A method for the prediction of GPCRs coupling specificity to G-proteins using refined profile Hidden Markov Models

Background  

G- Protein coupled receptors (GPCRs) comprise the largest group of eukaryotic cell surface receptors with great pharmacological interest. A broad range of native ligands interact and activate GPCRs, leading to signal transduction within cells. Most of these responses are mediated through the interaction of GPCRs with heterotrimeric GTP-binding proteins (G-proteins). Due to the information explosion in biological sequence databases, the development of software algorithms that could predict properties of GPCRs is important. Experimental data reported in the literature suggest that heterotrimeric G-proteins interact with parts of the activated receptor at the transmembrane helix-intracellular loop interface. Utilizing this information and membrane topology information, we have developed an intensive exploratory approach to generate a refined library of statistical models (Hidden Markov Models) that predict the coupling preference of GPCRs to heterotrimeric G-proteins. The method predicts the coupling preferences of GPCRs to G_s, G_i/o and G_q/11, but not G_12/13 subfamilies.     

Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells

Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.     

Plant signaling in stress: G-protein coupled receptors,heterotrimeric G-proteins and signal coupling via phospholipases

Plant growth and development are coordinalely controlled by several internal factors and environmental signals. To sense these environmental signals, the higher plants have evolved a complex signaling network, which may also cross talk with each other. Plants can respond to the signals as individual cells and as whole organisms. Various receptors including phytochromes, G-proteins coupled receptors (GPCR), kinase and hormone receptors play important role in signal transduction but very few have been characterized in plant system. The heterotrimeric G-proteins mediate the coupling of signal transduction from activated GPCR to appropriate downstream effectors and thereby play an important role in signaling. In this review we have focused on some of the recent work on G-proteins and two of the effectors, PLC and PLD, which have been shown to interact with Gα subunit and also discussed their role in abiotic stress tolerance.Key words: abiotic stress, G-protein couple receptor, heterotrimeric G-protein, phospholipases, plant receptors, signal transduction     

Molecular mechanisms of G-proteins coupling with membrane receptors and second messenger systems

G-proteins transmit the signals from hormone receptors onto intracellular effector systems which take part in production of the second messengers such as cAMP, IP3, DAG and Ca2+. Molecular mechanisms of G-protein participation in the coupling of the seven-domain receptors to adenylate cyclase, phospholipase C and channels for Ca2+ and K+ ions are discussed in this paper. G-protein is a heterotrimers built of alpha-, beta- and gamma-subunits, which dissociate onto alpha- and beta gamma-subunits during interaction with hormone-receptor complex. alpha-subunit as well as beta gamma-dimmer may interact with effector system that leads to acceleration or slowing down of second messengers formation. Molecular mechanisms of such regulatory signal diversification are described. Seven-domain receptors possess very high recognition specificity of G-proteins. It is defined by combination of both alpha- and beta gamma-subunits in the G-protein structure. There is well-defined interaction specificity of G-protein alpha-subunit with effector systems. Combinations of different beta- and gamma-subunits involved in complex formation define interaction specificity of G-protein beta gamma-complex with effector systems. The highest interaction specificity of receptors with G-proteins and G-proteins with effector systems is found during triple complex formations: receptor--G-protein--effector. Such specificity is stronger in living cells than in membrane preparations. It can be an evidence of intracellular factors influence on the processes of interaction of the proteins involved in transmembrane regulatory signal transduction.     

Predicting G-protein coupled receptors-G-protein coupling specificity based on autocross-covariance transform

Determining G-protein coupled receptors (GPCRs) coupling specificity is very important for further understanding the functions of receptors. A successful method in this area will benefit both basic research and drug discovery practice. Previously published methods rely on the transmembrane topology prediction at training step, even at prediction step. However, the transmembrane topology predicted by even the best algorithm is not of high accuracy. In this study, we developed a new method, autocross-covariance (ACC) transform based support vector machine (SVM), to predict coupling specificity between GPCRs and G-proteins. The primary amino acid sequences are translated into vectors based on the principal physicochemical properties of the amino acids and the data are transformed into a uniform matrix by applying ACC transform. SVMs for nonpromiscuous coupled GPCRs and promiscuous coupled GPCRs were trained and validated by jackknife test and the results thus obtained are very promising. All classifiers were also evaluated by the test datasets with good performance. Besides the high prediction accuracy, the most important feature of this method is that it does not require any transmembrane topology prediction at either training or prediction step but only the primary sequences of proteins. The results indicate that this relatively simple method is applicable. Academic users can freely download the prediction program at http://www.scucic.net/group/database/Service.asp.     

