Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor

The human adenosine A_2A receptor (A_2AR) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [θ]₂₂₂/[θ]₂₀₈ obtained by circular dichroism (CD) spectroscopy>1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Förster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A_2AR at position M193, which is located in the fifth TM domain, noticeably alters A_2AR monomer: dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A_2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.     

Assessment of the transmembrane domain structures in GPCR Dock 2013 models

The community-wide blind prediction of G-protein coupled receptor (GPCR) structures and ligand docking has been conducted three times and the quality of the models was primarily assessed by the accuracy of ligand binding modes. The seven transmembrane (TM) helices of the receptors were taken as a whole; thus the model quality within the 7TM domains has not been evaluated. Here we evaluate the 7TM domain structures in the models submitted for the last round of prediction – GPCR Dock 2013. Applying the 7?×?7 RMSD matrix analysis described in our prior work, we show that the models vary widely in prediction accuracy of the 7TM structures, exhibiting diverse structural differences from the targets. For the prediction of the 5-hydroxytryptamine receptors, the top 7TM models are rather close to the targets, which however are not ranked top by ligand-docking. On the other hand, notable deviations of the TMs are found in in the previously identified top docking models that closely resemble other receptors. We further reveal reasons of success and failure in ligand docking for the models. This current assessment not only complements the previous assessment, but also provides important insights into the current status of GPCR modeling and ligand docking.     

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.     

GPCR activation: protonation and membrane potential

GPCR proteins represent the largest family of signaling membrane proteins in eukaryotic cells. Their importance to basic cell biology, human diseases, and pharmaceutical interventions is well established. Many crystal structures of GPCR proteins have been reported in both active and inactive conformations. These data indicate that agonist binding alone is not suffi cient to trigger the conformational change of GPCRs necessary for binding of downstream G-proteins, yet other essential factors remain elusive. Based on analysis of available GPCR crystal structures, we identifi ed a potential conformational switch around the conserved Asp2.50, which consistently shows distinct conformations between inactive and active states. Combining the structural information with the current literature, we propose an energy-coupling mechanism, in which the interaction between a charge change of the GPCR protein and the membrane potential of the living cell plays a key role for GPCR activation.     

A hypothesis for GPCR activation

Growing evidence that rhodopsin (RD) and related G protein-coupled receptors form functional dimers/oligomers, followed by direct proof (using atomic force microscopy) that in the retina disc membrane RD associates into a paracrystalline network of rows of dimers, need models of the RD-transducin (Gt) complex that would envision an optimal RD dimer/oligomer able to satisfy all well-documented interactions with Gt. Of the models proposed so far, only a few refer to RD dimers and only one of them proposes a complex of Gt with an RD oligomer (Filipek S, Krzyko KA, Fotiadis D, Liang Y, Saperstein DA, Engel, A, Palczewski K Photochem Photobiol Sci 3: 628–638, 2004). This paper puts forward a hypothesis on another arrangement of RD monomers into the reported network of rows of dimers. Arguments for the compatibility of this set-up with interactions and activation of RD in the complex with Gt, in particular, with the well-documented movement of transmembrane helix 6 and cytosolic loop 3, which is vital for RD activation, are provided and discussed.This revised version was published online in June 2005 with corrections to the acknowledgements.     

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor,a human GPCR

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)₆ tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.     

