ABSTRACT
Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.
Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Receptors, Metabotropic Glutamate/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Heterotrimeric GTP-Binding Proteins/chemistry , Humans , Models, Molecular , Protein Multimerization , Receptors, Metabotropic Glutamate/chemistryABSTRACT
Allosteric modulators (AMs) are a promising avenue towards safe and selective drugs. AMs can interact selectively with unique domains distinct from the endogenous ligand binding site of receptors, up- or downregulating the response to receptor activation. Emphasis is placed in this article on the latest development in high-sensitivity technologies designed to identify AMs of G-protein coupled receptors. In addition to new pharmacological approaches, encouraging results in the crystal resolution of these targets enable use of more rational approaches to identification and optimization of AMs.
Subject(s)
Drug Discovery , Receptors, G-Protein-Coupled/metabolism , Allosteric Regulation , Animals , Receptors, G-Protein-Coupled/chemistryABSTRACT
Activation of G-protein-coupled receptors (GPCRs) results in a variety of cellular responses, such as binding to the same receptor of different ligands that activate distinct downstream cascades. Additional signaling complexity is achieved when two or more receptors are integrated into one signaling unit. Lateral receptor interactions can allosterically modulate the receptor response to a ligand, which creates a mechanism for tissue-specific fine tuning, depending on the cellular receptor coexpression pattern. GPCR homomers or heteromers have been explored widely for GPCR classes A and C but to lesser extent for class B. In the present study, we used bioluminescence resonance energy transfer (BRET) techniques, calcium flux measurements, and microscopy to study receptor interactions within the glucagon receptor family. We found basal BRET interactions for some of the receptor combinations tested that decreased upon ligand binding. A BRET increase was observed exclusively for the gastric inhibitory peptide (GIP) receptor and the glucagon-like peptide 1 (GLP-1) receptor upon binding of GLP-1 that could be reversed with GIP addition. The interactions of GLP-1 receptor and GIP receptor were characterized with BRET donor saturation studies, shift experiments, and tests of glucagon-like ligands. The heteromer displayed specific pharmacological characteristics with respect to GLP-1-induced ß-arrestin recruitment and calcium flux, which suggests a form of allosteric regulation between the receptors. This study provides the first example of ligand-induced heteromer formation in GPCR class B. In the body, the receptors are functionally related and coexpressed in the same cells. The physiological evidence for this heteromerization remains to be determined.
Subject(s)
Glucagon-Like Peptide 1/pharmacology , Receptors, G-Protein-Coupled/metabolism , Receptors, Glucagon/metabolism , Allosteric Regulation , Amino Acid Sequence , Cell Line , Endocytosis , Energy Transfer , Glucagon-Like Peptide 1/chemistry , Humans , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
OBJECTIVES: Despite a decreased mortality from sepsis, the absolute number of sepsis-related deaths has actually increased during the last years. At present, there are no biological markers available that can reliably assist early clinical diagnosis and the prompt initiation of therapy. This study investigated the changes in serum protein expression in a coecal ligature and puncture model of rat sepsis at 12, 24, and 48 hours after the induction of sepsis using differential proteomics. METHODS: Sixty-two male Wistar rats were randomly assigned to a sepsis group (coecal ligature and puncture; n = 46) or a sham group (n = 16). Surviving rats were killed 12 hour (n = 6), 24 hour (n = 9), or 48 hour (n = 4) after operation, and their serum lysates were subjected to two-dimensional gel electrophoresis and peptide mass fingerprinting. A systematic functional network mapping and molecular pathway analysis were performed using Ingenuity Pathways Analysis. RESULTS: Septic mortality was 58.7%, but no rat of the sham group was lost. Per gel, an average of 1,082 +/- 10 spots could be discriminated, of which 40 different protein spots were differentially expressed (p < 0.01). From the total of 40, the number of regulated protein spots was 13 (12 hour group) versus 10 (24 hour group) versus 18 (48 hour group). Ingenuity pathways analysis identified 10 of the differential proteins and allocated them to a pathway of tissue inflammation. CONCLUSIONS: The present study quantitatively detected several proteins differentially expressed in acute sepsis. Since a longer time-period was investigated and compared with previous studies, the results may offer new insights into septic organ dysfunction and altered protein pathways. The horizontal analysis of protein expression arrays and systematic biochemical pathways may represent an important new tool for the clinical assessment of septic conditions and support the development of early sepsis markers.
