Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers

Treatment of CHO cells expressing human Y receptors (Y₁, Y₂ or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi:, 2010).     

Effect of of ATP on Neurons of the Rat Intact Nodose Ganglion

Under intracellular recording, we studied the effect of ATP on nerve cells of the rat intact nodose ganglion. The resting membrane potential of the examined neurons was, on average, –60.3 ± 1.4 mV (n = 84); among such units, 88% were classified as C cells. Local application of 2 mM ATP to the surface of the ganglion using a modified laminar flow system led to depolarization of neurons by 7.1 ± 0.9 mV, on average (n = 19). A blocker of P2X receptors, PPADS (100 μM), suppressed these depolarization responses, decreasing their amplitude, on average, to 16 ± 3% (n = 3) of the initial value. The obtained data indicate that an overwhelming majority of neurons of the intact nodose ganglion possess functional P2X receptors on their membranes. The absence of the corresponding responses in a considerable part of neurons of intact spinal ganglia [13-15] was, apparently, determined by the fact that P2X receptors in the course of the described experiments had enough time to desensitize before ATP reached the effective concentration.     

The FPR2-induced rise in cytosolic calcium in human neutrophils relies on an emptying of intracellular calcium stores and is inhibited by a gelsolin-derived PIP<Subscript>2</Subscript>-binding peptide

Background  

The molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific G-protein-coupled cell surface receptors (GPCRs). Human neutrophils express two pattern recognition GPCRs, FPR1 and FPR2, which belong to the family of formyl peptide receptors. The high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. The aims of this study were to determine whether FPR2 triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived PIP₂-binding peptide, PBP10, selectively inhibits FPR2-mediated transient rise in intracellular Ca²⁺.     

The fourth intracellular domain of G-protein coupling receptors: helicity, basicity and similarity to opsins

The minimal size of the fourth intracellular domain of heptahelical G-protein coupling receptors (GPCRs) is close to 15 residues, and a juxtamembrane 15-residue segment is predicted as helical (Helix-8) in most of the receptors. Sequences of opsins, non-visual opsin-like (family A) GPCRs and Taste-2 receptors correspond with bovine rhodopsin at four positions in this tract. This is especially evident in monoamine receptors. In most GPCRs, the conserved juxtamembrane segment also has a large fraction of basic sidechains, and a considerable excess of cationic over anionic residues. The conservation is not dependent on the preferred G-protein α subunit or the overall length of the domain, indicating an additive speciation. In rod opsins and some A-GPCRs this segment has been shown to associate with the bilayer and to interact with G-proteins. The segment could also be involved in precoupling of receptors and transducers. These interactions could be helped by both the structural propensities and the high content of cationic sidechains.     

Identification of a gonadotropin-releasing hormone receptor orthologue in Caenorhabditis elegans

Background  

The Caenorhabditis elegans genome is known to code for at least 1149 G protein-coupled receptors (GPCRs), but the GPCR(s) critical to the regulation of reproduction in this nematode are not yet known. This study examined whether GPCRs orthologous to human gonadotropin-releasing hormone receptor (GnRHR) exist in C. elegans.     

The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide

Background  

The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites.     

P2 receptors in atherosclerosis and postangioplasty restenosis

Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction [1–3]. Extracellular nucleotides that are released from a variety of arterial and blood cells [4] can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells (SMC), which is known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis [5]. In addition, P2 receptors mediate many other functions, including platelet aggregation, leukocyte adherence, and arterial vasomotoricity. A direct pathological role of P2 receptors is reinforced by recent evidence showing that up-regulation and activation of P2Y₂ receptors in rabbit arteries mediates intimal hyperplasia [6]. In addition, up-regulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model [7] and in coronary arteries of diabetic dyslipidemic pigs [8]. It has been proposed that up-regulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis [9]. Therefore, particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels, which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty.     

A method for the prediction of GPCRs coupling specificity to G-proteins using refined profile Hidden Markov Models

Background  

G- Protein coupled receptors (GPCRs) comprise the largest group of eukaryotic cell surface receptors with great pharmacological interest. A broad range of native ligands interact and activate GPCRs, leading to signal transduction within cells. Most of these responses are mediated through the interaction of GPCRs with heterotrimeric GTP-binding proteins (G-proteins). Due to the information explosion in biological sequence databases, the development of software algorithms that could predict properties of GPCRs is important. Experimental data reported in the literature suggest that heterotrimeric G-proteins interact with parts of the activated receptor at the transmembrane helix-intracellular loop interface. Utilizing this information and membrane topology information, we have developed an intensive exploratory approach to generate a refined library of statistical models (Hidden Markov Models) that predict the coupling preference of GPCRs to heterotrimeric G-proteins. The method predicts the coupling preferences of GPCRs to G_s, G_i/o and G_q/11, but not G_12/13 subfamilies.     

