Types Of G-proteins Research Articles

3.6.5.2 Small monomeric GTPases in GtoPdb v.2023.1

Small G-proteins, are a family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate (GTP). They are a type of G-protein found in the cytosol that are homologous to the alpha subunit of heterotrimeric G-proteins, but unlike the alpha subunit of G proteins, a small GTPase can function independently as a hydrolase enzyme to bind to and hydrolyze a guanosine triphosphate (GTP) to form guanosine diphosphate (GDP). The best-known members are the Ras GTPases and hence they are sometimes called Ras subfamily GTPases.

Multiple Subthreshold GPCR Signals Combined by the G-Proteins Gαq and Gαs Activate the Caenorhabditis elegans Egg-Laying Muscles.

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gαq-coupled SER-1 and Gαs-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gαq or SER-7/Gαs alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or designer GPCRs in the muscle cells and found that their subthreshold signals can also combine to induce muscle activity. However, artificially inducing strong signaling through just one of these GPCRs can be sufficient to induce egg laying. Knocking down Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects that were stronger than those of a SER-1/SER-7 double knockout, indicating that additional endogenous GPCRs also activate the muscle cells. These results show that in the egg-laying muscles multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.SIGNIFICANCE STATEMENT How can neurons and other cells gather multiple independent pieces of information from the soup of chemical signals in their environment and compute an appropriate response? Most cells express >20 GPCRs that each receive one signal and transmit that information through three main types of G-proteins. We analyzed how this machinery generates responses by studying the egg-laying system of C. elegans, where serotonin and multiple other signals act through GPCRs on the egg-laying muscles to promote muscle activity and egg laying. We found that individual GPCRs within an intact animal each generate effects too weak to activate egg laying. However, combined signaling from multiple GPCR types reaches a threshold capable of activating the muscle cells.

Regulation of Diacylglycerol Content in Olfactory Neurons Determines Forgetting or Retrieval of Olfactory Memory in Caenorhabditis elegans.

Proper management of memories by forgetting and retrieval is essential for animals to adapt their behavior to changing environments. To elucidate the mechanisms underlying forgetting, we use olfactory learning to an attractive odorant, diacetyl, in Caenorhabditis elegans hermaphrodites as a model. In this learning paradigm, the TIR-1/JNK-1 pathway in AWC sensory neurons accelerates forgetting of the olfactory memory, which is stored as a sensory memory trace in AWA sensory neurons. Our genetic screening revealed that increased neuronal diacylglycerol in the olfactory neuronal circuit, by mutations in diacylglycerol kinase-1, egl-30 or goa-1, Gq and Go type G-proteins, suppresses the forgetting defect in the behavior of tir-1 mutants, although the calcium imaging analyses of the olfactory neurons revealed that the sensory memory trace to the odorant was maintained. In contrast, the expression of a gain-of-function goa-1 gene exclusively in AWC neurons caused a forgetting defect in behavior, although their sensory memory trace declined. Furthermore, the behavioral analysis of animals applied with diacylglycerol analog and measurement of diacylglycerol content by fluorescent imaging suggested that diacylglycerol content in AWC is important for the proper forgetting. These findings raise a possibility that diacylglycerol signaling plays a crucial role in determining whether to forget or to recall in olfactory learning.SIGNIFICANCE STATEMENT Forgetting and retrieval are important processes for proper management of memories, although the mechanisms underlying these processes remain largely unclear. We found that, in Caenorhabditis elegans, diacylglycerol signaling works in a forgetting mechanism downstream of TIR-1/JNK-1 pathway. Mutations that change diacylglycerol content in the olfactory neurons affect behavioral forgetting, although they did not alter the sensory memory trace. This suggests that diacylglycerol in specific neurons may determine the occurrence of retrieving, rather than modifying, the memory traces. Consistent with this hypothesis, application of diacylglycerol analog to animals suggests that diacylglycerol content until memory acquisition decides whether to retrieve or to forget the memory.

Orexin receptors: Targets and applications.