Subject(s)
Biomarkers/analysis , Metabolome/physiology , Proteome/analysis , Sepsis/diagnosis , Acute-Phase Proteins/genetics , Animals , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Image Processing, Computer-Assisted , Male , Metabolome/genetics , Peptide Mapping , Proteome/genetics , Rats , Rats, Wistar , Sepsis/bloodABSTRACT
Hemoglobin is the major protein in red blood cells and transports oxygen from the lungs to oxygen-demanding tissues, like the brain. Mechanisms that facilitate the uptake of oxygen in the vertebrate brain are unknown. In invertebrates, neuronal hemoglobin serves as intracellular storage molecule for oxygen. Here, we show by immunohistochemistry that hemoglobin is specifically expressed in neurons of the cortex, hippocampus, and cerebellum of the rodent brain, but not in astrocytes and oligodendrocytes. The neuronal hemoglobin distribution is distinct from the neuroglobin expression pattern on both cellular and subcellular levels. Probing for low oxygen levels in the tissue, we provide evidence that hemoglobin alpha-positive cells in direct neighborhood with hemoglobin alpha-negative cells display a better oxygenation than their neighbors and can be sharply distinguished from those. Neuronal hemoglobin expression is upregulated by injection or transgenic overexpression of erythropoietin and is accompanied by enhanced brain oxygenation under physiologic and hypoxic conditions. Thus we provide a novel mechanism for the neuroprotective actions of erythropoietin under ischemic-hypoxic conditions. We propose that neuronal hemoglobin expression is connected to facilitated oxygen uptake in neurons, and hemoglobin might serve as oxygen capacitator molecule.
Subject(s)
Cerebellum/metabolism , Cerebral Cortex/metabolism , Hemoglobins/biosynthesis , Hippocampus/metabolism , Neurons/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Cell Hypoxia , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Electrophoresis, Gel, Two-Dimensional , Erythropoietin/genetics , Erythropoietin/pharmacology , Female , Hippocampus/cytology , Hippocampus/drug effects , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/drug effects , Nitroimidazoles/pharmacology , Oxygen/metabolism , Rats , Rats, Wistar , Recombinant Proteins , Reverse Transcriptase Polymerase Chain Reaction , Species SpecificityABSTRACT
Hypoxic-ischemic damage is a major challenge for neuronal tissue. In the present study, we investigated the effects of anoxia and glucose deprivation on adult neural stem cells (NSCs) in vitro. We assessed glucose deprivation, anoxia and the combination of the latter separately. After 24 h of anoxia, cell numbers increased up to 60% compared to normoxic controls. Whereas nearly all normoxic cells incorporated the mitotic marker BrdU (99%), only 85% of the anoxic cells were BrdU-positive. Counting of interphase chromosomes showed 8-fold higher cell division activity after anoxia. The number of necrotic cells doubled after anoxia (14% compared to 7% after normoxia). Apoptosis was measured by two distinct caspases assays. Whereas the total caspase activity was reduced after anoxia, caspase 3/7 showed no alterations. Glucose deprivation and oxygen glucose deprivation both reduced cell viability by 56 and 53%, respectively. Under these conditions, total caspases activity doubled, but caspase 3/7 activity remained unchanged. Erythropoietin, which was reported as neuroprotective, did not increase cell viability in normoxia, but moderately under oxygen glucose deprivation by up to 6%. Erythropoietin reduced total caspase activity by nearly 30% under all the conditions, whereas caspase 3/7 activity was not affected. Our results show that anoxia increases proliferation and viability of adult NSCs, although a fraction of NSCs does not divide during anoxia. In conclusion, anoxia increased cell viability, cell number and proliferation in NSCs from the rat brain. Anoxia turned out to be a highly stimulating environmental for NSCs and NSCs died only when deprived of glucose. We conclude that the availability of glucose but not of oxygen is a crucial factor for NSC survival, regulating apoptotic pathways via caspases activity other than the caspases 3/7 pathway. Therefore, we conclude that NSCs are dying from glucose deprivation, not from hypoxic-ischemic damage.
