| NANOPORE BIOSENSOR FOR KINETICS OF REPARATIVE ANTIBODIES ON PLASMA MEMBRANES |
OH, SANG-HYUN |
UNIVERSITY OF MINNESOTA TWIN CITIES |
$414,019 |
| The capability to perform in vitro, label-free dynamic assays for membrane-bound antigens is a highly desired task, but is rarely achieved using standard commercialized technology such as BIAcoreTM. The problem is compounded for transmembrane proteins such as G protein coupled receptors (GPCR) because proteins in direct contact with a solid substrate, in particular with the gold substrate used in BIAcoreTM, often lose their functionality or denature. The nanopore- sensing architecture proposed here has the unique potential to overcome these challenges, since each nanopore sits on a glass substrate and forms a tiny well to confine the supported lipid membranes, while the surrounding gold film provides surface plasmon resonance effects to dynamically monitor binding of molecules onto the membrane. This proposal will validate these membrane biosensing concepts by characterizing the binding of therapeutic human monoclonal antibodies to candidate antigens. These human IgMs promote remyelination of demyelinated lesions and preserve axons. These are ideal molecules in which to test this system because the IgM antigen binding appears to require an intact membrane environment. A major challenge in moving these reparative IgMs to clinical trial is to understand the kinetics of binding to the cell-surface antigens. Our hypothesis and preliminary data suggests that the mAbs do not bind to a single membrane molecule, but to a signaling complex within lipid micro-domains (lipid rafts) of cells. If this complex is disrupted, mAb binding is eliminated. The IgMs maintain their cell specificity only when bound to intact plasma membranes. Fixation of any kind (methanol, formaldehyde, freezing) destroys the complex membrane antigen. When candidate antigens are presented in isolated form, the IgMs bind non-specifically to all or to none. Therefore, it is important to maintain the cell membrane antigens in their native state to preserve appropriate mAb binding kinetics. A new antigen screening technology is required to study these difficult but critical lipid and carbohydrate molecules of the plasma membrane. Unfortunately, there are no label-free kinetic screening and quantification methods to measure the binding affinity between cell plasma membranes and mAbs. The commercial BIAcore' instrument - currently the gold standard for measuring binding kinetics - works with purified molecules, primarily proteins, immobilized on a gold film substrate. However, this instrument is not suitable for quantification of interactions between mAbs and cell-surface antigens in their native membrane inserted state. We propose here to use a novel instrument, a nano-LAMP (LAser-illuminated Metallic Pore) array, to quantify the binding kinetics of mAbs to antigens anchored within a cell membrane at a high spatial resolution. We have validated this platform with membrane-free systems and with artificial membranes for binding kinetics measurements. The work proposed here will further optimize the platform by reconstituting oligodendrocytes and neuronal cell membranes on metallic nanopores to measure and quantify their binding affinity with human therapeutic IgMs, to identify candidate antigens. Once developed, this technology will likely prove important in the study of complex molecular interactions and signals transduced by cell receptors. As a future direction, we also propose the possibility of reconstituting free-standing lipid membranes hanging over a free-standing metallic nanopore substrate, incorporating transmembrane proteins such as GPCRs, and demonstrating the feasibility of kinetic sensing with an artificial membrane system that can integrate transmembrane proteins in contact with a buffer solution on both sides. PUBLIC HEALTH RELEVANCE: This proposal is designed to determine whether a nanoporous gold film detection platform can present cell antigens in manner that preserves the correct plasma membrane functionality similar to that of an intact cell. Standard binding assay Surface Plasmon Resonance (SPR) detectors such as BIAcore work well using only a single purified molecule, but cannot model the presentation of multiple molecules within a membrane. We propose to validate the nanopore SPR platform by measuring the interaction of human IgMs with membranes isolated from myelin, oligodendrocytes and neurons. These reparative IgMs clearly bind to cells of the nervous system, but attempts to identify their membrane antigens using conventional binding technology have been inconclusive. If successful, a nanopore based binding detector will be applicable to a wide variety of ligand/antigen binding studies for basic biology and drug discovery. |
| LUNG HRV: G-PROTEIN COUPLED SIGNALING INTERACTIONS IN ASTHMA |
LIGGETT, STEPHEN B |
