Vast majority of excitatory neurotransmission in mammalian central nervous system (CNS) is mediated by glutamatergic synapses. Synaptic plasticity is the mechanism for learning and memory. Drosophila neuromuscular junctions (NMJs) share many similarities with mammalian CNS excitatory synapses. Glutamate receptors (GluRs) in Drosophila neuromuscular synapses are conserved with mammalian non-NMDA receptors. Thus, it’s a suitable model for studying regulation of GluR expression. There are different types and subtypes of GluRs at synapses. So far, we know little about regulation of different GluR subtypes and its influence on synaptic plasticity. In this thesis, I studied the mechanism by which different GluR subtypes interact using Drosophila NMJ as a model. There are two GluR subtypes in the Drosophila NMJs, subtype A and B. By genetic analyses, I found homeostatic regulation of different GluR subtypes, i.e., upregulation or downregulation of one GluR subtype at NMJs can induce the opposite change of the other. However, the total synaptic GluR level doesn’t change. Further mRNA and total protein expression level analyses revealed that this homeostatic regulation happens at post-transcriptional level, and widely happens in different types of NMJ synapses. Synaptic efficacy is affected by both presynaptic neurotransmission and postsynaptic GluRs activity. By mutant analyses, I discovered that GluR subtype A increases but subtype B is stable no matter enhancement or inhibition of presynaptic neurotransmission. Through mutant and pharmacological analyses, I found synaptic GluR subtype A increases while subtype B reduces when activity of subtype A is inhibited. To further understand the regulation mechanism for this homeostatic regulation, I performed a genetic screen and found postsynaptic downregulation of dunce specifically induces a synaptic increase of GluR subtype A and a concomitant reduction of subtype B. dunce downregulates cAMP level. I further found that upre
GPCRs (G-protein coupled receptor) are widely expressed in organism as a signal transducing method. They carry over 80% of signal transduction works in cell to cell communication. The signal carried by extracellular compound is transduced into cytoplasm by the conformational change of transmembrane domain of GPCR and activate different cell activation by G protein or other related proteins to generate, improve or eliminate neural signals, transcription initiation or immune response. This extensive receptor family includes more than 800 members whom are all involved into critical physiology or pathology process, for example, Parkinson, Heart disease, Hypertension, Immune disorder, Cancer, Mental disease (Rask-Andersen et al., 2014). From 2011 to 2015, there are 33% of drugs listed on FDA approval are targeted on GPCRs (Santos et al., 2017).The Epinephrine is one kind of neurotransmitter secreted by adrenal glands. Epinephrine play an important role in fight or flight reaction by increasing blood flow to muscles, output of the heart, pupil dilation, and blood sugar. The signal of Epinephrine is transduced by G protein coupled adrenergic receptor. The adrenergic family can be divided into two sub-family: alpha and beta. Alpha family has six members: α1A, α1B, α1D (Gq coupled) and α2A, α2B, α2C (Gi coupled (Qin et al., 2008) ) and Beta family has three members: β1, β2 and β3. All beta members are coupled with Gs protein (Chen-Izu et al., 2000). Alpha 2A adrenergic receptor located at presynaptic membrane is coupled with Gi/o protein (B. Kobilka, 1991) and produce negative feedback mediation. Alpha 2A adrenergic receptor inhibit transmitter release after binding with norepinephrine secreted from presynaptic membrane.Becauseα2 family receptor is highly related to regulation of human blood pressure(Ruffolo et al., 1993), the whole family are selected as hypertension treatment target by many Pharmaceutical companies. One medicine Clonidine targeted at α2A recepto
G protein-coupled receptors (GPCRs) compose a large family of membrane receptors and are considered as the largest family of cell-surface receptors. As the mediated moleculars, GPCRs transduct extracellular signals into cells via binding of G protein. GPCRs are involved in various important physiological activities, including sense of sight, taste and smell, regulation of emotion, control of behavior, regulation of nervous system, endocrine system and immune system. Therefore, GPCRs have been widely used in pharmaceutical industry as the target molecular for drugs. Most of the GPCRs belong to the Rhodopsin family (Class A) in living organisms. In the Rhodopsin family, biogenic amine receptors, such as octopamine (OA) receptor, tyramine (TA) receptor, dopamine (DA) receptor, epinephrine receptor and serotonin receptor, are involved in various important vital movements in both vertebrates and invertebrates. Thus, biogenic amine receptors have gained considerable attention from many scientists. Biogenic amine receptors