The relaxin peptide family and their novel G-protein coupled receptors

Relaxin-1 is a heterodimeric peptide hormone primarily produced by the pregnant corpus luteum and/or placenta and is involved in many essential physiological processes centered on its action as a potent extracellular matrix (ECM) remodeling agent. Insulin-like peptide 3 (INSL3), also known as relaxin-like factor, is predominantly expressed in the Leydig cells of the testes and is an important mediator of testicular descent. The relaxin-1 equivalent peptide in humans is actually the product of the human RLN2 gene, human 2 (H2) relaxin. Recently identified and thought to be the ancestral relaxin, relaxin-3 is specifically expressed in the nucleus incertus of the mouse and rat brain and is most likely an important neuropeptide. Each of the hormones above act on cell membrane G-protein coupled receptors (GPCRs). The relaxin-1 receptor is leucine-rich repeat-containing GPCR 7 (LGR7) whereas INSL3 acts on the closely related LGR8. These receptors have large extra-cellular domains containing multiple leucine-rich repeats (LRRs) and a unique LDL receptor-like cysteine-rich motif (LDLR-domain). Relaxin-3 will bind and activate LGR7 with 50-fold lower activity than H2 relaxin. Two relaxin-3 selective GPCRs; somatostatin and angiotensin like peptide receptor (SALPR) and GPCR 142 were recently identified, these type I GPCRs are unrelated to LGR7 and LGR8. The discovery and characterisation of these receptors is greatly aiding the quest to unravel the mechanics of these important hormones, however with three other family members, insulin-like peptides 4–6 (INSL4, INSL5 and INSL6) with unknown functions and unidentified receptors, there is still much to be learnt about this hormone family.     

Peptide interactions with G-protein coupled receptors.

Peptide recognition by G-protein coupled receptors (GPCRs) is reviewed with an emphasis on the indirect approach used to determine the receptor-bound conformation of peptide ligands. This approach was developed in response to the lack of detailed structural information available for these receptors. Recent advances in the structural determination of rhodopsin (the GPCR of the visual system) by crystallography have provided a scaffold for homology modeling of the inactive state of a wide variety of GPCRs that interact with peptide messages. Additionally, the ability to mutate GPCRs and assay compounds of similar chemical structure to test a common binding site on the receptor provides a firm experimental basis for structure-activity studies. Recognition motifs, common in other well-studied systems such as proteolytic enzymes and major histocompatibility class receptors (MHC) are reviewed briefly to provide a basis of comparison. Finally, the development of true peptidomimetics is contrasted with nonpeptide ligands, discovered through combinatorial chemistry. In many systems, the evidence suggests that the peptide ligands bind at the interface between the transmembrane segments and the extracellular loops, while nonpeptide antagonists bind within the transmembrane segments. Plausible models of GPCRs and the mechanism by which they activate G-proteins on binding peptides are beginning to emerge.     

The activation mechanism of class-C G-protein coupled receptors

Class-C G-protein coupled receptors (GPCRs) represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), and the receptors for Ca(2+), some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, class-C receptors possess a heptahelical domain (HD) involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain (ECD) responsible for agonist recognition and binding. In addition, it is now well accepted that these receptors are dimers, either homo or heterodimers. This complex architecture raises a number of important questions. Here we will discuss our view of how agonist binding within the large ECD triggers the necessary change of conformation, or stabilize a specific conformation, of the heptahelical domain leading to G-protein activation. How ligands acting within the heptahelical domain can change the properties of these complex macromolecules.     

