TM-MOTIF: an alignment viewer to annotate predicted transmembrane helices and conserved motifs in aligned set of sequences

Multiple sequence alignments become biologically meaningful only if conserved and functionally important residues and secondary structural elements preserved can be identified at equivalent positions. This is particularly important for transmembrane proteins like G-protein coupled receptors (GPCRs) with seven transmembrane helices. TM-MOTIF is a software package and an effective alignment viewer to identify and display conserved motifs and amino acid substitutions (AAS) at each position of the aligned set of homologous sequences of GPCRs. The key feature of the package is to display the predicted membrane topology for seven transmembrane helices in seven colours (VIBGYOR colouring scheme) and to map the identified motifs on its respective helices /loop regions. It is an interactive package which provides options to the user to submit query or pre-aligned set of GPCR sequences to align with a reference sequence, like rhodopsin, whose structure has been solved experimentally. It also provides the possibility to identify the nearest homologue from the available inbuilt GPCR or Olfactory Receptor cluster dataset whose association is already known for its receptor type. AVAILABILITY: The database is available for free at mini@ncbs.res.in.     

Vasopressin V2 Receptor/Bioligand Interactions

Summary We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its agonists [Arg⁸]vasopressin (AVP) and [D-Arg⁸]vasopressin (DAVP), and the non-peptide antagonist OPC-31260. V2R controls antidiuresis and belongs to the superfamily of heptahelical transmembrane (7TM) G-protein-coupled receptors (GPCRs). The receptor was built, the ligands were docked and the structures relaxed using advanced molecular modeling techniques. Docked agonists and antagonists appear to prefer similar V2R compartments. A number of receptor amino acid residues are indicated, mainly in the TM3-TM7 helices, as potentially important in ligand binding. Many of these residues are invariant for either the GPCR superfamily or the subfamily of related (vasopressin V2R, V1aR and V1bR and oxytocin OR) receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for ligand affinity [Mouillac et al., J. Biol. Chem., 270 (1995) 25771].     

Vasopressin V2 Receptor/Bioligand Interactions

We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its agonists [Arg⁸]vasopressin (AVP) and [D-Arg⁸]vasopressin (DAVP), and the non-peptide antagonist OPC-31260. V2R controls antidiuresis and belongs to the superfamily of heptahelical transmembrane (7TM) G-protein-coupled receptors (GPCRs). The receptor was built, the ligands were docked and the structures relaxed using advanced molecular modeling techniques. Docked agonists and antagonists appear to prefer similar V2R compartments. A number of receptor amino acid residues are indicated, mainly in the TM3–TM7 helices, as potentially important in ligand binding. Many of these residues are invariant for either the GPCR superfamily or the subfamily of related (vasopressin V2R, V1aR and V1bR and oxytocin OR) receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for ligand affinity [Mouillac et al., J. Biol. Chem., 270 (1995) 25771].     

Dimerization of G-protein-coupled receptors: roles in signal transduction

Recently, many G-protein-coupled receptors (GPCRs) have been demonstrated to form constitutive dimers consisting of identical or distinct monomeric subunits. The discovery of GPCR dimerization has revealed a new level of molecular cross-talk between signalling molecules and may define a general mechanism that modulates the function of GPCRs under both physiological and pathological conditions. The heterodimerization between distinct GPCRs could be responsible for the generation of pharmacologically defined receptors for which no gene has been identified so far. Elucidating the role of dimerization in the activation processes of GPCRs will lead us to develop novel pharmaceutical agents that allosterically promote activation or inhibition of GPCR signalling.     

Statistical sequence analyses of G-protein-coupled receptors: structural and functional characteristics viewed with periodicities of entropy, hydrophobicity, and volume