can bind with biogenic amines. They exist in plants and animal, especially in many kinds of food. The molecular weight of biogenic amine is usually not very high. As the putative neurotransmitter, biogenic amines are involved in various important physiological activities, including growth, maturation, and reproduction. OA and its precursor TA are common biogenic amines in invertebrates. They are involved in numerous developmental process of invertebrates such as reproduction, maturation and metamorphosis, and play very important role in these vital process. Although OA was first discovered in octopus (mollusks), followed studies on OA, TA and related receptors have primarily been conducted in Ecdysozoa, especially in insects. While in mollusks such as bivalve molluscs, only limited reports are available and their physiological roles remain unclear. In this dissertation, we mainly conducted the physiological functional characterization of OA/TA during
Neuropeptide Y receptor type 1 (NPY1R), which belongs to G protein-coupled receptors(GPCR), is stimulated by neuropeptide Y. NPY1R has a broad distribution in human central nervous system and peripheral nervous system, and regulates many important physiological functions, such as food intake, memory, anxiety regulation, addiction, blood pressure regulation, sleep regulation, inflammation reaction, tissue growth, and energy metabolism. NPY1R has been studied as an important drug target, but there is no marketed drugs targeting the receptor yet. Structural information of NPY1R will deepen our understanding about the biological functions of this receptor and facilitates drug discovery of NPY1R. Many different types of ligands, such as small molecular antagonist, peptide antagonist and peptide agonists, have been tried to be co-crystallized with NPY1R. The crystal structure of the receptor-ligand complexes will provides insights into the mechanisms of NPY1R ligand recognition. We have successfully solved the crystal structures of NPY1R bound to two small molecular antagonists at 2.7 ? and 3.4 ? resolution. These structures reveal a ligand-binding pocket within the seven transmembrane helical bundle. The two antagonists strongly interact with the key residues in the NPY1R ligand-binding pocket, laying a foundation for NPY1R drug discovery.
G protein-coupled receptors (GPCRs) comprise the largest membrane protein superfamily, including more than 800 members. GPCRs are expressed in many tissues and play critical roles in cell signaling transduction. They are related to many diseases and considered as drug targets. Currently more than 40% of marketed drugs target on GPCRs. Class B GPCRs contain an extracellular domain (ECD) and seven-transmembrane domain (TMD), both of which are required to recognize their endogenous peptide ligands. Due to the lack of the full-length class B GPCR structure, the binding mode and activation mechanisms of class B GPCRs remain unknown. Glucagon receptor, belonging to class B GPCR family, is mainly expressed in the liver and kidney. Its natural ligand glucagon, a 29 amino acids peptide hormone, is produced by pancreas β cells. It increases the concentration of glucose by activating GCGR, including promoting gluconeogenesis and glycogenolysis. Inhibiting GCGR activation decreases blood glucose level, so GCGR has been considered as a potential drug target for type 2 diabetes. Determinataion of the full-length GCGR structure provides insights into signaling modulation and peptide recognition mechanisms of class B GPCRs, and lays a foundation for the drug discovery of diabetes. We solved the structure of the full-length GCGR in complex with an inhibitory antibody (mAb1) and an allosteric modulator (NNC0640), and another structure of GCGR bound to a glucagon analogue (NNC1702) at 3.0 ? resolution. In the mAb1-bound GCGR structure, the stalk region that connects ECD and TMD plays an important role in receptor activation and modulates peptide ligand binding. The peptide-bound structure reveals that the stalk region undergoes a conformational change upon peptide ligand binding. Its secondary structure changes from a β-strand to an α-helical. The stalk conformational change triggers the conformational rearrangement of the ECD relative to the TMD, leading to the close contacts be