Over the years, elucidating targets from the neural circuits that can be used to treat disorders pertaining to the nervous system and extending their scope to other systems have always proved interesting to researchers. The role of various peptides and neurotransmitters has been elucidated and is being developed as therapeutic targets. Out of these, orexins are neuropeptides produced in the hypothalamus that stimulate a specific type of G-Protein coupled receptors (GPCR) called orexin receptors and bring about various physiological and pathological roles. Orexin receptors are of interest not only because of their wide applications such as insomnia, obesity, and inflammatory disorders but also because of their contribution to promising aspects of drug discovery such as optogenetics and their tremendous growth from the stage of being orphans to orexins. This review will discuss in detail the structure of orexin receptors, their physiological role, and various applications in disease states adding a note on agonists and antagonists and finally summarizing the recent drug approvals in the field.

Knockdown of a β-Adrenergic-Like Octopamine Receptor Affects Locomotion and Reproduction of Tribolium castaneum.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a β-adrenergic-like octopamine receptor (TcOctβ2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctβ2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

Bitter Taste Receptors Are Expressed on Acute Myeloid Leukemia Cells and Their Activation Results in Quiescence Induction and Prevention of Migration

Acute Myeloid Leukemia (AML) is a clonal disease sprouting from a rare population of leukemic stem cells (LSCs). Over the past years, increasing interest is gaining the contribution that cell-extrinsic factors have in AML generation and maintenance. In this context, AML cell ability to sense the changes in the microenvironment is important for different cell functions, including the responsiveness to antineoplastic agents. Bitter taste receptors (T2Rs) are typical G-protein coupled receptors and are normally found on the surface of the tongue. Recent studies showed that T2Rs are widely expressed in various parts of human anatomy and have been shown to be involved in physiology of respiratory system, gastrointestinal tract and endocrine system thus suggesting a wider function in “sensing microenvironment”. So far, very few data about T2Rs expression in cancer cells are available. In particular, their expression and function in AML cells has not been investigated. Our aim was to investigate T2Rs expression and role in regulation of leukemic cell functionality.In the present work, for the first time, we showed that leukemia cell lines OCI-AML3, THP-1, KG1 and AML primary cells expressed several T2R subtypes (Fig.1A). The expression of TAS2R was associated with GPR-related downstream targets, including the beta subunit of gustducin and the PLC beta 2 (Fig.2B). Stimulation of leukemia cell lines with denatonium (DEN), a T2Rs agonist, induced intracellular Ca2+ concentration increase, thus demonstrating T2Rs functionality (Fig. 1C). To investigate whether bitter compound could modulate AML cell biological properties, we performed GEP analysis of DEN treated cells. GEP analysis identified a number of genes significantly modulated by denatonium treatment. Enrichment analysis identify significantly affected biological processes, among which, cell cycle/proliferation, apoptosis, migration/adhesion and DNA repair (Fig.1D). Specifically, leukemic cells stimulated with T2R agonist underwent down-regulation of genes involved in positive regulation of cell proliferation, migration, and cell-cycle. Whereas genes involved in cell adhesion and DNA repair were up-regulated. Functional assays confirmed molecular data. Indeed, depending on the extent of stimulation, TAS2R activation inhibited leukemia cell proliferation (Fig.2A) inducing cell cycle arrest in G0/G1 phase (Fig. 2B) or reduced cell viability inducing apoptosis (Fig. 2C), as demonstrated by caspase cascade activation and mitochondrial stress induction (Fig. 2D-E). Of note, a pronounced inhibitory effect of denatonium on leukemia cells motility, both spontaneous and in response to CXCL-12, was observed (Fig. 2F).Overall, our results indicate that in AML cells the activation of fully functional T2Rs is associated with quiescence induction and prevention of migration. These data may suggest a role for microenvironment “bitter” molecules in regulating leukemia cell functions, including lethal sensitivity to antineoplastic agents. [Display omitted] DisclosuresMartinelli:Pfizer: Consultancy; Celgene: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Roche: Consultancy.