Classifying G-protein coupled receptors with support vector machines

MOTIVATION: The enormous amount of protein sequence data uncovered by genome research has increased the demand for computer software that can automate the recognition of new proteins. We discuss the relative merits of various automated methods for recognizing G-Protein Coupled Receptors (GPCRs), a superfamily of cell membrane proteins. GPCRs are found in a wide range of organisms and are central to a cellular signalling network that regulates many basic physiological processes. They are the focus of a significant amount of current pharmaceutical research because they play a key role in many diseases. However, their tertiary structures remain largely unsolved. The methods described in this paper use only primary sequence information to make their predictions. We compare a simple nearest neighbor approach (BLAST), methods based on multiple alignments generated by a statistical profile Hidden Markov Model (HMM), and methods, including Support Vector Machines (SVMs), that transform protein sequences into fixed-length feature vectors. RESULTS: The last is the most computationally expensive method, but our experiments show that, for those interested in annotation-quality classification, the results are worth the effort. In two-fold cross-validation experiments testing recognition of GPCR subfamilies that bind a specific ligand (such as a histamine molecule), the errors per sequence at the Minimum Error Point (MEP) were 13.7% for multi-class SVMs, 17.1% for our SVMtree method of hierarchical multi-class SVM classification, 25.5% for BLAST, 30% for profile HMMs, and 49% for classification based on nearest neighbor feature vector Kernel Nearest Neighbor (kernNN). The percentage of true positives recognized before the first false positive was 65% for both SVM methods, 13% for BLAST, 5% for profile HMMs and 4% for kernNN.     

The role of G-protein coupled receptors and associated proteins in receptor tyrosine kinase signal transduction

It is well established that stimulation of G-protein coupled receptors (GPCRs) can activate signalling from receptor tyrosine kinases by a process termed transactivation. Indeed, in recent years, it has become apparent that transactivation is a general phenomenon that has been demonstrated for many unrelated GPCRs and receptor tyrosine kinases. In this case the GPCR/G-protein participation is up-stream of the receptor tyrosine kinase. Substantial research has addressed these findings but meanwhile another mechanism of cross talk has been slowly emerging. For over a decade, a growing body of evidence has demonstrated that numerous growth factors use G-proteins and attendant signalling molecules such as beta-arrestins that participate down-stream of the receptor tyrosine kinase to signal to effectors, such as p42/p44 MAPK. This review highlights this novel mechanism of cross talk between receptor tyrosine kinases and GPCRs, which is distinct from growth factor receptor transactivation by GPCRs.     

VIGR--a novel inducible adhesion family G-protein coupled receptor in endothelial cells

Using a signal sequence trap for selection of differentially expressed secretory and membrane proteins, we identified a novel member of the adhesion family of G-protein coupled receptors (GPCRs), termed vascular inducible GPCR (VIGR). VIGR contains C1r-C1s, Uegf and Bmp1 (CUB) and pentraxin (PTX)-like modules and a mucin-like spacer, followed by seven transmembrane domains. By surface biotinylation as well as by immunofluorescence analysis we demonstrate that endogenous, highly glycosylated VIGR is expressed on the cell surface of endothelial cells (ECs) upon LPS or thrombin treatment, and inducible expression is mediated by MAP kinases, but not NF-kappaB. We show that VIGR is selectively expressed in ECs derived from larger vessels, but not from microvessels. In summary, VIGR represents a novel GPCR of the adhesion family, which is unique in its long extra-cellular domain comprising CUB and PTX-like modules and in its inducibility by LPS and thrombin in a subset of ECs, suggesting an important function in cell-adhesion and potentially links inflammation and coagulation.     

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。     

A novel nuclear receptor superfamily member in Xenopus that associates with RXR, and shares extensive sequence similarity to the mammalian vitamin D3 receptor.

We report the isolation of xONR1, a novel member of the nuclear receptor superfamily from Xenopus laevis. xONR1 shares a high degree of amino acid sequence identity with the mammalian receptor for 1 alpha, 25-dihydroxy vitamin D3, particularly within the DNA-binding domain, although it does not bind this ligand. xONR1 DNA binding is stimulated by association with retinoid X receptor gamma (RXR gamma).     