UNIVERSITY OF MARYLAND BALTIMORE |
$401,500 |
| Human rhinovirus (HRV) infection causes at least 50% of asthma exacerbations. Airway epithelial cell (AEC) infection evokes the release of inflammatory, growth and bronchospastic factors, and other autocrine/paracrine mediators leading to generalized AEC and airway smooth muscle (ASM) 'pro-exacerbation' pathology. This includes altered AEC lining fluid/ion content, ASM hyperreactivity, and ASM resistance to relaxation by (-agonists, which are controlled by G-protein coupled receptors (GPCRs) on these cells. The heterogeneity of these asthmatic responses is, in part, thought to be dependent on the HRV strain (serotype). There are over 100 HRV strains, yet little is known about how genomic differences in strains impact asthma exacerbation phenotypes. We have very recently completed sequencing the genomes of all 99 reference HRV-A and -B serotypes from a banked historical repository. This revealed previously unknown aspects of HRV RNA and protein structure, phylogenetic relationships, recombination, and extensive diversity among the canonical serotypes. It also provided structure-based sequence alignments which are a scaffold for integration of additional HRVs into the phylogenetic tree. The broad long-term objectives of this revised proposal are to ascertain the genomic features of modern HRV strains that contribute to specific asthmatic airway GPCR phenotypes and their heterogeneity. This will be accomplished by three aims. In Aim 1, we will determine the complete genome sequences of HRVs from 200 modern clinical isolates using massively parallel sequencing methods. In Aim 2, this data will be integrated into our structure-based reference genomic scaffold so as to define genomic regions that are similar and dissimilar amongst the strains, providing a rigorous mechanism to select the HRVs for in vitro functional studies. In Aim 3, GPCR signaling phenotypes of HRVs in the context of AEC and ASM will be ascertained in cell culture models using high-throughput methods (Sub Aim 1). And, these signaling phenotypes will be correlated to HRV genome features using Bayesian techniques with internal and external validations (Sub Aim 2). Such studies will provide a genomic basis for those HRVs that do, and do not, evoke AEC and ASM phenotypic traits, and thus establish some of the mechanisms of heterogeneity of viral induced asthma exacerbations. These findings may provide diagnostic and prognostic information, and pharmacologic strategies, for managing the most common cause of asthma exacerbations. PUBLIC HEALTH RELEVANCE: Human rhinovirus (HRV) infection causes about 50% of asthma attacks (and COPD exacerbations). There is, though, substantial variability in the nature and severity of the clinical features of asthma attacks from HRV infections, for reasons that are not known. However, it is now clear that there are many distinct HRV strains, and this proposal will define which HRVs, and which parts of their genomes, impose changes in airway receptor function that contribute to the pathology and symptoms of asthmatic attacks during HRV infection. |
| CHEMINFORMATICS OF ALLOSTERIC MGLUR MODULATION PROMOTES THERAPEUTIC DEVELOPMENT |
MEILER, JENS |
VANDERBILT UNIVERSITY |
$387,385 |
| Selective potentiators of the metabotropic glutamate receptor subtype mGluR5 have exciting potential for development of novel treatment strategies for schizophrenia and other disorders that disrupt cognitive func-tion. The latest generation of selective mGluR5 potentiators is based on the lead compound CDPPB and features systemically active compounds with long half-lives that cross the blood-brain barrier. A high-throughput screen (HTS) for mGluR5 potentiators at Vanderbilt's screening center revealed a large and diverse set of about 1400 substances (1% hit rate) whose activity was validated in independent experiments. A previous exploratory research grant 'Novel Schizophrenia Therapeutics by Virtual High-Throughput Screening' (R21 MH082254) enabled testing of 813 compounds predicted through cheminformatics. 252 of these compounds were confirmed as active PAMs equaling an enrichment of >30 when compared with the original screen. The present proposal seeks to leverage these proof-of-principle results for the development of a tailored cheminformatics framework for drug discovery of allosteric modulators of brain GPCRs, apply these tools to inform an existing therapeutic discovery program of mGluR5 potentiators at Vanderbilt University, and disseminate the methods broadly through the NIH molecular libraries program. The central hypothesis of this proposal is that the complex relationship between chemical structure and biological activity of mGluR5 potentiators observed in this HTS can be used to generate a pharmacophore of the mGluR5 allosteric site. This map of steric and electronic features necessary for optimal interaction of modulators