G-protein-coupled receptors (GPCRs) play a crucial role in signal transduction and receive a wide variety of ligands. GPCRs are a major target in drug design, as nearly 50% of all contemporary medicines act on GPCRs. GPCRs are membrane proteins possessing a common structural feature, seven transmembrane helices. In order to design an effective drug to act on a GPCR, knowledge of the three-dimensional (3D) structure of the target GPCR is indispensable. However, as GPCRs are membrane bound, their 3D structures are difficult to obtain. Thus we conducted statistical sequence analyses to find information about 3D structure and ligand binding using the receptors' primary sequences. We present statistical sequence analyses of 270 human GPCRs with regard to entropy (Shannon entropy in sequence alignment), hydrophobicity and volume, which are associated with the alpha-helical periodicity of the accessibility to the surrounding lipid. We found periodicity such that the phase changes once in the middle of each transmembrane region, both in the entropy plot and in the hydrophobicity plot. The phase shift in the entropy plot reflects the variety of ligands and the generality of the mechanism of signal transduction. The two periodic regions in the hydrophobicity plot indicate the regions facing the hydrophobic lipid chain and the polar phospholipid headgroup. We also found a simple periodicity in the plot of volume deviation, which suggests conservation of the stable structural packing among the transmembrane helices.     

Heterotrimeric G-protein signaling in Arabidopsis: Puzzling G-protein-coupled receptor

Seven transmembrane G-protein-coupled receptors (GPCRs) are commonly used by eukaryotes to sense extracellular signals to switch on cellular responses through the activation of cognate heterotrimeric G-proteins. In Arabidopsis thaliana, GCR2 has been proposed as a GPCR for the plant hormone abscisic acid. On the other hand, biochemical analysis demonstrates that the sole Arabidopsis heterotrimeric G-protein α subunit, GPA1, is in the activated state (GTP-bound) by default, suggesting that the heterotrimeric G-proteins may act without any GPCRs.Key words: heterotrimeric G-proteins, GCR2, GPA1, G-protein-coupled receptor (GPCR), AtRGS1     

Kinetic diversity in G-protein-coupled receptor signalling

The majority of intracellular signalling cascades in higher eukaryotes are initiated by GPCRs (G-protein-coupled receptors). Hundreds of GPCRs signal through a handful of trimeric G-proteins, raising the issue of signal specificity. In the present paper, we illustrate a simple kinetic model of G-protein signalling. This model shows that stable production of significant amounts of free Galpha(GTP) (GTP-bound Galpha subunit) and betagamma is only one of multiple modes of behaviour of the G-protein system upon activation. Other modes, previously uncharacterized, are sustained production of betagamma without significant levels of Galpha(GTP) and transient production of Galpha(GTP) with sustained betagamma. The system can flip between different modes upon changes in conditions. This model demonstrates further that the negative feedback of receptor uncoupling or internalization, when combined with a positive feedback within the G-protein cycle, under a broad range of conditions results not in termination of the response but in relaxed oscillations in GPCR signalling. This variety of G-protein responses may serve to encode signal specificity in GPCR signal transduction.     

3D Modeling for TM Receptors: Algorithms and Validations

Summary This paper outlines the basic strategy to build 3D models of transmembrane G-protein coupled receptors (GPCRs) starting from their amino acid sequences in a ‘block-by-block’ manner: (i) locate possible TM helical fragments in the GPCR sequence; (ii) build 3D structures for these helices; (iii) arrange isolated helices across the membrane; (iv) calculate all pairwise helix-helix interactions; (v) assemble helical bundle(s); (vi) restore interhelical loops and N- and C-termini; and (vii) refine the entire 3D structure(s). Computer algorithms and preliminary results for most of the steps are discussed.     

Exploring a role for heteromerization in GPCR signalling specificity

The critical involvement of GPCRs (G-protein-coupled receptors) in nearly all physiological processes, and the presence of these receptors at the interface between the extracellular and the intracellular milieu, has positioned these receptors as pivotal therapeutic targets. Although a large number of drugs targeting GPCRs are currently available, significant efforts have been directed towards understanding receptor properties, with the goal of identifying and designing improved receptor ligands. Recent advances in GPCR pharmacology have demonstrated that different ligands binding to the same receptor can activate discrete sets of downstream effectors, a phenomenon known as 'ligand-directed signal specificity', which is currently being explored for drug development due to its potential therapeutic advantage. Emerging studies suggest that GPCR responses can also be modulated by contextual factors, such as interactions with other GPCRs. Association between different GPCR types leads to the formation of complexes, or GPCR heteromers, with distinct and unique signalling properties. Some of these heteromers activate discrete sets of signalling effectors upon activation by the same ligand, a phenomenon termed 'heteromer-directed signalling specificity'. This has been shown to be involved in the physiological role of receptors and, in some cases, in disease-specific dysregulation of a receptor effect. Hence targeting GPCR heteromers constitutes an emerging strategy to select receptor-specific responses and is likely to be useful in achieving specific beneficial therapeutic effects.     