The study of G-protein coupled receptor oligomerization with computational modeling and bioinformatics

To achieve a structural context for the analysis of G-protein coupled receptor (GPCR) oligomers, molecular modeling must be used to predict the corresponding interaction interfaces. The task is complicated by the paucity of detailed structural data at atomic resolution, and the large number of possible modes in which the bundles of seven transmembrane (TM) segments of the interacting GPCR monomers can be packed together into dimers and/or higher-order oligomers. Approaches and tools offered by bioinformatics can be used to reduce the complexity of this task and, combined with computational modeling, can serve to yield testable predictions for the structural properties of oligomers. Most of the bioinformatics methods take advantage of the evolutionary relation that exists among GPCRs, as expressed in their sequences and measurable in the common elements of their structural and functional features. These common elements are responsible for the presence of detectable patterns of motifs and correlated mutations evident from the alignment of the sequences of these complex biological systems. The decoding of these patterns in terms of structural and functional determinants can provide indications about the most likely interfaces of dimerization/oligomerization of GPCRs. We review here the main approaches from bioinformatics, enhanced by computational molecular modeling, that have been used to predict likely interfaces of dimerization/oligomerization of GPCRs, and compare results from their application to rhodopsin-like GPCRs. A compilation of the most frequently predicted GPCR oligomerization interfaces points to specific regions of TMs 4-6.     

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。     

Functional membrane diffusion of G-protein coupled receptors

G-protein-coupled receptor function involves interactions between the receptor, G-proteins and effectors in the cell plasma membrane. The main biochemical processes have been individually identified but the mechanisms governing the successive protein–protein interactions of this complex multi-molecular machinery have yet to be established. We discuss advances in understanding the functional dynamics of the receptor resulting from diffusion measurements, and in the context of the plasma membrane organization. Aurélie Baker and Aude Saulière contributed equally to this work. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors

Insect octopamine receptors carry out many functional roles traditionally associated with vertebrate adrenergic receptors. These include control of carbohydrate metabolism, modulation of muscular tension, modulation of sensory inputs and modulation of memory and learning. The activation of octopamine receptors mediating many of these actions leads to increases in the levels of cyclic AMP. However, to date none of the insect octopamine receptors that have been cloned have been convincingly shown to be capable of directly mediating selective and significant increases in cyclic AMP levels. Here we report on the identification and characterization of a novel, neuronally expressed family of three Drosophila G-protein coupled receptors that are selectively coupled to increases in intracellular cyclic AMP levels by octopamine. This group of receptors, DmOct beta1R (CG6919), DmOct beta2R (CG6989) and DmOct beta3R (CG7078) shows homology to vertebrate beta-adrenergic receptors. When expressed in Chinese hamster ovary cells all three receptors show a strong preference for octopamine over tyramine for the accumulation of cyclic AMP but show unique pharmacological profiles when tested with a range of synthetic agonists and antagonists. Thus, the pharmacological profile of individual insect tissue responses to octopamine might vary with the combination and the degree of expression of the individual octopamine receptors present.     

Structural models for dimerization of G-protein coupled receptors: the opioid receptor homodimers

Among the most exciting functional features of G-protein coupled receptors (GPCRs) that are coming into focus lately are those relating to the role and structural characteristics of their oligomerization (mostly homo- and heterodimers). The structural underpinnings of these novel functional insights are still not clear, as current experimental techniques have not yet succeeded in identifying the dimerization interfaces between GPCR monomers. Two computational approaches have recently been designed in our lab to provide reasonable three-dimensional (3D) molecular models of the transmembrane (TM) regions of GPCR dimers based on a combination of the structural information of receptor monomers and analyses of correlated mutations in receptor families. The modeling of GPCR heterodimers has been described recently. We present here a related approach for modeling of GPCR homodimers that identifies the interfaces in the most likely configurations of the complexes. The approach is illustrated for the three cloned opioid receptor subtypes (OPRD, OPRM, and OPRK).     

