首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 437 毫秒
1.
Wild isolates of Caenorhabditis elegans differ in their tendency to aggregate on food [1, 2]. Most quantitative variation in this behavior is explained by a polymorphism at a single amino acid in the G protein-coupled receptor NPR-1: gregarious strains carry the 215F allele, and solitary strains carry the 215V allele [2]. Although npr-1 regulates a behavioral syndrome with potential adaptive implications, the evolutionary causes and consequences of this natural polymorphism remain unclear. Here we show that npr-1 regulates two behaviors that can promote coexistence of the two alleles. First, gregarious and solitary worms differ in their responses to food such that they can partition a single, continuous patch of food. Second, gregarious worms disperse more readily from patch to patch than do solitary worms, which can cause partitioning of a fragmented resource. The dispersal propensity of both gregarious and solitary worms increases with density. npr-1-dependent dispersal is independent of aggregation and could be part of a food-searching strategy. The gregarious allele is favored in a fragmented relative to a continuous food environment in competition experiments. We conclude that the npr-1 polymorphism could be maintained by a trade-off between dispersal and competitive ability.  相似文献   

2.
3.
The ability to avoid noxious extremes of hot and cold is critical for survival and depends on thermal nociception. The TRPV subset of transient receptor potential (TRP) channels is heat activated and proposed to be responsible for heat detection in vertebrates and fruit flies. To gain insight into the genetic and neural basis of thermal nociception, we developed assays that quantify noxious heat avoidance in the nematode Caenorhabditis elegans and used them to investigate the genetic basis of this behavior. First, we screened mutants for 18 TRP channel genes (including all TRPV orthologs) and found only minor defects in heat avoidance in single and selected double and triple mutants, indicating that other genes are involved. Next, we compared two wild isolates of C. elegans that diverge in their threshold for heat avoidance and linked this phenotypic variation to a polymorphism in the neuropeptide receptor gene npr-1. Further analysis revealed that loss of either the NPR-1 receptor or its ligand, FLP-21, increases the threshold for heat avoidance. Cell-specific rescue of npr-1 implicates the interneuron RMG in the circuit regulating heat avoidance. This neuropeptide signaling pathway operates independently of the TRPV genes, osm-9 and ocr-2, since mutants lacking npr-1 and both TRPV channels had more severe defects in heat avoidance than mutants lacking only npr-1 or both osm-9 and ocr-2. Our results show that TRPV channels and the FLP-21/NPR-1 neuropeptide signaling pathway determine the threshold for heat avoidance in C. elegans.  相似文献   

4.
5.
6.
Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.  相似文献   

7.
Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.  相似文献   

8.
Whereas short neuropeptide F (sNPF) has already been reported to stimulate feeding behaviour in a variety of insect species, the opposite effect was observed in the desert locust. In the present study, we cloned a G protein-coupled receptor (GPCR) cDNA from the desert locust, Schistocerca gregaria. Cell-based functional analysis of this receptor indicated that it is activated by both known isoforms of Schgr-sNPF in a concentration dependent manner, with EC50 values in the nanomolar range. This Schgr-sNPF receptor constitutes the first functionally characterized peptide GPCR in locusts. The in vivo effects of the sNPF signalling pathway on the regulation of feeding in locusts were further studied by knocking down the newly identified Schgr-sNPF receptor by means of RNA interference, as well as by means of peptide injection studies. While injection of sNPF caused an inhibitory effect on food uptake in the desert locust, knocking down the corresponding peptide receptor resulted in an increase of total food uptake when compared to control animals. This is the first comprehensive study in which a clearly negative correlation is described between the sNPF signalling pathway and feeding, prompting a reconsideration of the diverse roles of sNPFs in the physiology of insects.  相似文献   

9.
Cloning and characterization of murine neuromedin U receptors   总被引:3,自引:0,他引:3  
Neuromedin U (NmU) is a neuropeptide involved in various physiological functions such as feeding behavior, muscle contractile activity, and regulation of intestinal ion transport. Recently, two human G protein-coupled receptors have been identified as NmU-specific receptors, NmU-R1 and NmU-R2, which share 55% amino acid identity. It is unclear however, which of the two receptors mediates responses to NmU observed in rodent models. Attempts to define the pharmacological profile of the two receptors are confounded by overlapping expression of the two receptors and a lack of subtype-selective compounds. In order to establish a basis to further our understanding of the function of these receptors, we cloned and characterized the mouse homologues of the two human NmU receptors. Mouse NmU-R1 and mouse NmU-R2 are 79 and 81% identical to their respective human homologues. Expression of NmU-R1 was mainly observed in testis, gastrointestinal (GI) tract, and immune system, while NmU-R2 was primarily expressed in brain tissues. Each mouse receptor was independently expressed in HEK293 cells and demonstrated a dose-dependent calcium flux in response to NmU-8, NmU-23 and NmU-25. In an attempt to identify a synthetic NmU peptide that would exhibit selectivity at one of the two receptors, we examined the functional activity of eight alanine-substituted NmU-8 peptides. These experiments demonstrated that alanine substitution at positions 5 and 7 affects the functional activity of the peptide at both receptors. The arginine residue at position 7 is required for NmU-8 activity at either receptor while alanine substitution at position 5 selectively affects the potency and the efficacy at mNmU-R1. These experiments validate the use of rodent models to characterize NmU function relative to humans and suggest that substitution at Arginine-5 of NmU-8 may provide a receptor selective peptide.  相似文献   