Relevance of rhodopsin studies for GPCR activation

Rhodopsin, the dim-light photoreceptor present in the rod cells of the retina, is both a retinal-binding protein and a G protein-coupled receptor (GPCR). Due to this conjunction, it benefits from an arsenal of spectroscopy techniques that can be used for its characterization, while being a model system for the important family of Class A (also referred to as “rhodopsin-like”) GPCRs. For instance, rhodopsin has been a crucial player in the field of GPCR structural biology. Until 2007, it was the only GPCR for which a high-resolution crystal structure was available, so all structure–activity analyses on GPCRs, from structure-based drug discovery to studies of structural changes upon activation, were based on rhodopsin. At present, about a third of currently available GPCR structures are still from rhodopsin. In this review, I show some examples of how these structures can still be used to gain insight into general aspects of GPCR activation. First, the analysis of the third intracellular loop in rhodopsin structures allows us to gain an understanding of the structural and dynamic properties of this region, which is absent (due to protein engineering or poor electron density) in most of the currently available GPCR structures. Second, a detailed analysis of the structure of the transmembrane domains in inactive, intermediate and active rhodopsin structures allows us to detect early conformational changes in the process of ligand-induced GPCR activation. Finally, the analysis of a conserved ligand-activated transmission switch in the transmembrane bundle of GPCRs in the context of the rhodopsin activation cycle, allows us to suggest that the structures of many of the currently available agonist-bound GPCRs may correspond to intermediate active states. While the focus in GPCR structural biology is inevitably moving away from rhodopsin, in other aspects rhodopsin is still at the forefront. For instance, the first studies of the structural basis of disease mutants in GPCRs, or the most detailed analysis of cellular GPCR signal transduction networks using a systems biology approach, have been carried out in rhodopsin. Finally, due again to its unique properties among GPCRs, rhodopsin will likely play an important role in the application of X-ray free electron laser crystallography to time-resolved structural biology in membrane proteins. Rhodopsin, thus, still remains relevant as a model system to study the molecular mechanisms of GPCR activation. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks.     

A single chemosensory GPCR is required for a concentration-dependent behavioral switching in C. elegans

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A structural model for ovine rhodopsin

An experimentally testable structure for ovine rhodopsin has been modelled from a combination of several secondary-structure prediction methods. The proposed structure agrees well with available experimental data. The model envisages seven transmembrane segments that are largely, but not entirely, α-helical. The prediction of roughly adjacent regions of more irregular structure within these segments (which could introduce significant changes in helix pitch or rotation) may provide a good working model for considering the structural mobility of the protein.     

Evolving cryo-EM structural approaches for GPCR drug discovery

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A constitutively active GPCR governs morphogenic transitions in Cryptococcus neoformans

Sex in fungi is driven by peptide pheromones sensed through seven‐transmembrane pheromone receptors. In Cryptococcus neoformans, sexual reproduction occurs through an outcrossing/heterothallic a ‐ sexual cycle or an inbreeding/homothallic – unisexual mating process. Pheromone receptors encoded by the mating‐type locus ( MAT ) mediate reciprocal pheromone sensing during opposite‐sex mating and contribute to but are not essential for unisexual mating. A pheromone receptor‐like gene, CPR2 , was discovered that is not encoded by MAT and whose expression is induced during a ‐ mating. cpr2 mutants are fertile but have a fusion defect and produce abnormal hyphal structures, whereas CPR2 overexpression elicits unisexual reproduction. When heterologously expressed in Saccharomyces cerevisiae , Cpr2 activates pheromone responses in the absence of any ligand. This constitutive activity results from an unconventional residue, Leu²²², in place of a conserved proline in transmembrane domain six; a Cpr2^L222P mutant is no longer constitutively active. Cpr2 engages the same G‐protein activated signalling cascade as the Ste3 a /α pheromone receptors, and thereby competes for pathway activation. This study established a new paradigm in which a naturally occurring constitutively active G protein‐coupled receptor governs morphogenesis in fungi.     

Theme and variations on kinetics of GPCR activation/deactivation

G protein-coupled receptors (GPCRs) initiate intracellular signaling pathways in response to physiologically and medically important extracellular ligands such as peptide and large glycoprotein hormones, neurotransmitters, sensory stimuli (odorant and taste molecules, light), calcium, l-amino acids, and are the target of many clinical drugs. The conversion of these extracellular stimuli into intracellular signals involves sequential and reversible reactions that initially take place at the plasma membrane. These reactions are mediated not only by dynamic interactions between ligands, receptors and heterotrimeric G proteins, but also by conformational changes associated with the activation/deactivation process of each protein. This review discusses the kinetic characteristics and rate-limiting reactions engaged in signal propagation that are involved in systems as diverse as neurotransmitter and hormonal signaling, and that have been recorded in live cells by Förster resonance energy transfer (FRET) approaches.     