Subject(s)
Cell Hypoxia/physiology , Hypoxia-Ischemia, Brain/metabolism , Neuronal Plasticity/physiology , Neurons/metabolism , Recovery of Function/physiology , Stem Cells/metabolism , Animals , Apoptosis/physiology , Bromodeoxyuridine , Caspases/metabolism , Cell Count , Cell Differentiation/physiology , Cell Proliferation , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Erythropoietin/pharmacology , Glucose/deficiency , Male , Mitosis/physiology , Necrosis/etiology , Necrosis/metabolism , Neurons/cytology , Neuroprotective Agents/pharmacology , Rats , Rats, Wistar , Signal Transduction/physiology , Stem Cells/cytologyABSTRACT
The cause of brain dysfunction during sepsis and septic encephalopathy is still under ongoing research. Sepsis induced changes in cerebral protein expression may play a significant role in the understanding of septic encephalopathy. The aim of the present study was to explore cerebral proteome alterations in septic rats. Fifty-six male Wistar rats were randomly assigned to a sepsis group (coecal ligature and puncture, CLP) or a control group (sham). Surviving rats were killed 24 or 48 hours after surgery and whole-brain lysates were used for two-dimensional gel electrophoresis and subsequent protein identification. Differentially expressed proteins were identified by mass spectrometry. Using the Ingenuity Pathways Analysis (IPA) tool, the relationship and interaction between the identified proteins was analyzed. Mortality was 53 % in septic rats. No rat of the control group was lost. More than 1,100 spots per gel were discriminated of which 29 different proteins were significantly (2-fold, P<0.01) changed: 24 proteins down-regulated after 24 hours; two proteins up-regulated and three down-regulated after 48 hours. IPA identified 11 of 35 differentially regulated proteins allocating them to an existing inflammatory pathway. In the analysis of septic rat brains, multiple differentially expressed proteins associated with metabolism, signaling, and cell stress can be identified via proteome analysis, that may help to understand the development of septic encephalopathy.
Subject(s)
Brain Chemistry/physiology , Energy Metabolism/physiology , Nerve Tissue Proteins/biosynthesis , Sepsis/metabolism , Animals , Cecum/pathology , Disease Progression , Electrophoresis, Gel, Two-Dimensional , Escherichia coli/metabolism , Image Processing, Computer-Assisted , Male , Neural Networks, Computer , Proteomics , Rats , Rats, Wistar , Sepsis/microbiology , Sepsis/mortality , Signal Transduction/physiologyABSTRACT
On the basis of its inhibition by SB216763, we identified the multifunctional enzyme Glycogen Synthase Kinase 3beta (GSK3beta) as a central regulator for differentiation and cell survival of adult neural stem cells. Detected by proteomic approaches, members of the Wnt/beta-catenin signaling pathway appear to participate in enhanced neuronal differentiation and activated transcription of beta-catenin target genes during GSK3beta inhibition, associated with decreased apoptosis.
Subject(s)
Cell Differentiation , Cell Proliferation , Cerebral Ventricles/cytology , Glycogen Synthase Kinase 3/physiology , Neurons/cytology , Stem Cells/cytology , Animals , Apoptosis , Electrophoresis, Gel, Two-Dimensional , Glycogen Synthase Kinase 3 beta , Neurons/enzymology , Proteomics/methods , Rats , Stem Cells/enzymology , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
In the brain, glucose is transported by GLUT1 across the blood-brain barrier and into astrocytes, and by GLUT3 into neurons. In the present study, the expression of GLUT1 and GLUT3 mRNA and protein was determined in adult neural stem cells cultured from the subventricular zone of rats. Both mRNAs and proteins were coexpressed, GLUT1 protein being 5-fold higher than GLUT3. Stress induced by hypoxia and/or hyperglycemia increased the expression of GLUT1 and GLUT3 mRNA and of GLUT3 protein. It is concluded that adult neural stem cells can transport glucose by GLUT1 and GLUT3 and can regulate their glucose transporter densities.