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.     

Structural biology of G protein‐coupled receptor signaling complexes

G protein‐coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one‐third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high‐resolution structures of GPCRs in complex with their signaling transducers, including G‐protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling‐biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high‐resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.     

Proton transfer-mediated GPCR activation

G-protein coupled receptors (GPCRs) play essential roles in signal transduction from the environment into the cell. While many structural features have been elucidated in great detail, a common functional mechanism on how the ligand-binding signal is converted into a conformational change on the cytoplasmic face resulting in subsequent activation of downstream effectors remain to be established. Based on available structural and functional data of the activation process in class-A GPCRs, we propose here that a change in protonation status, together with proton transfer via conserved structural elements located in the transmembrane region, are the key elements essential for signal transduction across the membrane.     

Conformational changes of G protein-coupled receptors during their activation by agonist binding

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Many of the over 1000 human GPCRs represent important pharmaceutical targets. However, despite high interest in this receptor family, no high-resolution structure of a human GPCR has been resolved yet. This is mainly due to difficulties in obtaining large quantities of pure and active protein. Until now, only a high-resolution x-ray structure of an inactive state of bovine rhodopsin is available. Since no structure of an active state has been solved, information of the GPCR activation process can be gained only by biophysical techniques. In this review, we first describe what is known about the ground state of GPCRs to then address questions about the nature of the conformational changes taking place during receptor activation and the mechanism controlling the transition from the resting to the active state. Finally, we will also address the question to what extent information about the three-dimensional GPCR structure can be included into pharmaceutical drug design programs.     

Studying heterotrimeric G-protein-linked signal transduction using replication-deficient adenoviruses

Plasma membrane-spanning G-protein-linked receptors transduce approximately 60% of all extracellular stimuli in higher animals. Many G-protein-linked receptor pathways are yet to be elucidated, with the receptor, G-protein or effector system as yet unidentified. In addition, many fundamental issues pertaining to G-protein signalling remain unresolved, such as the factors governing the specificity of G-protein receptor coupling and the control of signal amplitude in response to G-protein activation. In order to address some of these issues, the use of replication-deficient adenoviruses as gene transfer vectors for investigations of G-protein signalling has been developed, facilitating dissection of G-protein-linked signal transduction pathways in an extensive range of cultured cells, as well as in vivo. The present review focuses on the versatility and utility of adenoviruses for the investigation of signalling by heterotrimeric G-proteins and explores some of the recent advances in adenoviral technology as they relate to the study of signal transduction.     

Amplification of agonist stimulation of human G-protein-coupled receptor signaling in yeast

G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs.     

Heterodimerization of apelin receptor and neurotensin receptor 1 induces phosphorylation of ERK1/2 and cell proliferation via Gαq‐mediated mechanism

Dimerization of G protein‐coupled receptors (GPCRs) is crucial for receptor function including agonist affinity, efficacy, trafficking and specificity of signal transduction, including G protein coupling. Emerging data suggest that the cardiovascular system is the main target of apelin, which exerts an overall neuroprotective role, and is a positive regulator of angiotensin‐converting enzyme 2 (ACE2) in heart failure. Moreover, ACE2 cleaves off C‐terminal residues of vasoactive peptides including apelin‐13, and neurotensin that activate the apelin receptor (APJ) and neurotensin receptor 1 (NTSR1) respectively, that belong to the A class of GPCRs. Therefore, based on the similar mode of modification by ACE2 at peptide level, the homology at amino acid level and the capability of forming dimers with other GPCRs, we have been suggested that APJ and NTSR1 can form a functional heterodimer. Using co‐immunoprecipitation, BRET and FRET, we provided conclusive evidence of heterodimerization between APJ and NTSR1 in a constitutive and induced form. Upon agonist stimulation, hetrodimerization enhanced ERK_1/2 activation and increased proliferation via activation of Gq α‐subunits. These novel data provide evidence for a physiological role of APJ/NTSR1 heterodimers in terms of ERK_1/2 activation and increased intracellular calcium and induced cell proliferation and provide potential new pharmaceutical targets for cardiovascular disease.     

