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.     

Eleven new putative aminergic G-protein coupled receptors from Amphioxus (<Emphasis Type="Italic">Branchiostoma floridae</Emphasis>): identification,sequence analysis and phylogenetic relationship

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.     

Structure-based identification of binding sites,native ligands and potential inhibitors for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest family of cell-surface receptors involved in signal transmission. Drugs associated with GPCRs represent more than one fourth of the 100 top-selling drugs and are the targets of more than half of the current therapeutic agents on the market. Our methodology based on the internal coordinate mechanics (ICM) program can accurately identify the ligand-binding pocket in the currently available crystal structures of seven transmembrane (7TM) proteins [bacteriorhodopsin (BR) and bovine rhodopsin (bRho)]. The binding geometry of the ligand can be accurately predicted by ICM flexible docking with and without the loop regions, a useful finding for GPCR docking because the transmembrane regions are easier to model. We also demonstrate that the native ligand can be identified by flexible docking and scoring in 1.5% and 0.2% (for bRho and BR, respectively) of the best scoring compounds from two different types of compound database. The same procedure can be applied to the database of available chemicals to identify specific GPCR binders. Finally, we demonstrate that even if the sidechain positions in the bRho binding pocket are entirely wrong, their correct conformation can be fully restored with high accuracy (0.28 A) through the ICM global optimization with and without the ligand present. These binding site adjustments are critical for flexible docking of new ligands to known structures or for docking to GPCR homology models. The ICM docking method has the potential to be used to "de-orphanize" orphan GPCRs (oGPCRs) and to identify antagonists-agonists for GPCRs if an accurate model (experimentally and computationally validated) of the structure has been constructed or when future crystal structures are determined.     

Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents

G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2β are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.     

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.     

Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors

Summary Insect octopamine receptors are G-protein coupled receptors. They can be coupled to second messenger pathways to mediate either increases or decreases in intracellular cyclic AMP levels or the generation of intracellular calcium signals. Insect octopamine receptors were originally classified on the basis of second messenger changes induced in a variety of intact tissue preparations. Such a classification system is problematic if more than one receptor subtype is present in the same tissue preparation. Recent progress on the cloning and characterization in heterologous cell systems of octopamine receptors from Drosophila and other insects is reviewed. A new classification system for insect octopamine receptors into “α-adrenergic-like octopamine receptors (OctαRs)”, “β-adrenergic-like octopamine receptors (OctβRs)” and “octopamine/tyramine (or tyraminergic) receptors” is proposed based on their similarities in structure and in signalling properties with vertebrate adrenergic receptors. In future studies on the molecular basis of octopamine signalling in individual tissues it will be essential to identify the relative expression levels of the different classes of octopamine receptor present. In addition, it will be essential to identify if co-expression of such receptors in the same cells results in the formation of oligomeric receptors with specific emergent pharmacological and signalling properties.     

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families.     

Progestin, estrogen and androgen G-protein coupled receptors in fish gonads

The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.     

Prediction and classification of human G-protein coupled receptors based on support vector machines

A computational system for the prediction and classification of human G-protein coupled receptors （GPCRs） has been developed based on the support vector machine （SVM） method and protein sequence information. The feature vectors used to develop the SVM prediction models consist of statistically significant features selected from single amino acid, dipeptide, and tripeptide compositions of protein sequences. Furthermore, the length distribution difference between GPCRs and non-GPCRs has also been exploited to improve the prediction performance. The testing results with annotated human protein sequences demonstrate that this system can get good performance for both prediction and classification of human GPCRs.     

Plasmon-waveguide resonance spectroscopy: a new tool for investigating signal transduction by G-protein coupled receptors

Plasmon-waveguide resonance (PWR) spectroscopy provides a highly sensitive method for characterizing the kinetics, affinities and conformational changes involved in ligand binding to G-protein coupled receptors, without the need for radioactive or other labeling strategies. In the case of the cloned delta-opioid receptor from human brain incorporated into a lipid bilayer, we have shown that affinities determined in this way are consistent with those measured by standard binding procedures using membranes or whole cells containing the receptors, and that the spectral and kinetic properties of the binding processes allow facile distinction between agonist, inverse agonist, and antagonist ligands. We have also shown by direct measurements that G-protein binding affinities and the ability to undergo GTP/GDP exchange are dependent upon the type of ligand pre-bound to the receptor. PWR spectroscopy thus provides a powerful new approach to investigating signal transduction in biological membrane systems.     

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.     