Classification of amine type G-protein coupled receptors with feature selection

G-protein coupled receptors (GPCRs) are involved in various physiological processes. Therefore, classification of amine type GPCRs is important for proper understanding of their functions. Though some effective methods have been developed, it still remains unknown how many and which features are essential for this task. Empirical studies show that feature selection might address this problem and provide us with some biologically useful knowledge. In this paper, a feature selection technique is introduced to identify those relevant features of proteins which are potentially important for the prediction of amine type GPCRs. The selected features are finally accepted to characterize proteins in a more compact form. High prediction accuracy is observed on two data sets with different sequence similarity by 5-fold cross-validation test. The comparison with a previous method demonstrates the efficiency and effectiveness of the proposed method.     

Nine new human Rhodopsin family G-protein coupled receptors: identification, sequence characterisation and evolutionary relationship

We report nine new members of the Rhodopsin family of human G protein-coupled receptors (GPCRs) found by searches in the genome databases. BLAST searches and phylogenetic analyses showed that only four of the receptors are closely related to previously characterised GPCRs, GPR150 and GPR154 to oxytocin/vasopressin receptors, GPR152 to CRTH2/FPRs and GPR165 to GPR72/NPYR. Four of the receptors, GPR139, GPR146, GPR153 and GPR162, have one other orphan GPCRs as close relative while GPR148 lacks close relatives. We have identified in total 37 orthologues for the new receptors, primarily from rat, mouse, chicken, fugu and zebrafish. GPR162 and GPR139 are remarkably well conserved while GPR148 seems to be evolving rapidly. Analyses using expressed sequence tags (ESTs) indicate that all the new receptors except GPR153 have the CNS as a major site of expression.     

PREDICT modeling and in-silico screening for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are a major group of drug targets for which only one x-ray structure is known (the nondrugable rhodopsin), limiting the application of structure-based drug discovery to GPCRs. In this paper we present the details of PREDICT, a new algorithmic approach for modeling the 3D structure of GPCRs without relying on homology to rhodopsin. PREDICT, which focuses on the transmembrane domain of GPCRs, starts from the primary sequence of the receptor, simultaneously optimizing multiple 'decoy' conformations of the protein in order to find its most stable structure, culminating in a virtual receptor-ligand complex. In this paper we present a comprehensive analysis of three PREDICT models for the dopamine D2, neurokinin NK1, and neuropeptide Y Y1 receptors. A shorter discussion of the CCR3 receptor model is also included. All models were found to be in good agreement with a large body of experimental data. The quality of the PREDICT models, at least for drug discovery purposes, was evaluated by their successful utilization in in-silico screening. Virtual screening using all three PREDICT models yielded enrichment factors 9-fold to 44-fold better than random screening. Namely, the PREDICT models can be used to identify active small-molecule ligands embedded in large compound libraries with an efficiency comparable to that obtained using crystal structures for non-GPCR targets.     

Modelling of the activation of G-protein coupled receptors: drug free constitutive receptor activity

G-protein coupled receptors (GPCRs) form a crucial component of approximately 80% of hormone pathways. In this paper, the most popular mechanism for activation of GPCRs—the shuttling mechanism—is modelled mathematically. An asymptotic analysis of this model clarifies the dynamics of the system in the absence of drug, in particular which reactions dominate during the different timescales. Equilibrium analysis of the model demonstrates the model’s ability to predict constitutive receptor activity.     

Adaptive evolution of G-protein coupled receptor genes

The phylogeny and patterns of nucleotide substitutions in the visual pigment genes, adrenergic receptor genes, muscarinic receptor genes, and in the human mas oncogene were studied by comparing their DNA sequences. The evolutionary tree obtained shows that the visual pigment genes and mas oncogene form one cluster and that the receptor genes form another. In the evolution of rhodopsin genes, synonymous substitutions outnumber nonsynonymous substitutions. This is consistent with the neutral theory of molecular evolution. However, the early evolutionary stages of alpha- and beta-adrenergic and muscarinic receptors are notable for significantly more nonsynonymous substitutions than synonymous substitutions, suggesting the acquisition of novel functional adaptations. Variable rates of nonsynonymous changes in different domains of these proteins reveal DNA segments that might have been important in their functional adaptations.