A machine learning based method for the prediction of G protein-coupled receptor-binding PDZ domain proteins

G protein-coupled receptors (GPCRs) are part of multi-protein networks called ‘receptosomes’. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.     

基于遗传算法特征选择的HBV再激活分类预测模型

探讨原发性肝癌患者精确放疗后乙型肝炎病毒(hepatitis b virus,HBV)再激活的危险特征和分类预测模型。提出基于遗传算法的特征选择方法,从原发性肝癌数据的初始特征集中选择HBV再激活的最优特征子集。建立贝叶斯和支持向量机的HBV再激活分类预测模型,并预测最优特征子集和初始特征集的分类性能。实验结果表明,基于遗传算法的特征选择提高了HBV再激活分类性能,最优特征子集的分类性能明显优于初始特征子集的分类性能。影响HBV再激活的最优特征子集包括:HBV DNA水平,肿瘤分期TNM,Child-Pugh,外放边界和全肝最大剂量。贝叶斯的分类准确性最高可达82.89%,支持向量机的分类准确性最高可达83.34%。     

FoldGPCR: Structure prediction protocol for the transmembrane domain of G protein‐coupled receptors from class A

Building reliable structural models of G protein‐coupled receptors (GPCRs) is a difficult task because of the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template‐based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1–3 Å Cα RMSD of the native structure remains a major challenge. Here, we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter‐residue contacts between the template and target, and exploits an all‐atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor‐ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human β₂‐adrenergic receptor (β₂AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison with the X‐ray crystal structure of the β₂AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix‐helix packing interactions that distinguish rhodopsin from β₂AR. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Stable interactions between the transmembrane domains of the adenosine A2A receptor

G-protein-coupled receptors (GPCRs) must properly insert and fold in the membrane to adopt a stable native structure and become biologically active. The interactions between transmembrane (TM) helices are believed to play a major role in these processes. Previous studies in our group showed that specific interactions between TM helices occur, leading to an increase in helical content, especially in weakly helical TM domains, suggesting that helix–helix interactions in addition to helix–lipid interactions facilitate helix formation. They also demonstrated that TM peptides interact in a similar fashion in micelles and lipid vesicles, as they exhibit relatively similar thermal stability and α-helicity inserted in SDS micelles to that observed in liposomes. In this study, we perform an analysis of pairwise interactions between peptides corresponding to the seven TM domains of the human A_2A receptor (A_2AR). We used a combination of Förster resonance energy transfer (FRET) measurement and circular dichroism (CD) spectroscopy to detect and analyze these interactions in detergent micelles. We found that strong and specific interactions occur in only seven of the 28 possible peptide pairs. Furthermore, not all interactions, identified by FRET, lead to a change in helicity. Our results identify stabilizing contacts that are likely related to the stability of the receptor and that are consistent with what is known about the three-dimensional structure and stability of rhodopsin and the β₂ adrenergic receptor.     

果蝇来源的GPCR Methuselah G蛋白偶联信号转导通路研究

Methuselah(MTH)是果蝇来源的GPCR中的一员,它的突变可延长果蝇平均寿命并提高果蝇对外界胁迫因素的耐受性。但目前对MTH在细胞水平的信号转导研究鲜有报道。该研究用稳定表达MTH的HEK293细胞株,对与该受体偶联的G蛋白选择性做了研究。首先,用免疫荧光染色、Western blot及钙流实验验证了MTH在HEK293/Myc-MTH细胞表面能稳定表达,且具有正常生物学活性;MTH受体被其配体N-stunted活化后所引起细胞内钙的上升不能被PTX预处理抑制,提示活化的MTH可能通过与Gq/11而非Gi/o蛋白相偶联;进一步研究发现,MTH激活后不显著改变细胞中的cAMP水平,表明MTH不与Gs和Gi/o相偶联;MTH被激活后可引起ERK磷酸化。这些结果提示:MTH可能是Gq/11蛋白的偶联受体,为进一步研究MTH的下游信号转导和生物学功能奠定了基础。