Wnt3a stimulation elicits G-protein-coupled receptor properties of mammalian Frizzled proteins

Receptors of the Fz (Frizzled) family initiate Wnt ligand-dependent signalling controlling multiple steps in organism development and carcinogenesis. Fz proteins possess seven transmembrane domains, and their signalling depends on heterotrimeric G-proteins in various organisms; however, Fz proteins constitute a distinct group within the GPCR (G-protein-coupled receptor) superfamily, and Fz signalling can be G-protein-independent in some experimental setups, leading to concerns about the GPCR nature of these proteins. In the present study, we demonstrate that mammalian Fz proteins act as GPCRs on heterotrimeric G(o/i) proteins. Addition of the Wnt3a ligand to rat brain membranes or cultured cells elicits Fz-dependent guanine-nucleotide exchange on G(o/i) proteins. These responses were sensitive to a Wnt antagonist and to pertussis toxin, which decouples the G(o/i) proteins from their receptors through covalent modification. The results of the present study provide the long-awaited biochemical proof of the GPCR nature of Fz receptors.     

G-protein-coupled receptor dynamics: dimerization and activation models compared with experiment

Our previously derived models of the active state of the β2-adrenergic receptor are compared with recently published X-ray crystallographic structures of activated GPCRs (G-protein-coupled receptors). These molecular dynamics-based models using experimental data derived from biophysical experiments on activation were used to restrain the receptor to an active state that gave high enrichment for agonists in virtual screening. The β2-adrenergic receptor active model and X-ray structures are in good agreement over both the transmembrane region and the orthosteric binding site, although in some regions the active model is more similar to the active rhodopsin X-ray structures. The general features of the microswitches were well reproduced, but with minor differences, partly because of the unexpected X-ray results for the rotamer toggle switch. In addition, most of the interacting residues between the receptor and the G-protein were identified. This analysis of the modelling has also given important additional insight into GPCR dimerization: re-analysis of results on photoaffinity analogues of rhodopsin provided additional evidence that TM4 (transmembrane helix 4) resides at the dimer interface and that ligands such as bivalent ligands may pass between the mobile helices. A comparison, and discussion, is also carried out between the use of implicit and explicit solvent for active-state modelling.     

Crystal structure of rhodopsin: implications for vision and beyond

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.     

Structure-based identification of binding sites,native ligands and potential inhibitors for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest family of cell-surface receptors involved in signal transmission. Drugs associated with GPCRs represent more than one fourth of the 100 top-selling drugs and are the targets of more than half of the current therapeutic agents on the market. Our methodology based on the internal coordinate mechanics (ICM) program can accurately identify the ligand-binding pocket in the currently available crystal structures of seven transmembrane (7TM) proteins [bacteriorhodopsin (BR) and bovine rhodopsin (bRho)]. The binding geometry of the ligand can be accurately predicted by ICM flexible docking with and without the loop regions, a useful finding for GPCR docking because the transmembrane regions are easier to model. We also demonstrate that the native ligand can be identified by flexible docking and scoring in 1.5% and 0.2% (for bRho and BR, respectively) of the best scoring compounds from two different types of compound database. The same procedure can be applied to the database of available chemicals to identify specific GPCR binders. Finally, we demonstrate that even if the sidechain positions in the bRho binding pocket are entirely wrong, their correct conformation can be fully restored with high accuracy (0.28 A) through the ICM global optimization with and without the ligand present. These binding site adjustments are critical for flexible docking of new ligands to known structures or for docking to GPCR homology models. The ICM docking method has the potential to be used to "de-orphanize" orphan GPCRs (oGPCRs) and to identify antagonists-agonists for GPCRs if an accurate model (experimentally and computationally validated) of the structure has been constructed or when future crystal structures are determined.     

Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons

Olfactory receptors (ORs) are a large family of proteins involved in the recognition and discrimination of numerous odorants. These receptors belong to the G-protein coupled receptor (GPCR) hyperfamily, for which little structural data are available. In this study we predict the binding site residues of OR proteins by analyzing a set of 1441 OR protein sequences from mouse and human. The central insight utilized is that functional contact residues would be conserved among pairs of orthologous receptors, but considerably less conserved among paralogous pairs. Using judiciously selected subsets of 218 ortholog pairs and 518 paralog pairs, we have identified 22 sequence positions that are both highly conserved among the putative orthologs and variable among paralogs. These residues are disposed on transmembrane helices 2 to 7, and on the second extracellular loop of the receptor. Strikingly, although the prediction makes no assumption about the location of the binding site, these amino acid positions are clustered around a pocket in a structural homology model of ORs, mostly facing the inner lumen. We propose that the identified positions constitute the odorant binding site. This conclusion is supported by the observation that all but one of the predicted binding site residues correspond to ligand-contact positions in other rhodopsin-like GPCRs.     

The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila

Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins). These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans. The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies. How methuselah functions in controlling ageing is not clear. A first essential step towards the understanding of methuselah function is to determine the ligands of Methuselah. Here we report the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B. Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress. We conclude that the Stunted-Methuselah system is involved in the control of animal ageing.     

Roles of G-protein-coupled receptor dimerization

The classical idea that G-protein-coupled receptors (GPCRs) function as monomeric entities has been unsettled by the emerging concept of GPCR dimerization. Recent findings have indicated not only that many GPCRs exist as homodimers and heterodimers, but also that their oligomeric assembly could have important functional roles. Several studies have shown that dimerization occurs early after biosynthesis, suggesting that it has a primary role in receptor maturation. G-protein coupling, downstream signalling and regulatory processes such as internalization have also been shown to be influenced by the dimeric nature of the receptors. In addition to raising fundamental questions about GPCR function, the concept of dimerization could be important in the development and screening of drugs that act through this receptor class. In particular, the changes in ligand-binding and signalling properties that accompany heterodimerization could give rise to an unexpected pharmacological diversity that would need to be considered.     

Comparison of class A and D G protein-coupled receptors: common features in structure and activation

All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast alpha-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.     

Interferon beta-1a counteracts effects of activation on the expression of G-protein-coupled receptor kinases 2 and 3, beta-arrestin-1, and regulators of G-protein signalling 2 and 16 in human mononuclear leukocytes

Activation regulates the responsiveness of G-protein-coupled receptors (GPCRs) on T cells, and modifications in the activity of GPCRs characterize lymphocytes from some immune disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Some lines of evidence suggest that such an effect is connected with the altered expression of some GPCRs regulatory proteins. Herein we demonstrate that phitoemagglutinin (PHA)-induced activation leads to differential expression of G-protein-coupled receptor kinase (GRK) 2, GRK3, beta-arrestin-1, regulators of G-protein signalling (RGS) 2, and RGS16 and decreases responsiveness of mononuclear leukocytes (MNL) to the beta-adrenergic agonist isoproterenol. Interferon beta-1a (IFN beta-1a), which is known to ameliorate the course of MS, counteracts the activation-induced effects on the expression of these GPCR regulatory proteins in MNL. Furthermore, IFN beta-1a quenches the effects of PHA on the isoproterenol-induced accumulation of cyclic AMP (cAMP). We suggest that regulation of GPCRs responsiveness may be a relevant property of IFN beta-1a in MS.     

Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface

Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer’s disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hβ₂AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.

Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. This cryo-electron microscopy study shows how galanin interacts with one of its three human receptor proteins, GALR2, and reveals the basis of the selectivity of this GPCR for Gq.     

Structural insights into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are the largest family of eukaryotic plasma membrane receptors, and are responsible for the majority of cellular responses to external signals. GPCRs share a common architecture comprising seven transmembrane (TM) helices. Binding of an activating ligand enables the receptor to catalyze the exchange of GTP for GDP in a heterotrimeric G protein. GPCRs are in a conformational equilibrium between inactive and activating states. Crystallographic and spectroscopic studies of the visual pigment rhodopsin and two beta-adrenergic receptors have defined some of the conformational changes associated with activation.