ABSTRACT
Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP-2R), a class-B G protein-coupled receptor. The GLP-2R antagonist GLP-2(3-33) has relatively high partial agonistic activity, and there are as yet no ideal known potent GLP-2R antagonists. We therefore prepared several truncated forms of human GLP-2 and characterized them by binding and reporter assays to find antagonists more potent than GLP-2(3-33). We found that GLP-2(11-33) was the most potent orthosteric GLP-2R antagonist, with binding activity almost equal to those of GLP-2 and GLP-2(3-33) and weaker intrinsic agonistic activity than GLP-2(3-33). GLP-2(11-33) retained weak agonistic activity toward human, cynomolgus monkey, dog, and Syrian hamster GLP-2Rs. However, it had no agonistic activity toward rat GLP-2R. GLP-2(11-33) potentiated the agonistic activity of an ago-allosteric modulator of GLP-2R, compound 1 (N-[1-(2,5-dichlorothiophen-3-yl)-2-(phenylsulfanyl)ethylidene]hydroxylamine), synergistically toward human GLP-2R. In the case of rat GLP-2R, GLP-2(11-33) decreased the agonistic activity of compound 1, although GLP-2 and GLP-2(3-33) increased this activity additively. These findings suggest that the binding sites of the ago-allosteric modulator and GLP-2 overlap, at least in rat GLP-2R. GLP-2(11-33) is a novel, useful tool for analyzing the mode of action of agonists and ago-allosteric modulators of GLP-2R.
Subject(s)
Glucagon-Like Peptide 2/chemistry , Peptide Fragments/chemistry , Receptors, Glucagon/agonists , Receptors, Glucagon/antagonists & inhibitors , Alkaline Phosphatase/metabolism , Allosteric Regulation , Animals , Cricetinae , Cyclic AMP/metabolism , Dogs , Glucagon-Like Peptide 2/genetics , Glucagon-Like Peptide 2/pharmacology , Glucagon-Like Peptide-2 Receptor , HEK293 Cells , Humans , Hydroxylamine/chemical synthesis , Hydroxylamine/pharmacology , Kinetics , Macaca fascicularis , Peptide Fragments/genetics , Peptide Fragments/pharmacology , Protein Binding , Rats , Receptors, Glucagon/metabolism , Species Specificity , Thiophenes/chemical synthesis , Thiophenes/pharmacologyABSTRACT
The effect of the intracerebroventricular (i.c.v.) injection of relaxin-3 (RLX3) was evaluated using anxiety-related behavioral tests in rats. RLX3-injected animals showed normal locomotion activity in a habituated environment and declined anxiety cognition in the elevated plus maze test and the shock probe-burying test. The measurement of spontaneous locomotor activity in a novel environment also suggested that RLX3 reduced the stress response. To elucidate the regulatory mechanisms of the downstream signaling pathways underlying RLX3 activity and its relation to anxiolytic and hyperphagic behavior phenotypes, RLX3-i.c.v.-injected rat hypothalamic responses were examined using a microarray analysis. Ingenuity Pathway Analysis software listed the phenotype-relating genes and they showed characteristic expression patterns in the rat hypothalamus. When peptidome data sets for the same listed genes was analyzed using a semi-quantitative approach, the expressions of two neuropeptides were found to have increased. One of these neuropeptides, oxytocin (Oxt), exhibited increased expression in both the microarray and the peptidomic analysis, and a Western blot analysis validated the mass spectrometry results. A cross-omics data analysis is useful for predicting downstream signaling pathways, and the anxiolytic-like behavior of RLX3 may be mediated by an oxytocin signaling pathway in rats. These results suggest that RLX3 acts as an anxiolytic peptide and that the downstream pathways mediated by its receptors may be potential candidates for the treatment of anxieties in the future.
Subject(s)
Anxiety/drug therapy , Behavior, Animal/drug effects , Nerve Tissue Proteins/metabolism , Relaxin/metabolism , Stress, Physiological/drug effects , Animals , Anxiety/physiopathology , Behavior, Animal/physiology , Hypothalamus/metabolism , Injections, Intraventricular , Maze Learning , Microarray Analysis , Nerve Tissue Proteins/administration & dosage , Neuropeptides/isolation & purification , Neuropeptides/metabolism , Oxytocin/metabolism , Rats , Relaxin/administration & dosage , Signal TransductionABSTRACT
Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP- 2R), a class-B G protein-coupled receptor (GPCR) coupled with Gα(s). Few small-molecule agonists had been reported for class-B GPCRs, but we recently reported the first scaffold compounds of ago-allosteric modulators for human GLP-2R. Methyl 2-{[(2Z)-2-(2,5-dichlorothiophen- 3-yl)-2-(hydroxyimino)ethyl]sulfanyl}benzoate (compound 1) and its de-esterified derivative (compound 2) induced placental alkaline phosphatase (PLAP) activity in HEK293 cells overexpressing human GLP-2R and PLAP driven by cAMP response element. In this study, we observed that rat, Syrian hamster, and dog GLP-2Rs also responded to compounds 1 and 2 in the same reporter system. However, no agonistic activity of the compounds toward mouse GLP-2R was detected. Mutagenesis studies showed that mutant human GLP-2Rs with Pro392Leu substitution of mouse GLP-2R for human GLP-2R amino acid residues nullified the PLAP activity of compound 2, although these mutant receptors responded to GLP-2. This finding suggests that the Pro392 residue of human GLP-2R is essential for the agonistic activity of compound 2.