Opioid drugs play an important role in clinical analgesia by targeting opioid receptors. Opioid receptors belong to the superfamily of seven transmembrane-spanning G protein-coupled receptors (GPCRs) which contain μ-opioid receptor (MOR), the major target of opioids extensively used in clinical. Although multiple clinical opioid drugs share the same target, they perform different drug potencies and properties. Repeated administration of opioids causes drug tolerance in patients. However, the regulatory mechanisms of distinct opioid drug effects and the basic cellular mechanisms of drug tolerance remain to be fully investigated.Previous studies reported that δ-opioid receptor (DOR), another type of opioid receptor, could interact with MOR and form MOR/DOR heteromer which underwent endocytosis together. When treated with DOR specific agonist Deltorphin I, MORs and DORs were co-internalized and processed in the degradation pathway leading to receptor down-regulation. While after treatment with MOR specific agonist DAMGO, MORs and DORs were also co-internalized, but not processed for further degradation. Moreover, both deletion of DOR and disruption of the interaction between MOR and DOR could lead to the reduction of morphine tolerance. Therefore, DOR is involved in the development of MOR-mediated morphine tolerance.Here we firstly studied the effects on the internalization of MOR/DOR heteromers induced by MOR-targeting specific opioid drugs, including non-clinical opioids: DAMGO, DALDA, endomorphin 1, endomorphin 2, and clinical opioids: morphine, fentanyl, methadone and tramadol. We transfected human embryonic kidney (HEK) 293 cells or cultured primary sensory neurons with plasmids expressing tagged MORs and DORs, then treated cells with different opioid drugs. In both cell types, MOR specific opioids caused surface MOR internalization with DOR, the extent of which was positively correlated to the drug concentration. Most applied opioid drugs could induce the co-in
G protein-coupled receptor (GPCR) is the largest receptor superfamily in human and relates to lots of human diseases. More than 40% of marketed drugs target on GPCR, indicating its great research value. Chemokine receptor CCR5 belongs to the GPCR superfamily and mainly expresses on the surface of leukocytes. CCR5 is involved in many biological functions, including the cell migration and proliferation of T cells, monocytes/macrophages as well as inflammation and immune responses. CCR5 also plays an important role in the process of type I human immunodeficiency virus (HIV-1) infection, leading to the acquired immunodeficiency syndrome (AIDs). The viral envelope glycoproteins gp120 and gp41 bind to host cell surface glycoprotein receptor CD4 and a co-receptor during HIV-1 infection, leading to the membrane fusion of the virus and host cell. Chemokine receptors CCR5 and CXCR4 are two main co-receptors on host cell, facilitating different strains of HIV-1 entry. The crystal structures of CXCR4 in complex with two different antagonists have been determined in 2010. These structures provide fundamental structural information for molecular mechanism of HIV-1 invasion. Structural studies of CCR5 can provide new insights into the interaction mode between HIV-1 with human cells as well as structural basis for development of new anti-HIV-1 drugs targeting on CCR5. We performed structural studies on CCR5 complexes with small molecular ligands and protein ligands, respectively. With extensive construct screening, protein expression, purification and crystallization condition optimization, the crystal structure of CCR5 bound to an anti-HIV-1 drug, maraviroc, was solved at 2.7 ? resolution. The structure reveals the allosteric modulation mechanism of maraviroc, and provides clues about anti-HIV-1 drug design. The related data was published in Science in 2013. We further carried out structural studies of CCR5 in complex with virus protein gp120 and host receptor CD4. We got stable C
Mineralocorticoid receptor (MR) and its ligand Aldosterone play a very important role in cardiovascular system. Clinical trials have demonstrated that MR antagonists effectively decreased the morbidity and mortality among patients with heart failure. However, the usage of MR antagonists, spironolactone and eplerenone, remain suboptimal at least partially because of their side effects. Delineating the underlying mechanisms may facilitate better usage of spironolactone and eplerenone and help to develop new generation of MR antagonists.Chronic inflammation is an important feature of heart failure and hypertrophic remodeling. We assumed that MR in immune cells may promote cardiac hypertrophy and dysfunction. Therefore, we respectively generated myeloid-specific and T cell-specific MR knockout (MMRKO and TMRKO) mice, and subjected them to murine model of cardiac hypertrophy.We found that MMRKO attenuated pressure overload (POL)-induced cardiac hypertrophy and remodeling. In mechanism, we found MR mediated inflammatory response in Macrophages. MR directly regulated the expression of Sgk1, which affects transcriptional factors AP-1 and NF-κB in macrophages. Similarly, we found that TMRKO could also inhibit POL-induced cardiac hypertrophy and remodeling. We also determined that MR directly regulated T cell activation, which had an effect on cardiac inflammation.Our studies implied that MR blockade in myeloid and T cells is a feasible approach to treat pathological cardiac hypertrophy and heart failure, which may effectively circumvent the side effects of systemic MR blockade. Our studies further demonstrated that modifying cardiac inflammation is a promising method for heart failure treatment.