with mGluR5 will not only inform our understanding of the allosteric modulation of brain GPCRs. The methods proposed overcome limitations of present cheminformatics techniques by enabling identification of novel chemotypes through virtual screening (scaffold hoping), and allowing design of focused libraries in hit-to- lead optimization of novel schizophrenia therapeutics. The generalizbility of the approach will be tested through application on negative modulators of mGluR5, a potential novel treatment strategy of fragile X syndrome, a CNS disorder associated with autism spectrum disorders (ASD) among multiple other symptoms. The developed applications will be made freely and readily accessible for academic research. The employed QSAR models require no crystal structure of the target brain GPCR. Hence the method can be readily applied to membrane proteins-such as GPCRs-which are target of 40-50% of modern medicinal drugs. PUBLIC HEALTH RELEVANCE: Ligands for specific mGluR subtypes have potential for treatment of a wide variety of neurological and psychiatric disorders. We will use computational methods identify potentiators of mGluR5, compounds that have exciting potential as treatment strategy for schizophrenia. In silico hit compounds will be experimentally validated and enter hit-to-lead optimization. |
| PHYSIOLOGICAL REGULATION OF MLCK IN INTACT ARTERIES |
WIER, WITHROW GIL |
UNIVERSITY OF MARYLAND BALTIMORE |
$370,000 |
| Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) are the major regulators of cross-bridge cycling and force generation in vascular smooth muscle. The overall goal of the proposed research is to gain new information on the role of these molecules (particularly MLCK) in controlling arterial contraction in normal function, and in a model of salt dependent hypertension (DOCA-salt model, deoxycorticosterone acetate and high dietary NaCl intake). Direct examination of MLCK activity in isolated arteries and in the arteries of living animals (i.e. in vivo) will be achieved through the use of (transgenic) 'biosensor' mice that express an optical (FRET) MLCK activity sensor. Regulation of MLCP in isolated arteries will be studied by quantifying threonine-855 phosphorylation of myosin phosphatase targeting subunit (MYPT1). Initial fluorescence imaging studies in isolated arteries (Aims 1 & 2) will reveal the activation of MLCK and regulation of MLCP in relation to 1) myogenic tone and 2) certain G-protein coupled receptors (GPCR) that are known to be important in hypertension. Myogenic tone (MT) is a key smooth muscle function that is involved in maintenance of arterial pressure, and in the response to tissue over-perfusion in initial stages of salt-induced hypertension. Therefore, Aim 1 is to quantify the dynamic and long-term (hours) activation of MLCK and regulation of MLCP as pressure is changed over the range of 10 to 150 mm Hg in isolated arteries. Aim 2 is to quantify MLCK activation, and MLCP inhibition, accomplished by two key classes of GPCR: 1) those coupled primarily to Gq/11, and 2) those also coupled strongly to G12/13. The latter have been implicated particularly in salt-induced hypertension and may utilize strong inhibition of MLCP, in addition to activation of MLCK. The influence of MT on GPCR induced signaling will also be studied since new data indicates that it affects contractile signaling of GPCR in ways not yet fully appreciated. Aim 3 will build on the knowledge gained in the isolated arteries , but will utilize in vivo imaging (i.e. intravital FRET microscopy) of arteries in anesthetized biosensor animals to quantify the role of MLCK in the increased vasoconstriction that occurs in DOCA-salt hypertension. In this final Aim, two current, competing, hypotheses will be examined: 1) that DOCA-salt hypertension is importantly maintained by circulating factors acting through G12/13 coupled GPCR and therefore involves strong inhibition of MLCP, rather than exclusive activation of MLCK, and 2) that salt-dependent hypertension involves mainly endogenous Na+ pump ligands (natriuretic factors) that contract smooth muscle by increasing [Ca2+] and thus act mainly through MLCK, rather than inhibition of MLCP. Summary: The research is intended to provide a detailed, quantitative, dynamic description of the activation and regulation of MLCK and MLCP in normal and hypertensive arteries in response to physiological stimuli, including transmural pressure and GPCR signaling. It will provide the first direct evidence, from arteries in the living animal, on the role of MLCK in salt-induced hypertension. PUBLIC HEALTH RELEVANCE: The proposed research is intended to provide basic information on the activity and regulation of an enzyme (myosin light chain kinase, MLCK) that is critical to contraction of arteries, both in normal physiology and in high blood pressure (hypertension). Arteries exist in a contracted state in order to maintain blood pressure. The amount of contraction changes rapidly in response to activity of the nervous system and hormones. This research will utilize a mouse model of salt-induced hypertension to provide specific new information on the role of MLCK in high blood pressure. |