Endogenous and Synthetic Regulators of the Peripheral Components of the Hypothalamo-Hypophyseal-Gonadal and -Thyroid Axes

Activity in the peripheral components of the hypothalamo-hypophyseal-gonadal and -thyroid axes is regulated by hypophyseal hormones – gonadotropins and thyrotropic hormones (thyroid-stimulating hormone, TSH), which are secreted by specialized cells in the adenohypophysis. Luteinizing hormone (LH) and its homolog chorionic gonadotropin (CG) realize their steroidal effects by binding to LH/CG receptors on the surfaces of Leydig cells in the testes and theca cells and granulosa cells in mature follicles in the ovaries. Follicle-stimulating hormone (FSH) binds FSH receptors on Sertoli cells in the testes and granulosa cells in primordial and maturing follicles in the ovaries, controlling the processes of folliculogenesis, spermatogenesis, and steroidogenesis. TSH, via activation of TSH receptors, stimulates the synthesis of thyroid hormones by thyrocytes in the thyroid gland. Gonadotropins (LH, CG, and FSH) and TSH, which bind with high affinity to the extracellular domains of specific G protein-coupled receptors, directly activate various signal cascades operating via different types of G-proteins and β-arrestins. Recombinant gonadotropins and gonadotropins extracted from natural sources and used for the treatment of reproductive dysfunction and as assisted reproduction technologies have a number of drawbacks, which has led to the development of peptide and low molecular weight regulators of LH/CG and FSH receptors which interact with allosteric sites on the transmembrane or cytoplasmic domains of the receptors. Wide perspectives in the regulation of reproductive functions and the control of fertility are opened up by the use of adipokines, peptides of the insulin and relaxin families, and the antidiabetic drug metformin, which not only regulate and modify the responses of the gonads to gonadotropins, but also themselves influence steroidogenesis and gamete maturation. In the case of TSH receptors, the most acute problem is that of reversing increases in their activity in autoimmune and oncological diseases of the thyroid gland and in endocrine ophthalmopathy. The greatest potential in this direction lies in the ongoing development of low molecular weight inverse agonists and neutral antagonists, which interact with an allosteric site located in the transmembrane domain of the TSH receptor. The present review addresses contemporary advances in the development and study of endogenous and synthetic regulators and modulators of gonadotrophic and TSH receptors, along with their influences on the peripheral components of the hypothalamo-hypophyseal-gonadal and -thyroid axes.

CXCR7: a \u03b2-arrestin-biased receptor that potentiates cell migration and recruits \u03b2-arrestin2 exclusively through G\u03b2\u03b3 subunits and GRK2

BackgroundSome chemokine receptors referred to as atypical chemokine receptors (ACKRs) are thought to non-signaling decoys because of their inability to activate typical G-protein signaling pathways. CXCR7, also known as ACKR3, binds to only two chemokines, SDF-1α and I-TAC, and recruits β-arrestins. SDF-1α also binds to its own conventional receptor, CXCR4, involving in homeostatic modulation such as development and immune surveillance as well as pathological conditions such as inflammation, ischemia, and cancers. Recently, CXCR7 is suggested as a key therapeutic target together with CXCR4 in such conditions. However, the molecular mechanisms underlying cellular responses and functional relation with CXCR7 and CXCR4 have not been elucidated, despite massive studies. Therefore, we aimed to reveal the molecular networks of CXCR7 and CXCR4 and compare their effects on cell migration.MethodsBase on structural complementation assay using NanoBiT technology, we characterized the distinct mechanisms underlying β-arrestin2 recruitment by both CXCR4 and CXCR7. Crosslinking and immunoprecipitation were conducted to analyze complex formation of the receptors. Gene deletion using CRISPR and reconstitution of the receptors were applied to analysis of ligand-dependent ERK phosphorylation and cell migration. All experiments were performed in triplicate and repeated more than three times. Unpaired Student’s t-tests or ANOVA using PRISM5 software were employed for statistical analyses.ResultsLigand binding to CXCR7 does not result in activation of typical signaling pathways via Gα subunits but activation of GRK2 via βγ subunits and receptor phosphorylation with subsequent β-arrestin2 recruitment. In contrast, CXCR4 induced Gαi activation and recruited β-arrestin2 through C-terminal phosphorylation by both GRK2 and GRK5. SDF-1α-stimulated ERK phosphorylation was facilitated by CXCR4, but not CXCR7. Heterodimerization of CXCR4 and CXCR7 was not confirmed in this study, while homodimerization of them was verified by crosslinking experiment and NanoBiT assay. Regarding chemotaxis, SDF-1α-stimulated cell migration was mediated by both CXCR4 and CXCR7.ConclusionThis study demonstrates that SDF-1α-stimulated CXCR7 mediates β-arrestin2 recruitment via different molecular networking from that of CXCR4. CXCR7 may be neither a simple scavenger nor auxiliary receptor but plays an essential role in cell migration through cooperation with CXCR4.