Cellular dielectric spectroscopy: a powerful new approach to label-free cellular analysis

The past decade has seen a number of significant changes in identifying higher quality lead compounds earlier in the drug discovery process. Cell-based assay technologies yielding high-content information have emerged to achieve this goal. Although most of these systems are based on fluorescence detection, this article describes the development and application of an innovative cellular assay technology based on radio frequency spectrometry and bioimpedance measurements. Using this technique, the authors have discovered a link between cellular bioimpedance changes and receptor-mediated signal transduction events. By performing dielectric spectroscopy of cells across as pectrum of frequencies (1 KHz to 110 MHz), a series of receptor-specific, frequency-dependent impedance patterns is collected. These raw data patterns are used to determine the identity of the cellular receptor-signaling pathway being tested and to quantify stimulation endpoints and kinetics. The authors describe the application of this technology to the analysis of ligand-induced cellular responses mediated by the 3 major classes of G-protein-coupled receptors (GPCRs) and protein tyrosine kinase receptors. This single assay platform can be used with ease to monitor G(s), G(i), and G(q) GPCRs without the need for chimeric or promiscuous G-proteins, fluorophors, or tagged proteins. In contrast to other methods of monitoring cellular signal transduction, this approach provides high information content in a simplified, noninvasive, and biologically relevant fashion.     

Conformational Changes of G Protein‐Coupled Receptors During Their Activation by Agonist Binding

Abstract

The superfamily of G protein‐coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Many of the over 1000 human GPCRs represent important pharmaceutical targets. However, despite high interest in this receptor family, no high‐resolution structure of a human GPCR has been resolved yet. This is mainly due to difficulties in obtaining large quantities of pure and active protein. Until now, only a high‐resolution x‐ray structure of an inactive state of bovine rhodopsin is available. Since no structure of an active state has been solved, information of the GPCR activation process can be gained only by biophysical techniques. In this review, we first describe what is known about the ground state of GPCRs to then address questions about the nature of the conformational changes taking place during receptor activation and the mechanism controlling the transition from the resting to the active state. Finally, we will also address the question to what extent information about the three‐dimensional GPCR structure can be included into pharmaceutical drug design programs.     

Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins

G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.     

Signaling property study of adhesion G-protein-coupled receptors

Adhesion G-protein-coupled receptors (GPCR) are special members of GPCRs with long N-termini containing multiple domains. We overexpressed our collection of receptors together with G-proteins in mammalian cell lines and measured the concentrations of intracellular signaling molecules, such as inositol phosphate and cAMP. Our results show that a subset of tested adhesion GPCRs has constitutive activities and is capable of coupling to a variety of G-proteins. In addition, we have identified a small molecule compound that specifically activates one of the subfamily members, GPR97, and the activation was confirmed by an independent GTPγS assay. These findings suggest classical GPCR screening assays could be applied to de-orphanize these receptors, and provide pharmacological tools to improve understanding of the physiological functions of these receptors.     

Identification of cholesterol recognition amino acid consensus (CRAC) motif in G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Membrane cholesterol has been reported to have an important role in the function of a number of GPCRs. Several structural features of proteins, believed to result in preferential association with cholesterol, have been recognized. Cholesterol recognition/interaction amino acid consensus (CRAC) sequence represents such a motif. Many proteins that interact with cholesterol have been shown to contain the CRAC motif in their sequence. We report here the presence of CRAC motifs in three representative GPCRs, namely, rhodopsin, the β(2)-adrenergic receptor, and the serotonin(1A) receptor. Interestingly, the function of these GPCRs has been previously shown to be dependent on membrane cholesterol. The presence of CRAC motifs in GPCRs indicates that interaction of cholesterol with GPCRs could be specific in nature. Further analysis shows that CRAC motifs are inherent characteristic features of the serotonin(1A) receptor and are conserved over natural evolution. These results constitute the first report of the presence of CRAC motifs in GPCRs and provide novel insight in the molecular nature of GPCR-cholesterol interaction.     

gpDB: a database of GPCRs, G-proteins, effectors and their interactions

gpDB is a publicly accessible, relational database, containing information about G-proteins, G-protein coupled receptors (GPCRs) and effectors, as well as information concerning known interactions between these molecules. The sequences are classified according to a hierarchy of different classes, families and subfamilies based on literature search. The main innovation besides the classification of G-proteins, GPCRs and effectors is the relational model of the database, describing the known coupling specificity of GPCRs to their respective alpha subunits of G-proteins, and also the specific interaction between G-proteins and their effectors, a unique feature not available in any other database. AVAILABILITY: http://bioinformatics.biol.uoa.gr/gpDB CONTACT: shamodr@biol.uoa.gr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.