| PHARMACEUTICAL THERAPY FOR SEASONAL AFFECTIVE DISORDER |
TRAUTMAN, JAY K |
PHOTOSWITCH BIOSCIENCES, INC. |
$349,999 |
| Seasonal Affective Disorder (SAD) is one of the most common mood disorders, affecting 1-3% of the population in temperate climates, predominantly women. Currently, there are no specific drug therapies for SAD. Patients are prescribed general anti-depressants or bright light therapy. SAD symptoms include low mood, loss of interest, difficulty concentrating, loss of energy and fatigue. In addition, SAD patients tend to have an increased appetite with associated weight gain and carbohydrate cravings, sweets in particular, in the afternoon or evening. There is often an intense daytime drowsiness in spite of increased sleep duration. SAD constitutes an unmet medical need. The prevalence of SAD and the absence of specific, efficacious and safe pharmacotherapy warrant initiation of a project to discover a therapeutic based in the biology the disease. The end goal of the proposed work is the development a pharmaceutical treatment for SAD by targeting the melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGC) found in the mammalian retina. The pathway is biologically validated in that bright light therapy, which stimulates the ipRGCs, is efficacious in patients with SAD, and patients having a particular melanopsin mutation have been found to be ~6x more likely to have SAD. The pathway has at its apex, melanopsin, a G-protein coupled receptor (GPCR) that is only expressed in the target cells. A drug activating melanopsin should be able to entrain and shift the circadian pacemaker in the suprachiasmatic nucleus and thus would be invaluable as a drug to treat SAD, as well as jet lag, insomnia related to shift work, and sleep timing disorders. The proposed Specific Aims are (1) demonstrate luminescence reporter assay readout of activation of human melanopsin in 293T cells; (2) optimize the reporter system in several cell types; (3) identify by microarray analysis other, potentially better, reporters and evaluate their performance in the luminescence assay; and (4) adapt and validate the optimized assay from Aims 1-3 for a high-throughput screen for melanopsin agonists. There is, to our knowledge, no pharmaceutical precedent to this approach. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from a disease called Seasonal Affective Disorder (SAD). People with SAD exhibit classical symptoms of depression during the winter months, associated with the prolonged periods of darkness as are found in higher latitudes. Increasing evidence points to a disruption of the light-dark cycling that is inherent in humans as the primary cause of SAD. We are developing drugs that target the source of the light-dark cycling; cells in the eye that do not contribute to normal vision but detect whether it is night or day. Our therapy should be better than existing treatments, which are non-specific, as we are targeting the primary source of the disease. |
| RADIOLYTIC FOOTPRINTING METHODS FOR STRUCTURAL MASS SPECTROMETRY |
CHANCE, MARK R |
CASE WESTERN RESERVE UNIVERSITY |
$282,600 |
| We have developed innovative methods of structural mass spectrometry based on hydroxyl radical modification of proteins. These structural mass spectrometry methods have recently gained widespread acceptance and are ripe for further development. In this proposal we will: increase the sensitivity of protein footprinting methods ~1000 fold; integrate docking approaches with protein footprinting data to probe the structure of protein complexes and develop methods to examine the dynamics of water in proteins using footprinting. Our preliminary data has shown feasibility for the examination of the G- protein coupled receptor rhodopsin in its ground and photo-activated states using increased x-ray flux density and shorter exposure times. Within Aim 1 we will use increased x-ray flux density to further explore the structural and solvent dynamics accompanying GPCR activation and the structural mechanism of signaling that mediates information to downstream signaling proteins. Our guiding hypothesis is that highly conserved structural motifs that include bound waters are reorganized to provide a highly controlled signaling channel. In Aim 2 we will further develop a novel O18 water labeling- radiolysis technique to examine the locations and dynamics of structural waters and the exchange properties of bulk water in multiple biological states of interest for rhodopsin and actin. In Aim 3 we will perfect targeted MS approaches to detect low abundance modifications in protein footprinting experiments