Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells

Protein-protein interactions are fundamental processes for manybiological systems including those involving the superfamily ofG-protein coupled receptors (GPCRs). When addressing keyquestions concerning the regulation of GPCR-protein complexes andtheir functional significance, the development and refinement ofnon-invasive techniques to study these interactions will be ofgreat value. One such technique, bioluminescence resonanceenergy transfer (BRET), is a recently described biophysicalmethod that represents a powerful tool with which to measureprotein-protein interactions in live cells, in real time. Thisminireview highlights the impact that evolving techniques such asBRET have had on the study of dynamic protein interactionsinvolving GPCRs. In particular, the application of BRET to thestudy of protein interactions involving the receptors forhypothalamic peptide hormones, thyrotropin-releasing hormone(TRH) and gonadotropin-releasing hormone (GnRH), will bediscussed. Using these receptors, BRET has successfully beenused to demonstrate formation of both agonist-dependent andindependent GPCR-GPCR complexes (oligomerization) and theagonist-dependent interaction of GPCRs with their intracellularadaptor protein partners, the arrestins. In summary, BRET is ahighly sensitive method that will not only aid in advancing ourunderstanding of GPCR signalling and trafficking but could alsopotentially lead to the development of novel therapeutics thattarget these GPCR-protein complexes.     

Lamotrigine inhibits TRESK regulated by G-protein coupled receptor agonists

Dorsal root ganglion (DRG) neurons express mRNAs for numerous two-pore domain K⁺ (K_2P) channels and G-protein coupled receptors (GPCR). Recent studies have shown that TRESK is a major background K⁺ channel in DRG neurons. Here, we demonstrate the pharmacological properties of TRESK, including GPCR agonist-induced effects on DRG neurons. TRESK mRNA was highly expressed in DRG compared to brain and spinal cord. Similar to cloned TRESK, native TRESK was inhibited by acid and arachidonic acid (AA), but not zinc. Native TRESK was also activated by GPCR agonists such as acetylcholine, glutamate, and histamine. The glutamate-activated TRESK was blocked by lamotrigine in DRG neurons. In COS-7 cells transfected with mouse TRESK, 30 μM lamotrigine inhibited TRESK by ∼50%. Since TRESK is target of modulation by acid, AA, GPCR agonists, and lamotrigine, it is likely to play an active role in the regulation of excitability in DRG neurons.     

孤儿G蛋白偶联受体研究进展

孤儿G蛋白偶联受体的研究意味着发现其尚未了解的内源性配体,是后基因组时代功能基因组学研究的热点之一,对生命科学的发展具有深 影响。本文介绍孤儿G蛋白偶联受体的概念、研究策略及其应用。     

G蛋白偶联受体119——治疗糖尿病的新靶点

G蛋白偶联受体119(GPR119)与激动剂结合后,通过cAMP信号转导途径,促进葡萄糖依赖性胰岛素和肠肽激素的分泌,是新一代的治疗2型糖尿病药物靶点。本文对GPR119的组织学分布、生理学作用、内源性配体以及小分子激动剂作一简要的介绍。     

Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.     

Modelling the activation of G-protein coupled receptors by a single drug

In this paper, the most popular proposed mechanism for activation of G-protein coupled receptors (GPCRs) - the shuttling mechanism - is modelled mathematically. An asymptotic analysis of this model clarifies the dynamics of the system in the presence of a drug, in particular identifying which reactions dominate during the different timescales. The modelling also reveals challenging behaviour in the form of a peak response. This new mechanism gives simple explanations for complex, possibly misunderstood, behaviour.     

Fast fourier transform-based support vector machine for prediction of G-protein coupled receptor subfamilies

Although the sequence information on G-protein coupled receptors （GPCRs） continues to grow, many GPCRs remain orphaned （i.e. ligand specificity unknown） or poorly characterized with little structural information available, so an automated and reliable method is badly needed to facilitate the identification of novel receptors. In this study, a method of fast Fourier transform-based support vector machine has been developed for predicting GPCR subfamilies according to protein＇s hydrophobicity. In classifying Class B, C, D and F subfamilies, the method achieved an overall Matthew＇s correlation coefficient and accuracy of 0.95 and 93.3%, respectively, when evaluated using the jackknife test. The method achieved an accuracy of 100% on the Class B independent dataset. The results show that this method can classify GPCR subfamilies as well as their functional classification with high accuracy. A web server implementing the prediction is available at http：//chem.scu.edu.cn/blast/Pred-GPCR.     

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin_1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin_1A receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin_1A receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin_1A receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.     

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies,in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A_2A receptor (A_2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A_2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A_2AR controls. The A_2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.     

Using BRET to study chemical compound-induced disruptions of the p53-HDM2 interactions in live cells

Modification of protein-protein interactions (PPIs) holds promise for novel rational drug design. Disrupting or modifying protein interactions offers new challenges in terms of chemical compound libraries and techniques for compound validation. As proteins interact with several partners in different allosteric conformation in a pathological and tissue-specific fashion, it is difficult to predict the in vivo effect of PPI acting compounds identified by in vitro screening assays. It is therefore desirable to develop techniques that rapidly allow cell-based validation of protein interacting compounds. The binding of the p53 tumor suppressor to the HDM2 E3 ubiquitin ligase is important for controlling p53 activity, and several compounds, such as Nutlin-3, have been designed to bind a hydrophobic pocket in the N-terminus of HDM2 to prevent the interaction with p53 to stabilize and activate downstream p53 pathways. We have used the p53-HDM2 interaction as a model system to explore the bioluminescence resonance energy transfer (BRET) technique for validating compounds that disrupt PPIs in living cells.     