COVID-19 transmission through host cell directed network of GPCR.

COVID-19 transmission through host cell directed network of GPCR.

3.6.5.2 Small monomeric GTPases (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Small G-proteins, are a family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate (GTP). They are a type of G-protein found in the cytosol that are homologous to the alpha subunit of heterotrimeric G-proteins, but unlike the alpha subunit of G proteins, a small GTPase can function independently as a hydrolase enzyme to bind to and hydrolyze a guanosine triphosphate (GTP) to form guanosine diphosphate (GDP). The best-known members are the Ras GTPases and hence they are sometimes called Ras subfamily GTPases.

Synchronization of pancreatic islets by periodic or non-periodic muscarinic agonist pulse trains.

Pulsatile insulin secretion into the portal vein from the many pancreatic islets of Langerhans is critical for efficient glucose homeostasis. The islets are themselves endogenous oscillators, but since they are not physically coupled it is not obvious how their oscillations are synchronized across the pancreas. It has been proposed that synchronization of islets is achieved through periodic activity of intrapancreatic ganglia, and indeed there are data supporting this proposal. Postganglionic nerves are cholinergic, and their product, acetylcholine, can influence islet β-cells through actions on M3 muscarinic receptors which are coupled to Gq type G-proteins. In addition, the neurons secrete several peptide hormones that act on β-cell receptors. The data supporting synchronization via intrapancreatic ganglia are, however, limited. In particular, it has not been shown that trains of muscarinic pulses are effective at synchronizing islets in vitro. Also, if as has been suggested, there is a ganglionic pacemaker driving islets to a preferred frequency, no neural circuitry for this pacemaker has been identified. In this study, both points are addressed using a microfluidic system that allows for the pulsed application of the muscarinic agonist carbachol. We find that murine islets are entrained and synchronized over a wide range of frequencies when the carbachol pulsing is periodic, adding support to the hypothesis that ganglia can synchronize islets in vivo. We also find that islet synchronization is very effective even if the carbachol pulses are applied at random times. This suggests that a neural pacemaker is not needed; all that is required is that islets receive occasional coordinated input from postganglionic neurons. The endogenous rhythmic activity of the islets then sets the frequency of the islet population rhythm, while the input from ganglia acts only to keep the islet oscillators in phase.

Bitter Taste Receptors System Is Expressed and Functional in Both HSCs and Leukemic Cells

