeutic target for neurodegenerative diseases

GPR17, a previously orphan receptor responding to both uracil nucleotides and cysteinyl-leukotrienes, has been proposed as a novel promising target for human neurodegenerative diseases. Here, in order to specifically identify novel potent ligands of GPR17, we first modeled in silico the receptor by using a multiple template approach, in which extracellular loops of the receptor, quite complex to treat, were modeled making reference to the most similar parts of all the class-A GPCRs crystallized so far. A high-throughput virtual screening exploration of GPR17 binding site with more than 130,000 lead-like compounds was then applied, followed by the wet functional and pharmacological validation of the top-scoring chemical structures. This approach revealed successful for the proposed aim, and allowed us to identify five agonists or partial agonists with very diverse chemical structure. None of these compounds could have been expected 'a priori' to act on a GPCR, and all of them behaved as much more potent ligands than GPR17 endogenous activators.

Complementary roles of the DRY motif and C-terminus tail of GPCRS for G protein coupling and β-arrestin interaction

β-arrestin mediates the desensitization of GPCRs and acts as an adaptor molecule to recruit the receptor complex to clathrin-rich regions. Class-A GPCRs subsequently dissociate from β-arrestin but class-B GPCRs internalize with β-arrestin in the endocytic vesicles. Here the dopamine D2 and D3 receptors, which have similar structural features but different intracellular trafficking properties, were used in an attempt to better understand the structural requirements for the classification of GPCRs. The C-terminus tail of the vasopressin type-2 receptor was added to the ends of D2R and D3R to increase their affinity to β-arrestin. A point mutation was introduced into the DRY motif to change their basal activation levels. Among a battery of constructs in which the C-terminus tail and/or DRY motif was altered, class-B behavior was observed with the constructs whose affinities for β-arrestin were increased complementarily and their signaling was either maintained or regained. In conclusion, the DRY motif and C-terminal tail of the GPCRs determine complementarily their intracellular trafficking behavior by regulating the affinity to β-arrestin and G protein coupling.