Structural features of the G-protein/GPCR interactions

Background

The details of the functional interaction between G proteins and the G protein coupled receptors (GPCRs) have long been subjected to extensive investigations with structural and functional assays and a large number of computational studies.

Scope of review

The nature and sites of interaction in the G-protein/GPCR complexes, and the specificities of these interactions selecting coupling partners among the large number of families of GPCRs and G protein forms, are still poorly defined.

Major conclusions

Many of the contact sites between the two proteins in specific complexes have been identified, but the three dimensional molecular architecture of a receptor-Gα interface is only known for one pair. Consequently, many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered.

General significance

In the context of current structural data we review the structural details of the interfaces and recognition sites in complexes of sub-family A GPCRs with cognate G-proteins, with special emphasis on the consequences of activation on GPCR structure, the prevalence of preassembled GPCR/G-protein complexes, the key structural determinants for selective coupling and the possible involvement of GPCR oligomerization in this process.     

The Ig VH complementarity-determining region 3-containing Rb9 peptide,inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the V_HCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca²⁺ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.     

NemChR-DB: a database of parasitic nematode chemosensory G-protein coupled receptors

Nematode Chemosensory G-Protein Coupled Receptors have expanded within nematodes, where they play important roles in foraging and host-seeking behaviour. Nematode Chemosensory G-Protein Coupled Receptors are most highly expressed during free-living stages when chemosensory signalling is required for host detection and nematode activation in various parasitic nematodes, and therefore position Nematode Chemosensory G-Protein Coupled Receptors at the transition from infective to parasitic stages, making them important regulators to study in terms of host-seeking and host specificity. To facilitate the analysis of Nematode Chemosensory G-Protein Coupled Receptors, here we describe an integrative database of nematode chemoreceptors called NemChR-DB. This database enables users to study diverse parasitic nematode chemoreceptors, functionally explore sequence entries through structural and literature-based annotations, and perform cross-species comparisons.     

The sixth transmembrane region of a pheromone G-protein coupled receptor,Map3, is implicated in discrimination of closely related pheromones in Schizosaccharomyces pombe

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycete fungi including yeasts, mating between cells of opposite mating type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focused on the stringency of pheromone receptors. The fission yeast Schizosaccharomyces pombe has two mating types, Plus (P) and Minus (M). Here, we investigated the stringency of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the activation by the pheromone. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are key residues important for discrimination of closely related M-factors. Thus, the differences in these two GPCRs might reflect the significantly distinct stringency/flexibility of their respective pheromone/receptor systems; nevertheless, species-specific pheromone recognition remains incomplete.     

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.     

Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET_A/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.     

Dynamics of apelin receptor/G protein coupling in living cells

During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of Gαi₂ or Gαi₃ through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi₁ subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demonstrated that stimulation of APJ with apelin-13 causes activation of Gαi₂, Gαi₃, Gαo, Gαq; among which Gαi₂, Gαi₃ were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.     

Regulatory role of C-terminus in the G-protein coupling of the metabotropic glutamate receptor 1

The signaling property of metabotropic glutamate receptor 1alpha (mGlu1alpha) is different from that of short-form splice variants. This could be caused by the exposure of a cluster of positively charged amino acid residues, RRKK, in the proximal C-tail which is thought to be masked by the long C-tail of mGlu1alpha. We found that the RRKK residues, when exposed, attenuate Gq coupling and decrease the basal activity and the surface expression of mGlu1, in agreement with previous results. Moreover, these residues abolish the Gi/o coupling of mGlu1, but do not affect glutamate-induced dimeric rearrangement and protein kinase A-dependent modulation of mGlu1. These results suggest that the RRKK residues do not inhibit the conformational change upon glutamate binding and protein accessibility to the intracellular loops where G-protein coupling occurs, but rather act as an inhibitory domain against G-protein coupling in a different manner depending on the type of G protein.     

The RGS proteins add to the diversity of soybean heterotrimeric G-protein signaling

Regulator of G-protein signaling (RGS) proteins are a family of highly diverse, multifunctional proteins that function primarily as GTPase accelerating proteins (GAPs). RGS proteins increase the rate of GTP hydrolysis by Gα proteins and essentially regulate the duration of active signaling. Recently, we have identified two chimeric RGS proteins from soybean and reported their distinct GAP activities on individual Gα proteins. A single amino acid substitution (Alanine 357 to Valine) of RGS2 is responsible for differential GAP activity. Surprisingly, most monocot plant genomes do not encode for a RGS protein homolog. Here we discuss the soybean RGS proteins in the context of their evolution in plants, their relatedness to non-plant RGS protein homologs and the effect they might have on the heterotrimeric G-protein signaling mechanisms. We also provide experimental evidence to show that the interaction interface between plant RGS and Gα proteins is different from what is predicted based on mammalian models.