Characterization of oxylipins and dioxygenase genes in the asexual fungus Aspergillus niger

Background  

Aspergillus niger is an ascomycetous fungus that is known to reproduce through asexual spores, only. Interestingly, recent genome analysis of A. niger has revealed the presence of a full complement of functional genes related to sexual reproduction [1]. An example of such genes are the dioxygenase genes which in Aspergillus nidulans, have been shown to be connected to oxylipin production and regulation of both sexual and asexual sporulation [2–4]. Nevertheless, the presence of sex related genes alone does not confirm sexual sporulation in A. niger.     

Use of weighted reference panels based on empirical estimates of ancestry for capturing untyped variation

Many association methods use a subset of genotyped single nucleotide polymorphisms (SNPs) to capture or infer genotypes at other untyped SNPs. We and others previously showed that tag SNPs selected to capture common variation using data from The International HapMap Consortium (Nature 437:1299–1320, 2005), The International HapMap Consortium (Nature 449:851–861, 2007) could also capture variation in populations of similar ancestry to HapMap reference populations (de Bakker et al. in Nat Genet 38:1298–1303, 2006; González-Neira et al. in Genome Res 16:323–330, 2006; Montpetit et al. in PLoS Genet 2:282–290, 2006; Mueller et al. in Am J Hum Genet 76:387–398, 2005). To capture variation in admixed populations or populations less similar to HapMap panels, a “cosmopolitan approach,” in which all samples from HapMap are used as a single reference panel, was proposed. Here we refine this suggestion and show that use of a “weighted reference panel,” constructed based on empirical estimates of ancestry in the target population (relative to available reference panels), is more efficient than the cosmopolitan approach. Weighted reference panels capture, on average, only slightly fewer common variants (minor allele frequency > 5%) than the cosmopolitan approach (mean r ² = 0.977 vs. 0.989, 94.5% variation captured vs. 96.8% at r ² > 0.8), across the five populations of the Multiethnic Cohort, but entail approximately 25% fewer tag SNPs per panel (average 538 vs. 718). These results extend a recent study in two Indian populations (Pemberton et al. in Ann Hum Genet 72:535–546, 2008). Weighted reference panels are potentially useful for both the selection of tag SNPs in diverse populations and perhaps in the design of reference panels for imputation of untyped genotypes in genome-wide association studies in admixed populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

GPCR signalling to the translation machinery

G protein-coupled receptors (GPCRs) are involved in most physiological processes, many of them being engaged in fully differentiated cells. These receptors couple to transducers of their own, primarily G proteins and β-arrestins, which launch intracellular signalling cascades. Some of these signalling events regulate the translational machinery to fine-tune general cell metabolism or to alter protein expression pattern. Though extensively documented for tyrosine kinase receptors, translational regulation by GPCRs is still poorly appreciated. The objective of this review paper is to address the following questions: i) is there a “GPCR signature” impacting on the translational machinery, and ultimately on the type of mRNA translated? ii) are the regulatory networks involved similar as those utilized by tyrosine kinase receptors? In particular, we will discuss the specific features of translational control mediated by GPCRs and highlight the intrinsic properties of GPCRs these mechanisms could rely on.     

A robust antibody discovery platform for difficult-to-express G protein-coupled receptors

G protein-coupled receptors (GPCRs) are in the spotlight as drug targets due to the fact that multiple research results have verified the correlation between the activation of GPCRs and disease indications. This is because the GPCRs are present across the cell membranes, which interact with either extracellular ligands or other types of compartments and simultaneously mediate intracellular signaling. Despite the importance of the GPCRs as drug targets, they are too difficult to express in soluble forms. Currently, the difficulty of preparing functional GPCRs and the lack of efficient antibody screening methods are the most challenging steps in the discovery of antibodies targeting GPCRs. In this study, we developed a powerful platform that facilitates isolating GPCR-specific antibodies by obviating difficulties in GPCR preparation. The strategies include (i) conjugation of the P9 peptide, an envelope protein of Pseudomonas phi6, to the N-terminus of GPCRs to improve the expression level of the GPCRs in Escherichia coli, (ii) stabilization of the GPCRs in their active forms with amphiphilic poly-γ-glutamate (APG) to shield the seven hydrophobic transmembrane domains, and (iii) further limiting the size of the APG complex to improve the chance to isolate antibodies targeting the proteins-of-interest. Capitalizing on the above strategies, we could prepare GPCR proteins in their active forms as facile as other general-soluble antigen proteins. Furthermore, this protocol was validated to be successful in discovering three individual GPCR-specific antibodies targeting glucagon-like peptide-1 receptor, C-X-C chemokine receptor type 4, and prostaglandin E2 receptor 4 in this study.     