Subject(s)
Glucagon-Like Peptide 2/pharmacology , Receptors, Glucagon/agonists , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Dogs , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Expression Regulation/drug effects , Genes, Reporter , Glucagon-Like Peptide-2 Receptor , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Molecular Sequence Data , Mutation , Rats , Receptors, Glucagon/chemistry , Receptors, Glucagon/genetics , Sequence Alignment , Species SpecificityABSTRACT
Glucagon-like peptide 2 (GLP-2) is an intestinotropic peptide that binds to GLP-2 receptor (GLP-2R), a class-B G protein-coupled receptor (GPCR). Few synthetic agonists have been reported so far for class-B GPCRs. Here, we report the first scaffold compounds of ago-allosteric modulators for human GLP-2R, derived from methyl 2-{[(2Z)-2-(2,5-dichlorothiophen-3-yl)-2-(hydroxyimino)ethyl]sulfanyl}benzoate (compound 1).
Subject(s)
Benzoates/pharmacology , Receptors, Glucagon/agonists , Thiophenes/pharmacology , Benzoates/chemical synthesis , Benzoates/chemistry , Dose-Response Relationship, Drug , Glucagon-Like Peptide-2 Receptor , Humans , Molecular Structure , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistryABSTRACT
Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.
Subject(s)
Body Weight/drug effects , Relaxin/analogs & derivatives , Animals , Body Weight/physiology , Eating/drug effects , Eating/physiology , Humans , Injections, Intraventricular , Insulin/blood , Leptin/blood , Male , Motor Activity/drug effects , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/physiology , Obesity/drug therapy , Obesity/etiology , Obesity/physiopathology , Rats , Rats, Wistar , Recombinant Proteins/administration & dosage , Relaxin/administration & dosage , Relaxin/antagonists & inhibitors , Relaxin/physiology , Signal Transduction/drug effectsABSTRACT
ErbB2 and alpha6 integrin have been implicated in malignancy of breast cancer cells. Here we have determined the influence of alpha6beta1 integrin on erbB2 signaling in anchorage-independent growth, using MDA-MB435 breast cancer cells. Firstly, we transfected the cells with erbB2 cDNA, and isolated cells with high or low levels of alpha6beta1 integrin by cell sorting (alpha6H-ErbB and alpha6L-ErbB). We found that an erbB ligand, heregulin beta1, enhanced growth activity of alpha6L-ErbB cells, but not alpha6H-ErbB cells. Secondly, we established cells expressing a beta4 integrin deletion mutant (beta4-deltacyt), which selectively inhibited alpha6beta1 integrin expression and adhesion to laminin-1. Again, heregulin beta1 enhanced the growth of erbB2 cDNA-transfected beta4-deltacyt cells, but not mock cells. Western blot analysis revealed that heregulin beta1 stimulated phosphorylation of Akt and its downstream molecules, GSK3beta and p70S6kinase, and that the extent of phosphorylation was greater in ErbB2/beta4-deltacyt cells than ErbB2/mock cells. Furthermore, we found that the erbB2 cytoplasmic domain was truncated in ErbB2/mock cells, which was independent of ligand stimulation and adhesion, and was suppressed by proteasome inhibitors. These results suggest that alpha6beta1 integrin inhibits erbB2 signals by inducing proteasome-dependent proteolytic cleavage of the erbB2 cytoplasmic domain, and may thereby contribute to the regulation of tumor growth.
Subject(s)
Breast Neoplasms/metabolism , Cysteine Endopeptidases/metabolism , Integrin alpha6beta1/metabolism , Multienzyme Complexes/metabolism , Receptor, ErbB-2/metabolism , Breast Neoplasms/genetics , Cysteine Endopeptidases/genetics , Female , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Integrin alpha6beta1/genetics , Multienzyme Complexes/genetics , Mutation , Neuregulin-1/metabolism , Proteasome Endopeptidase Complex , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/genetics , Signal Transduction/physiology , Transfection , Tumor Cells, CulturedABSTRACT
The HNK-1 carbohydrate epitope is expressed on a series of cell adhesion molecules and some glycolipids in the nervous system. Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of the HNK-1 carbohydrate epitope. In this study, we isolated cDNA and genomic clones encoding the mouse glucuronyltransferase-S involved in the biosynthesis of the HNK-1 carbohydrate epitope and determined the structural organization of the gene. The deduced amino acid sequence of mouse GlcAT-S consists of 324 amino acids and has a type II membrane topology. The predicted amino acid sequence of mouse GlcAT-S is 98.1% identical to that of rat GlcAT-S. Northern blot analysis revealed that the mouse GlcAT-S transcript is specifically expressed in the nervous system. Moreover, the mouse GlcAT-S gene is composed of four exons spanning over more than 25 kilobase pairs. Southern blot analysis and chromosomal mapping indicated that the mouse GlcAT-S gene is a single copy gene and it was mapped to the A4-B region of mouse chromosome 1.