Endoplasmic reticulum retention,degradation, and aggregation of olfactory G-protein coupled receptors

The mammalian olfactory G-protein coupled receptor family is comprised of hundreds of proteins that mediate odorant binding and initiate signal transduction cascades leading to the sensation of smell. However, efforts to functionally express olfactory receptors and identify specific odorant ligand–olfactory receptor interactions have been severely impeded by poor olfactory receptor surface expression in heterologous systems. Therefore, experiments were performed to elucidate the cellular mechanism(s) responsible for inefficient olfactory receptor cell surface expression. We determined that the mouse odorant receptors mI7 and mOREG are not selected for export from the ER and therefore are not detectable at the Golgi apparatus or plasma membrane. Specifically, olfactory receptors interact with the ER chaperone calnexin, are excluded from ER export sites, do not accumulate in ER–Golgi transport intermediates at 15 °C, and contain endoglycosidase H-sensitive oligosaccharides, consistent with olfactory receptor exclusion from post-ER compartments. A labile pool of ER-retained olfactory receptors are post-translationally modified by polyubiquitination and targeted for degradation by the proteasome. In addition, olfactory receptors are sequestered into ER aggregates that are degraded by autophagy. Collectively, these data demonstrate that poor surface expression of olfactory receptors in heterologous cells is attributable to a combination of ER retention due to inefficient folding and poor coupling to ER export machinery, aggregation, and degradation via both proteasomal and autophagic pathways Plasmids .     

Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling

Background

The classic paradigm of heterotrimeric G-protein signaling describes a heptahelical, membrane-spanning G-protein coupled receptor that physically interacts with an intracellular Gα subunit of the G-protein heterotrimer to transduce signals. G-protein coupled receptors comprise the largest protein superfamily in metazoa and are physiologically important as they sense highly diverse stimuli and play key roles in human disease. The heterotrimeric G-protein signaling mechanism is conserved across metazoa, and also readily identifiable in plants, but the low sequence conservation of G-protein coupled receptors hampers the identification of novel ones. Using diverse computational methods, we performed whole-proteome analyses of the three dominant model plant species, the herbaceous dicot Arabidopsis thaliana (mouse-eared cress), the monocot Oryza sativa (rice), and the woody dicot Populus trichocarpa (poplar), to identify plant protein sequences most likely to be GPCRs.

Results

Our stringent bioinformatic pipeline allowed the high confidence identification of candidate G-protein coupled receptors within the Arabidopsis, Oryza, and Populus proteomes. We extended these computational results through actual wet-bench experiments where we tested over half of our highest ranking Arabidopsis candidate G-protein coupled receptors for the ability to physically couple with GPA1, the sole Gα in Arabidopsis. We found that seven out of eight tested candidate G-protein coupled receptors do in fact interact with GPA1. We show through G-protein coupled receptor classification and molecular evolutionary analyses that both individual G-protein coupled receptor candidates and candidate G-protein coupled receptor families are conserved across plant species and that, in some cases, this conservation extends to metazoans.

Conclusion

Our computational and wet-bench results provide the first step toward understanding the diversity, conservation, and functional roles of plant candidate G-protein coupled receptors.     

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.     

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

Lipid-facing correlated mutations and dimerization in G-protein coupled receptors.

A correlated mutation analysis has been performed on the aligned protein sequences of a number of class A G-protein coupled receptor families, including the chemokine, neurokinin, opioid, somatostatin, thyrotrophin and the whole biogenic amine family. Many of the correlated mutations are observed flanking or neighbouring conserved residues. The correlated residues have been plotted onto the transmembrane portion of the rhodopsin crystal structure. The structure shows that a significant proportion of the correlated mutations are located on the external (lipid-facing) region of the helices. The occurrence of these highly correlated patterns of change amongst the external residues suggest that they are sites for protein-protein interactions. In particular, it is suggested that the correlated residues may be involved in either large conformational changes, the formation of heterodimers or homodimers (which may be domain swapped) or oligomers required for activation or internalization. The results are discussed in the light of the subtype-specific heterodimerization observed for the chemokine, opioid and somatostatin receptors.     

Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D

Recently three orphan G-protein coupled receptors, RAIG1, GPRC5B and GPRC5C, with homology to members of family C (metabotropic glutamate receptor-like) have been identified. Using the protein sequences of these receptors as queries we identified overlapping expressed sequence tags which were predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane. The four human receptor subtypes, which we assign to group 5 of family C GPCRs, show 31-42% amino acid sequence identity to each other and 20-25% sequence identity to the transmembrane domains of metabotropic glutamate receptor subtypes 2 and 3 and other family C members. In contrast to the remaining family C members, the group 5 receptors have short amino terminal domains of some 30-50 amino acids. GPRC5D was shown to be clustered with RAIG1 on chromosome 12p13.3 and like RAIG1 and GPRC5B to consist of three exons, the first exon being the largest containing all seven transmembrane segments. GPRC5D mRNA is widely expressed in the peripheral system but all four receptors show distinct expression patterns. Interestingly, mRNA levels of all four group 5 receptors were found in medium to high levels in the kidney, pancreas and prostate and in low to medium levels in the colon and the small intestine, whereas other organs only express a subset of the genes. In an attempt to delineate the signal transduction pathway(s) of the orphan receptors, a series of chimeric receptors containing the amino terminal domain of the calcium sensing receptor or metabotropic glutamate receptor subtype 1, and the seven transmembrane domain of the orphan receptors were constructed and tested in binding and functional assays.