Acute Myeloid Leukemia (AML) is a clonal disease sprouting from a rare population of leukemic stem cells. Over the past years, increasing interest is gaining the contribution that cell-extrinsic factors have in AML generation and maintenance. In this context, the ability of leukemia cells to detect changes in the microenvironment is important in responsiveness to environmental fluctuations.Bitter taste receptors (T2Rs) are typical G-protein coupled receptors and are normally found on the surface of the tongue. Recent studies showed that T2Rs are widely expressed in various parts of human anatomy and have been shown to be involved in physiology of respiratory system, gastrointestinal tract and endocrine system. thus suggesting a wider function in “sensing microenvironment”. We recently reported that AML cell lines OCI-AML3, THP-1, and AML primary cells expressed fully functional T2Rs subtypes. Gene expression profile analysis showed that after T2Rs activation, leukemic cell lines underwent down-regulation of genes involved in positive regulation of cell proliferation, migration, and cell-cycle. Whereas genes involved in cell adhesion and DNA repair were up-regulated. Functional assays supported these results (Blood 2017 130:3949).In the present work, we further investigated the role of T2Rs in BM microenvironment by extending the analysis to AML primary samples and to normal hematopoietic stem cells (HSCs). Similarly to AML cell lines, T2Rs activation with high dose of agonist induced a reduction of cell viability associated to apoptosis induction, while non-toxic doses reduced cell migration and clonogenic capacity. In addition, T2Rs stimulation with agonist makes AML cell lines more prone to oxidative and metabolic stress. Leukemia cells displayed a quiescent phenotype in response to T2Rs activation suggesting that mitochondrial activity is significantly limited by T2Rs agonist treatment. Since no data are available on the presence and the function of T2Rs on normal hematopoietic stem cell counterpart, we characterized T2Rs expression on CD34+ cell isolated from healthy donor. CD34+ cells express several T2Rs subtype without significant differences compare to AML cells. Their activation with high dose of agonist reduced HSCs viability inducing apoptosis, while non-cytotoxic doses reduced clonogenic capacity and promoted migration.Given the effect of T2Rs activation on crucial AML cell function, we tested the therapeutical potential of T2R agonist with and without conventional chemioterapic agent. Interestingly we observed that T2Rs agonist have a synergistic effect with cytarabine, reducing leukemia cell viability when combined with ARA-C compared to their use as single compound. The combination allowed to reach a high toxicity using lower doses of chemotherapic agent.Overall our results indicate that T2Rs receptor system is expressed and functional in both leukemic cells and HSCs. In particular, in AML cells T2Rs activation is associated with quiescence induction and prevention of migration. T2Rs stimulation modulates HSCs function but their role need to be further deepen. These data may suggest a role for microenvironment “bitter” molecules in regulating normal and leukemic hematopoiesis. DisclosuresCavo:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1–4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1–3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

How the ubiquitous GPCR receptor family selectively activates signalling pathways.

G-protein-coupled receptors activate different G-protein types to trigger divergent signalling pathways. Four structures of receptor–G-protein complexes shed light on this selectivity. G-protein-coupled receptors activate different G-protein types to trigger divergent signalling pathways. Four structures of receptor–G-protein complexes shed light on this selectivity.

Striatal Gαolf/cAMP Signal-Dependent Mechanism to Generate Levodopa-Induced Dyskinesia in Parkinson's Disease.

The motor symptoms of Parkinson’s disease (PD) result from striatal dopamine (DA) deficiency due to a progressive degeneration of nigral dopaminergic cells. Although DA replacement therapy is the mainstay to treat parkinsonian symptoms, a long-term daily administration of levodopa often develops levodopa-induced dyskinesia (LID). LID is closely linked to the dysregulation of cyclic adenosine monophosphate (cAMP) signaling cascades in the medium spiny neurons (MSNs), the principal neurons of the striatum, which are roughly halved with striatonigral MSNs by striatopallidal MSNs. The olfactory type G-protein α subunit (Gαolf) represents an important regulator of the cAMP signal activities in the striatum, where it positively couples with D1-type dopamine receptor (D1R) and adenosine A2A receptor (A2AR) to increase cAMP production in the MSNs. Notably, D1Rs are primarily expressed in striatonigral MSNs, whereas D2Rs and A2ARs are expressed in striatopallidal MSNs. Based on the evidence obtained from parkinsonian mice, we hypothesized that in the DA-denervated striatum with D1R hypersensitivity, a repeated and pulsatile exposure to levodopa might cause a usage-induced degradation of Gαolf proteins in striatal MSNs, resulting in increased and decreased levels of Gαolf protein in the striatonigral and striatopallidal MSNs, respectively. As a principal cause for generating LID, this might lead to an increased responsiveness to levodopa exposure in both striatonigral and striatopallidal MSNs. Our hypothesis reinforces the long-standing concept that LID might result from the reduced activity of the striatopallidal pathway and has important clinical implications.