Information criterion-based clustering with order-restricted candidate profiles in short time-course microarray experiments

Background  

Time-course microarray experiments produce vector gene expression profiles across a series of time points. Clustering genes based on these profiles is important in discovering functional related and co-regulated genes. Early developed clustering algorithms do not take advantage of the ordering in a time-course study, explicit use of which should allow more sensitive detection of genes that display a consistent pattern over time. Peddada et al. [1] proposed a clustering algorithm that can incorporate the temporal ordering using order-restricted statistical inference. This algorithm is, however, very time-consuming and hence inapplicable to most microarray experiments that contain a large number of genes. Its computational burden also imposes difficulty to assess the clustering reliability, which is a very important measure when clustering noisy microarray data.     

Manipulating assimilate availability provides insight into the genes controlling grain size in sorghum

Variation in grain size, a major determinant of grain yield and quality in cereal crops, is determined by both the plant’s genetic potential and the available assimilate to fill the grain in the absence of stress. This study investigated grain size variation in response to variation in assimilate supply in sorghum using a diversity panel (n = 837) and a backcross-nested association mapping population (n = 1421) across four experiments. To explore the effects of genetic potential and assimilate availability on grain size, the top half of selected panicles was removed at anthesis. Results showed substantial variation in five grain size parameters with high heritability. Artificial reduction in grain number resulted in a general increase in grain weight, with the extent of the increase varying across genotypes. Genome-wide association studies identified 44 grain size quantitative trait locus (QTL) that were likely to act on assimilate availability and 50 QTL that were likely to act on genetic potential. This finding was further supported by functional enrichment analysis and co-location analysis with known grain number QTL and candidate genes. RNA interference and overexpression experiments were conducted to validate the function of one of the identified gene, SbDEP1, showing that SbDEP1 positively regulates grain number and negatively regulates grain size by controlling primary branching in sorghum. Haplotype analysis of SbDEP1 suggested a possible role in racial differentiation. The enhanced understanding of grain size variation in relation to assimilate availability presented in this study will benefit sorghum improvement and have implications for other cereal crops.     

A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q

Only two genome-wide association (GWA) screens have been published for melanoma (Nat Genet 47:920–925, 2009; Nat Genet 40:838–840, 2008). Using a unique approach, we performed a genome-wide association study in 156 related melanoma cases from 34 high-risk Utah pedigrees. Genome-wide association analysis was performed on nearly 500,000 markers; we compared cases to 2,150 genotypically matched samples from Illumina’s iControls database. We performed genome-wide association with EMMAX software, which is designed to account for population structure, including relatedness between cases. Three SNPs exceeded a genome-wide significance threshold of p < 5 × 10⁻⁸ on chromosome arm 10q25.1 (rs17119434, rs17119461, and rs17119490), where the most extreme p value was 7.21 × 10⁻¹². This study represents a new and unique approach to predisposition gene identification; and it is the first genome-wide association study performed in related cases in high-risk pedigrees. Our approach illustrates an example of using high-risk pedigrees for the identification of new melanoma predisposition variants.     

Shaping bursting by electrical coupling and noise

Gap-junctional coupling is an important way of communication between neurons and other excitable cells. Strong electrical coupling synchronizes activity across cell ensembles. Surprisingly, in the presence of noise synchronous oscillations generated by an electrically coupled network may differ qualitatively from the oscillations produced by uncoupled individual cells forming the network. A prominent example of such behavior is the synchronized bursting in islets of Langerhans formed by pancreatic β-cells, which in isolation are known to exhibit irregular spiking (Sherman and Rinzel, Biophys J 54:411–425, 1988; Sherman and Rinzel, Biophys J 59:547–559, 1991). At the heart of this intriguing phenomenon lies denoising, a remarkable ability of electrical coupling to diminish the effects of noise acting on individual cells. In this paper, building on an earlier analysis of denoising in networks of integrate-and-fire neurons (Medvedev, Neural Comput 21 (11):3057–3078, 2009) and our recent study of spontaneous activity in a closely related model of the Locus Coeruleus network (Medvedev and Zhuravytska, The geometry of spontaneous spiking in neuronal networks, submitted, 2012), we derive quantitative estimates characterizing denoising in electrically coupled networks of conductance-based models of square wave bursting cells. Our analysis reveals the interplay of the intrinsic properties of the individual cells and network topology and their respective contributions to this important effect. In particular, we show that networks on graphs with large algebraic connectivity (Fiedler, Czech Math J 23(98):298–305, 1973) or small total effective resistance (Bollobas, Modern graph theory, Graduate Texts in Mathematics, vol. 184, Springer, New York, 1998) are better equipped for implementing denoising. As a by-product of the analysis of denoising, we analytically estimate the rate with which trajectories converge to the synchronization subspace and the stability of the latter to random perturbations. These estimates reveal the role of the network topology in synchronization. The analysis is complemented by numerical simulations of electrically coupled conductance-based networks. Taken together, these results explain the mechanisms underlying synchronization and denoising in an important class of biological models.     