10.
The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M(3) receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M(3) receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M(3) receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs.  相似文献   

11.
12.
Phe-Met-Arg-Phe-NH2 (FMRFamide)-like peptides (FLPs) are the largest neuropeptide family in animals, particularly invertebrates. FLPs are characterized by a C-N-terminal gradient of decreasing amino acid conservation. Neuropeptide receptor 1 (NPR-1) is a G-protein coupled receptor (GPCR), which has been shown to be a strong regulator of foraging behavior and aggregation responses in Caenorhabditis elegans. Recently, ligands for NPR-1 were identified as neuropeptides coded by the precursor genes flp-18 and flp-21 in C. elegans. The flp-18 gene encodes eight FLPs including DFDGAMPGVLRF-NH2 and EMPGVLRF-NH2. These peptides exhibit considerably different activities on NPR-1, with the longer one showing a lower potency. We have used nuclear magnetic resonance and biological activity to investigate structural features that may explain these activity differences. Our data demonstrate that long-range electrostatic interactions exist between N-terminal aspartates and the C-terminal penultimate arginine as well as N-terminal hydrogen-bonding interactions that form transient loops within DFDGAMPGVLRF-NH2. We hypothesize that these loops, along with peptide charge, diminish the activity of this peptide on NPR-1 relative to that of EMPGVLRF-NH2. These results provide some insight into the large amino acid diversity in FLPs.  相似文献   

13.
Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. We cloned a PBAN receptor (Plx-PBANr) gene from the female pheromone gland of the diamondback moth, Plutella xylostella (L.). Plx-PBANr encodes 338 amino acids and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains, indicating a typical G protein-coupled receptor. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. Taken together, the identified PBAN receptor is functional in PBAN signaling via calcium secondary messenger, which leads to activation of pheromone biosynthesis and male attraction.  相似文献   

14.
The second messenger 3', 5'-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.  相似文献   

15.
16.
Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Here we show that allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of Caenorhabditis elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations.  相似文献   

17.
The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBANR2K) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca2+ mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca2+ mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide.  相似文献   

18.
To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K(m) for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.  相似文献   

19.
Recently, an orphan G protein coupled receptor (GPCR) termed NPGPR was described. A shorter variant of this receptor lacking exon 1 was shown to have subnanomolar affinity for neuropeptide FF (NPFF), a pain modulatory peptide, and therefore was named NPFF(2) receptor. Here, we characterize the full-length cloned NPGPR and identify a novel short form lacking exon 2 with a differential pattern of mRNA abundance in several tissues and organs. The NPGPR is most similar to the recently cloned neuropeptide FF (NPFF) receptor which lacks exon 1, but also shows high homology to the orexin and neuropeptide Y (NPY) receptor families, two neuropeptides involved in food intake regulation. Therefore, we used binding studies to examine the interaction of NPFF, orexin and NPY with the NPGPR. [125I] NPFF was displaced by NPFF with an IC(50) of 14.7 +/- 8.8 nM, whereas [125I] Orexin B was displaced by Orexin B with an IC(50) of 415 +/- 195 nM. We conclude that orexins interact with the NPGPR and that the affinity of NPFF for NPGPR is approximately 100-fold lower than for the NPFF2 receptor. We postulate that NPGPR is a splice variant of the family of NPFF receptors and displays a binding profile different from the other members of the NPFF receptor family due to the presence of exon 1. In order to evaluate whether NPGPR levels are affected by the feeding status, we examined the mRNA level using real-time PCR in two feeding models, i.e. before and after diet-induced body weight increase as well as after chronic food restriction in rats. However, hypothalamic NPGPR mRNA was unchanged in both models. Therefore, our evidence does not support the hypothesis that NPGPR is involved in feeding regulation.  相似文献   

20.
The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT1 receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT1 receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28(1.32)–Ile-53(1.57) fragment of TMD1 and Leu-143(4.40)–Phe-170(4.67) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C(1.34)-AT1 and T33C(1.37)-AT1 and TMD4 mutant V169C(4.66)-AT1. Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT1) background, we found that V169C(4.66)-AT1 remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C(4.64)-AT1 was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30(1.34) and Thr-33(1.37), as well as residues Arg-167(4.64) and Val-169(4.66) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT1 receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号