Dopamine-Induced Changes in Gαolf Protein Levels in Striatonigral and Striatopallidal Medium Spiny Neurons Underlie the Genesis of l-DOPA-Induced Dyskinesia in Parkinsonian Mice.

The dopamine precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), exerts powerful therapeutic effects but eventually generates l-DOPA-induced dyskinesia (LID) in patients with Parkinson’s disease (PD). LID has a close link with deregulation of striatal dopamine/cAMP signaling, which is integrated by medium spiny neurons (MSNs). Olfactory type G-protein α subunit (Gαolf), a stimulatory GTP-binding protein encoded by the GNAL gene, is highly concentrated in the striatum, where it positively couples with dopamine D1 (D1R) receptor and adenosine A2A receptor (A2AR) to increase intracellular cAMP levels in MSNs. In the striatum, D1Rs are mainly expressed in the MSNs that form the striatonigral pathway, while D2Rs and A2ARs are expressed in the MSNs that form the striatopallidal pathway. Here, we examined the association between striatal Gαolf protein levels and the development of LID. We used a hemi-parkinsonian mouse model with nigrostriatal lesions induced by 6-hydroxydopamine (6-OHDA). Using quantitative immunohistochemistry (IHC) and a dual-antigen recognition in situ proximity ligation assay (PLA), we here found that in the dopamine-depleted striatum, there appeared increased and decreased levels of Gαolf protein in striatonigral and striatopallidal MSNs, respectively, after a daily pulsatile administration of l-DOPA. This leads to increased responsiveness to dopamine stimulation in both striatonigral and striatopallidal MSNs. Because Gαolf protein levels serve as a determinant of cAMP signal-dependent activity in striatal MSNs, we suggest that l-DOPA-induced changes in striatal Gαolf levels in the dopamine-depleted striatum could be a key event in generating LID.

Role of a tachykinin-related peptide and its receptor in modulating the olfactory sensitivity in the oriental fruit fly, Bactrocera dorsalis (Hendel)

Insect tachykinin-related peptide (TRP), an ortholog of tachykinin in vertebrates, has been linked with regulation of diverse physiological processes, such as olfactory perception, locomotion, aggression, lipid metabolism and myotropic activity. In this study, we investigated the function of TRP (BdTRP) and its receptor (BdTRPR) in an important agricultural pest, the oriental fruit fly Bactrocera dorsalis. BdTRPR is a typical G-protein coupled-receptor (GPCR), and it could be activated by the putative BdTRP mature peptides with the effective concentrations (EC50) at the nanomolar range when expressed in Chinese hamster ovary cells. Consistent with its role as a neuromodulator, expression of BdTRP was detected in the central nervous system (CNS) of B. dorsalis, specifically in the local interneurons with cell bodies lateral to the antennal lobe. BdTRPR was found in the CNS, midgut and hindgut, but interestingly also in the antennae. To investigate the role of BdTRP and BdTRPR in olfaction behavior, adult flies were subjected to RNA interference, which led to a reduction in the antennal electrophysiological response and sensitivity to ethyl acetate in the Y-tube assay. Taken together, we demonstrate the impact of TRP/TRPR signaling on the modulation of the olfactory sensitivity in B. dorsalis. The result improve our understanding of olfactory processing in this agriculturally important pest insect.