Yohimbine as a Starting Point to Access Diverse Natural Product-Like Agents with Re-programmed Activities against Cancer-Relevant GPCR Targets

G protein-coupled receptors (GPCRs) constitute the largest protein superfamily in the human genome. GPCRs play key roles in mediating a wide variety of physiological events including proliferation and cancer metastasis. Given the major roles that GPCRs play in mediating cancer growth, they present promising targets for small molecule therapeutics. One of the principal goals of our lab is to identify complex natural products (NPs) suitable for ring distortion, or the dramatic altering of the inherently complex architectures of NPs, to rapidly generate an array of compounds with diverse molecular skeletal systems. The overarching goal of our ring distortion approach is to re-program the biological activity of select natural products and identify new compounds of importance to the treatment of disease, such as cancer. Described herein are the results from biological screens of diverse small molecules derived from the indole alkaloid yohimbine against a panel of GPCRs involved in various diseases. Several analogues displayed highly differential antagonistic activities across the GPCRs tested. We highlight the re-programmed profile of one analogue, Y7g, which exhibited selective antagonistic activities against AVPR2 (IC₅₀ = 459 nM) and OXTR (IC₅₀ = 1.16 µM). The activity profile of Y7g could correlate its HIF-dependent anti-cancer activity to its GPCR antagonism since these receptors are known to be upregulated in hypoxic cellular environments. Our findings demonstrate that the ring distortion of yohimbine can lead to the identification of new compounds capable of interacting with distinct cancer-relevant targets.     

An improved classification of G-protein-coupled receptors using sequence-derived features

Background  

G-protein-coupled receptors (GPCRs) play a key role in diverse physiological processes and are the targets of almost two-thirds of the marketed drugs. The 3 D structures of GPCRs are largely unavailable; however, a large number of GPCR primary sequences are known. To facilitate the identification and characterization of novel receptors, it is therefore very valuable to develop a computational method to accurately predict GPCRs from the protein primary sequences.     

Evolution of the class C GPCR Venus flytrap modules involved positive selected functional divergence

Background  

Class C G protein-coupled receptors (GPCRs) represent a distinct group of the GPCR family, which structurally possess a characteristically distinct extracellular domain inclusive of the Venus flytrap module (VFTM). The VFTMs of the class C GPCRs is responsible for ligand recognition and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs). An extensive phylogenetic investigation of the VFTMs was conducted by analyzing for functional divergence and testing for positive selection for five typical groups of the class C GPCRs. The altered selective constraints were determined to identify the sites that had undergone functional divergence via positive selection. In order to structurally demonstrate the pattern changes during the evolutionary process, three-dimensional (3D) structures of the GPCR VFTMs were modelled and reconstructed from ancestral VFTMs.     

Antarctic associations: the parasitic relationship between the gastropod <Emphasis Type="Italic">Bathycrinicola tumidula</Emphasis> (Thiele, 1912) (Ptenoglossa: Eulimidae) and the comatulid <Emphasis Type="Italic">Notocrinus virilis</Emphasis> Mortensen, 1917 (Crinoidea: Notocrinidae) in the Ross Sea

The first case of parasitic association between an eulimid mollusc (Gastropoda, Ptenoglossa) and a comatulid (Echinodermata: Crinoidea) is reported for Antarctica. The mollusc involved in the association is Eulima tumidula Thiele, 1912, which has now been ascribed to the genus Bathycrinicola Bouchet & Warén, 1986, never recognized before in Antarctica. This genus is present only in the NE Atlantic Ocean and the Mediterranean Sea, and encompass species which are specific parasites of the sessile stalked crinoids of the family Bathycrinidae. However, in Antarctica, Bathycrinicola tumidula (Thiele, 1912) exploits the endemic vagile comatulid Notocrinus virilis Mortensen, 1917, and attains the largest known dimensions (∼1 cm) for a Bathycrinicola species. The absence of suitable Bathycrinidae host in modern Antarctic benthic assemblages, as well as the long paleontological history of the genus Notocrinus in Antarctica, suggest a possible ‘host-switch’ phenomenon. This event could reasonably have occurred when many species underwent considerable bathymetric shifts, during the dramatic climatic changes that affected Antarctica.