Classification of G proteins and prediction of GPCRs-G proteins coupling specificity using continuous wavelet transform and information theory

The coupling between G protein-coupled receptors (GPCRs) and guanine nucleotide-binding proteins (G proteins) regulates various signal transductions from extracellular space into the cell. However, the coupling mechanism between GPCRs and G proteins is still unknown, and experimental determination of their coupling specificity and function is both expensive and time consuming. Therefore, it is significant to develop a theoretical method to predict the coupling specificity between GPCRs and G proteins as well as their function using their primary sequences. In this study, a novel four-layer predictor (GPCRsG_CWTIT) based on support vector machine (SVM), continuous wavelet transform (CWT) and information theory (IT) is developed to classify G proteins and predict the coupling specificity between GPCRs and G proteins. SVM is used for construction of models. CWT and IT are used to characterize the primary structure of protein. Performance of GPCRsG_CWTIT is evaluated with cross-validation test on various working dataset. The overall accuracy of the G proteins at the levels of class and family is 98.23 and 85.42%, respectively. The accuracy of the coupling specificity prediction varies from 74.60 to 94.30%. These results indicate that the proposed predictor is an effective and feasible tool to predict the coupling specificity between GPCRs and G proteins as well as their functions using only the protein full sequence. The establishment of such an accurate prediction method will facilitate drug discovery by improving the ability to identify and predict protein-protein interactions. GPCRsG_CWTIT and dataset can be acquired freely on request from the authors.     

Prediction of GPCR-G protein coupling specificity using features of sequences and biological functions

Understanding the coupling specificity between G protein-coupled receptors （GPCRs） and specific classes of G proteins is important for further elucidation of receptor functions within a cell. Increasing information on GPCR sequences and the G protein family would facilitate prediction of the coupling properties of GPCRs. In this study, we describe a novel approach for predicting the coupling specificity between GPCRs and G proteins. This method uses not only GPCR sequences but also the functional knowledge generated by natural language processing, and can achieve 92.2% prediction accuracy by using the C4.5 algorithm. Furthermore, rules related to GPCR-G protein coupling are generated. The combination of sequence analysis and text mining improves the prediction accuracy for GPCR-G protein coupling specificity, and also provides clues for understanding GPCR signaling.     

D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.     

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.     

Purification and identification of G protein-coupled receptor protein complexes under native conditions

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.     

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.     

A constitutively active GPCR retains its G protein specificity and the ability to form dimers

G protein-coupled receptors (GPCRs) are cell surface proteins which help to regulate the physiology of all the major organ systems within higher eukaryotes. They are stimulated by multiple ligands and activate a range of effector molecules to bring about changes in cell behaviour. The use of constitutively active mutants (CAMs) of GPCRs has enabled a better understanding of receptor activation as CAMs exhibit ligand-independent signalling negating the use of ligands. Here we introduce the fission yeast Schizosaccharomyces pombe as a host for producing CAMs, by describing the isolation and characterization of constitutive mutants of the P-factor receptor (Mam2). One mutant Mam2[P261L] contained a single-amino-acid substitution (Pro261 to Leu) within a region of high homology in GPCRs. Substitution of this proline leads to an 18-fold increase in ligand-independent signalling. We utilized Mam2[P261L] to investigate CAM activity by demonstrating that Mam2[P261L] is efficiently trafficked to the cell surface where it can form fully functional oligomeric complexes with the native receptor. Mam2[P261L] also retains the G protein specificity (RG-profile) of the native receptor and only induces constitutive signalling in the same G proteins. Finally, evidence is provided to indicate that CAM activity results from a reduction in the kinetics of G protein binding. This is the first time that S. pombe has been utilized for isolating and characterizing CAMs and the techniques employed will complement the current systems available for studying these important receptors.     

A concept for G protein activation by G protein-coupled receptor dimers: the transducin/rhodopsin interface.

G protein-coupled receptors (GPCRs) are ubiquitous and essential in modulating virtually all physiological processes. These receptors share a similar structural design consisting of the seven-transmembrane alpha-helical segments. The active conformations of the receptors are stabilized by an agonist and couple to structurally highly conserved heterotrimeric G proteins. One of the most important unanswered questions is how GPCRs couple to their cognate G proteins. Phototransduction represents an excellent model system for understanding G protein signaling, owing to the high expression of rhodopsin in rod photoreceptors and the multidisciplinary experimental approaches used to study this GPCR. Here, we describe how a G protein (transducin) docks on to an oligomeric GPCR (rhodopsin), revealing structural details of this critical interface in the signal transduction process. This conceptual model takes into account recent structural information on the receptor and G protein, as well as oligomeric states of GPCRs.     

GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes

The extent and temporal characteristics of G protein-coupled receptor (GPCR) signaling are shaped by the regulator of G protein signaling (RGS) proteins, which promote G protein deactivation. With hundreds of GPCRs and dozens of RGS proteins, compartmentalization plays a key role in establishing signaling specificity. However, the molecular details and mechanisms of this process are poorly understood. In this paper, we report that the R7 group of RGS regulators is controlled by interaction with two previously uncharacterized orphan GPCRs: GPR158 and GPR179. We show that GPR158/179 recruited RGS complexes to the plasma membrane and augmented their ability to regulate GPCR signaling. The loss of GPR179 in a mouse model of night blindness prevented targeting of RGS to the postsynaptic compartment of bipolar neurons in the retina, illuminating the role of GPR179 in night vision. We propose that the interaction of RGS proteins with orphan GPCRs promotes signaling selectivity in G protein pathways.     