Molecular characterizations of natalisin and its roles in modulating mating in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Initially, natalisin (NTL) was identified from three holometabolous insect species, Drosophila melanogaster, Tribolium castaneum and Bombyx mori, and was documented to regulate reproductive behaviours in D. melanogaster and T. castaneum. In this study, we report the sequences of the NTL precursor and its receptor (NTLR) from an important agricultural pest, Bactrocera dorsalis (Hendel). NTLR is a typical G-protein coupled receptor and phylogenetic analysis showed that B. dorsalis NTLR was closely related to insect natalisin receptors from other species. A functional assay of NTLR transiently expressed in Chinese hamster ovary cells showed that it was activated by putative natalisin mature peptides in a concentration-dependent manner, with 50% effective concentrations (EC50 ) at nanomolar or micromolar levels. As indicated by quantitative real-time PCR, both NTL and NTLR had the highest expression in the central nervous system of B. dorsalis compared with the other tested tissues. Three pairs of adult brain neurones of B. dorsalis were identified with immunohistochemical antibody staining against D. melanogaster NTL4, and in situ hybridization with specific DNA probes. Moreover, RNA interference mediated by double-stranded RNA injection in adults provided evidence for the important roles of NTL in regulating both male and female mating frequencies in this fly.

Bacterial expression and/or solid phase peptide synthesis of 20-40 amino acid long polypeptides and miniproteins, the case study of Class B GPCR ligands.

By using two different synthetic techniques several polypeptides interacting with Class B type G-protein coupled receptors were prepared. These polypeptides of different lengths (20 ≤ amino acids ≤ 40), structural and aggregation properties, were prepared both by solid phase peptide synthesis (SPPS) and E.coli bacterial expression. Their purity, synthetic yields, by-products and (15)N/(13)Clabelling characteristics were compared as function of i) the applied method, ii) amino acid length and iii) folding propensities. Their tentative yields, costs and "environmental footprints" were analyzed and found as follows. For unlabelled and short polypeptides (n= 20 aa.) the method of choice is the less environmentally friendly however, quick and effective SPPS. If the polypeptide is (un)folded and/or has no aggregation propensity, then SPPS gives relatively good yield (e.g. 14 ± 4%) and a pure product (>97%). For aggregating polypeptides production yields drop for both methods 4 ± 2% (SPPS) and 2 ± 1% (E. coli), respectively. For longer (n≥ 30 aa.) macromolecules (e.g. miniproteins) bacterial expression efficacy gets higher. Moreover biotechnology is "greener", the resulting in raw material is purer (2.8 ± 1.5 mg). All these advantages for at a lower cost: ~4 €/aa. If isotopic labelling is needed for heteronuclear NMR measurements, bacterial expression is the sole option, due to the high cost of (15)N/(13)C labelled Fmoc(Boc)-L-aa-OH starting materials needed for SPPS. In E.coli uniformly double-labelled, pure polypeptides can be obtained for less than 5-700 €/mg, regardless of the length of the polypeptide chain. Thus, chemists are encouraged to use E.coli expression systems when adequate to make not only proteins but polypeptides and miniproteins as well.

Renal Protection by Genetic Deletion of the Atypical Chemokine Receptor ACKR2 in Diabetic OVE Mice.

In diabetic nephropathy (DN) proinflammatory chemokines and leukocyte infiltration correlate with tubulointerstitial injury and declining renal function. The atypical chemokine receptor ACKR2 is a chemokine scavenger receptor which binds and sequesters many inflammatory CC chemokines but does not transduce typical G-protein mediated signaling events. ACKR2 is known to regulate diverse inflammatory diseases but its role in DN has not been tested. In this study, we utilized ACKR2−/− mice to test whether ACKR2 elimination alters progression of diabetic kidney disease. Elimination of ACKR2 greatly reduced DN in OVE26 mice, an established DN model. Albuminuria was significantly lower at 2, 4, and 6 months of age. ACKR2 deletion did not affect diabetic blood glucose levels but significantly decreased parameters of renal inflammation including leukocyte infiltration and fibrosis. Activation of pathways that increase inflammatory gene expression was attenuated. Human biopsies stained with ACKR2 antibody revealed increased staining in diabetic kidney, especially in some tubule and interstitial cells. The results demonstrate a significant interaction between diabetes and ACKR2 protein in the kidney. Unexpectedly, ACKR2 deletion reduced renal inflammation in diabetes and the ultimate response was a high degree of protection from diabetic nephropathy.