G- protein coupled receptors (GPCRs) consitute a large membrane protein superfamily. The receptors of this family all share a conserved 7 transmembrane (TM) α helices fold, as such they are also called 7TMs. They are involved in numerous signal transduction pathways in the cell, leading to a series of physiological and pathological reactions. Many diseases are related to the abnormal expression or regulation of GPCRs, accounting for targets for about 30% of drugs on the market. According to sequence and structure similarity all GPCRs can be divided into 5 family-Rhodopsin (Class A), Secretin /Adhesion (Class B), Glutamate (Class C), Frizzled/Taste2 (Class F). Frizzled7 receptor, belonging to the Frizzled family or Class F GPCRs, is an important component of the Wnt/β-catenin signaling pathway. Frizzled7 gene is an oncogene, the high expression of Frizzled7 can lead to abnormal activation of Wnt/β-catenin signaling pathway and lead to the occurrence of cancer. Frizzled7 is abnormally expressed in many different cancer tissues and is a potential target for cancer treatment. Injection of Frizzled7 antagnoist antibodies can inhibit tumor growth, sheding light on the promising role of Frizzled7-targeted cancer therapy. Further exploration of Frizzled7 signaling axis requires structural and functional understanding of this noncanonical GPCR. One of the major challenges for Frizzled7 related studies is the lack of tool ligand or compounds that could serve as molecular probe. Furthermore, Frizzled7 protein like many other GPCRs, is very unstable without ligand bound. Therefore, it is of high priority to screen Frizzled7 ligands to stabilize the receptor and facilitate its structural investigation.Apelin receptor is widely distributed in the cardiovascular system, brain, and other tissues. It plays an important regulatory role in human cardiovascular function. When activated apelin receptor can enhance myocardial contractility, reduce blood pressure and is a promising tar
Olfactory and gustatory system plays a crucial role in the survival and reproduction process of insects. Insects through its sensitive chemical receptors identify the various chemicals to complete foraging, mating and ovipositing site selection and other processes. Insect gustatory system tells non-volatile nutrients and toxic substances in the environment. Gustatory sensory neurons (GSNs) are widely distributed in the insect periphery sensilla. GSNs stimulated by sugar, amino acids, and deterrent compounds can cause different behavioral responses. Gustatory receptors (Grs).The cotton bollworm, Helicoverpa armigera (Hubner) and the Oriental tobacco budworm, Helicoverpa assulta (Guenée) are two sympatric species, but their host ranges are quite different. H. armigera is a typical polyphagous species, while H. assulta is a oligophagous species. In order to reveal differences in host-plant selection of the two species and find the new ways for controlling cotton bollworm and tobacco budworm in the crop field, we employ a comparative study to identify the Grs of the two species and analyze the function of these Grs.Grs have been identified in Drosophila and other model insects. The nucleotide similarity of the Grs with the same function in general is less than 33% in the insect species. For the common gene sequence matrix of Grs is short, it is very difficult to clone the Grs by using degenerate primers. In this study, we first took an approach of transcriptome to obtain the putative Grs gustatory organs. Secondly, we obtained the full-length coding sequence through the rapid-amplification of cDNA ends technology (RACE) if it is necessary. Thirdly, we identified the ligand of the putative Grs by the heterologous expression system, Xenopus oocyte. Finally, we located the expression of the receptors by in situ hybridization (FISH). The results communication system evolution of possible molecular mechanism. The result have a complementary role for models organism. In the