The receptor activity modifying protein family of G protein coupled receptor accessory proteins

Receptor diversity for the calcitonin peptide family is created by the interaction of two 7-transmembrane proteins--the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CL-R)--with the receptor activity modifying protein (RAMP) family. The discovery of heterodimeric complexes of these proteins heralded a new era in the study of G protein coupled receptors (GPCRs), whereby receptor phenotype is no longer governed by just the GPCR. In this article, recent advances in the study of RAMPs are discussed--from our current understanding of the molecular basis of RAMP-receptor interaction to a broader role for RAMPs outside the calcitonin receptor family.     

A conformational trigger for activation of a G protein by a G protein-coupled receptor

G protein-coupled receptors (GPCRs) are a family of seven transmembrane helical proteins that initiate a cellular response to an environmental signal. Once activated by an extracellular signal, GPCRs trigger the intracellular signal transduction cascade by activating a heterotrimeric G protein. The interaction between the G protein and the receptor, which triggers the signal transduction, is the focus of intense interest. Three-dimensional structures of the ground state of only one GPCR, rhodopsin, are currently available, but since the G protein cannot bind to this structure, these structures did not lead to an understanding of the activation process. The recent publication of an excited state structure for the same GPCR (and comparison to the ground state structures), in conjunction with other recent biochemical data, provides new insight into G protein activation. We find that the structure data and the biochemical data, for the first time, point to a specific mode of interaction between the G protein and the receptor. Furthermore, we find that transducin (G(t)) must alter its conformation to bind to the activated receptor; the "lock and key" fit heretofore expected is likely not the correct model. We suggest that a conformational distortion, driven by the energy of binding, is induced in G(t) when it binds to the activated receptor. The conformational change in turn enables the exchange of GTP for GDP and the dissociation of the subunits. This is an example of "induced fit" originally proposed by Koshland to describe enzyme-substrate interactions.     

G protein selectivity is regulated by multiple intracellular regions of GPCRs

GTP-binding protein coupled receptors (GPCRs) bind to a vast diversity of extracellular ligands to regulate a wide variety of physiological responses. Upon binding of extracellular ligands, these seven-transmembrane-spanning receptor molecules couple to one or several subtypes of G protein which reside at the intracellular side of the plasma membrane to trigger intracellular signaling events. Amid the large structural diversity at the intracellular regions of GPCRs, there are only 18 different subtypes of G protein belonging to four subfamilies. The question of how GPCRs select and activate a single or multiple G protein subtype(s) has been the topic of intense investigations. This review will attempt to summarize the available data on the structural determinants in GPCRs that regulate the selectivity of G protein activation. The available data suggest that G protein can be activated by structurally diverse cationic alpha-helical structures with no obvious homology in primary sequence. The selectivity of receptor-G protein coupling is maintained by a combination of two functional domains at the intracellular region. One is the 'activation domain' which can activate multiple G protein subtypes, while the other is the 'selectivity domain' which restricts the coupling to the desired signaling pathway(s). A slight change in the conformation at these two functional domains can affect the fidelity of G protein selectivity. This hypothesis can account for the vast structural diversity of GPCRs which link a fascinating variety of extracellular inputs, yet couple to a limited number of intracellular signaling pathways.     

5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19

The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor–Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.     

Regulation of G protein signaling by the 70 kDa heat shock protein

G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha_2A adrenergic receptor (α_2A-R) by the ubiquitous stress-inducible 70 kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α_2A-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α_2A-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α_2A-R-catalyzed [³⁵S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine_1A receptor (5-HT_1A-R). In heat-stressed CHO cells, the α_2A-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α_2A-R compared to the 5-HT_1A-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.     

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.     

Detergent-free solubilisation & purification of a G protein coupled receptor using a polymethacrylate polymer

G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both G_q and G_i1 heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.     

Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.     

Coupling interaction between thromboxane A2 receptor and alpha-13 subunit of guanine nucleotide-binding protein

G protein-coupled receptors (GPCRs) form a large superfamily of membrane proteins that play an essential role in modulating many vital physiological events, such as cell communication, neurotransmission, sensory perception, and chemotaxis. Understanding of the 3D (dimensional) structures of these receptors and their binding interactions with G proteins will help in the design of drugs for the treatment of GPCR-related diseases. By means of the approach of structural bioinformatics, the 3D structures of human alpha-13 subunit of guanine nucleotide-binding protein (G alpha 13) and human thromboxane A2 (TXA2) receptor were developed. The former plays an important role in the control of cell growth that may serve as a prototypical G protein; the latter is a target for nitric oxide-mediated desensitization that may serve as a prototypical GPCR. On the basis of the 3D models, their coupling interactions were investigated via docking studies. It has been found that the two proteins are coupled with each other mainly through the interaction between the minigene of G alpha 13 and the 3rd intracellular loop of the TXA2 receptor, consistent with the existing deduction in the literatures. However, it has also been observed via a close view that some residues of the TXA2 receptor that are sequentially far away but spatially quite close to the loop region are also involved in forming hydrogen bonds with the minigene of G alpha 13. These findings may provide useful information for conducting mutagenesis and reveal the molecular mechanism how the human TXA2 receptor interact with G alpha 13 to activate intracellular signaling. The findings may also provide useful insights for stimulating new therapeutic approaches by manipulating the interaction of the receptor with the relevant G proteins.     

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.