to enhance the number of amino acids side chains routinely detected by these experiments. In Aim 4 we will develop computational methods of docking that incorporate footprinting data in structure determination. Our hypothesis is that use of footprinting data will drive correct selection of the correct structure among competing docking solutions that are of comparable energies. Optimized algorithmic approaches will be used to model complexes of myosin or cofilin with actin and complexes of rhodopsin and transducin. PUBLIC HEALTH RELEVANCE: G-protein coupled receptors are the targets of close to half of all drugs; a better understanding of their activation mechanisms would have a major impact on health and disease. Advanced technologies to examine the structure and dynamics of all proteins are needed in biology and medicine to better correlate structure and function. |
| DEVELOPING METHODS FOR CRYSTALLIZING FAMILY C GPCRS |
FENG, DAN |
CONFOMETRX, INC. |
$250,000 |
| The goal of this proposal is to develop methods to obtain high-resolution crystal structures of Family C G-protein-coupled receptors (GPCRs). These structures would greatly facilitate the development of effective and highly selective allosteric drug molecules for the treatment of psychiatric and neurological disorders. GPCRs are characterized by the presence of seven membrane-spanning 1-helical segments separated by alternating intracellular and extracellular loop regions. GPCRs in vertebrates are commonly divided into five families by sequence and structural similarity, specified as Rhodopsin (Family A), Secretin (Family B), Glutamate (Family C), Adhesion, and Frizzled/Taste2. Family C GPCRs are structurally distinct from Family A receptors. While they share the same 7 TM topology, there is no sequence homology with Family A receptors. Family C receptors have a large extracellular, amino terminal ligand binding site consisting of a bilobed venus flytrap domain. However, it has been possible to modulate the activity of several Family C receptors by small molecule drugs that bind directly to the 7TM bundle and regulate receptor activity allosterically. The 7TM bundle is an ideal drug target because there is less sequence conservation between closely related receptor subtypes than observed for the native hormone binding site located in the Venus flytrap domain. Despite very active academic and industrial research efforts on drug discovery for GPCRs over the past two decades, the number of new GPCR drugs has been disappointing. One of the major bottlenecks in drug development has been the lack of high-resolution structural information on GPCRs for both identifying and optimizing leads. A recent advance in crystallization technology for family A GPCRs was developed in the laboratory of ConfometRx co-founder Brian Kobilka: generating GPCR-T4 Lysozyme (GPCR-T4L) fusion proteins. This technology has been applied to the high-resolution structures of two Family A GPCRs (the 22AR and Adenosine A2a receptor), opening new opportunities for structure-based design of drugs. We propose to adapt this technology to Family C receptors, using the metabotropic glutamate receptors (mGluRs) as a model system. The mGluRs are expressed primarily in the central nervous system and are potential therapeutic targets for the treatment of several neuropsychiatric disorders. In Phase I of this SBIR, we aim to determine the feasibility of using the GPCR-T4L technology to express and purify functional mGluR-T4L fusion proteins. In Phase II, we will proceed with the crystallization trials and structure determination and will use this information to initiate the development of a new class of mGluRs drugs. The methodology developed for generating mGluR-T4L proteins should be readily applicable to other Family C GPCRs. This proposal is in response to the NIH PA-06-375, Novel Tools for Investigating Brain-derived GPCRs in Mental Health Research, as well as Roadmap initiatives on the Structural Biology of Membrane Proteins. PUBLIC HEALTH RELEVANCE: We propose to develop methods to determine the three-dimensional structures of Family C G-protein-coupled receptors (GPCRs), which are known to play essential roles in regulating normal neuronal function in the central nervous system. Their diverse physiologic roles make them viable therapeutic targets for the treatment of several psychiatric and neurological disorders including anxiety disorders, schizophrenia, depression, Parkinson's disease, and Alzheimer's disease among others. High-resolution crystal structures of Family C GPCRs will provide valuable tools for the development of more effective and selective drugs. |
| GPCR ARCHITECTURE |
HERRICK-DAVIS, KATHARINE |
ALBANY MEDICAL COLLEGE |
$237,000 |
| G-protein coupled receptors (GPCR) are present in most if not all cells in the human body and play vital roles in cell communication and survival. They are targets for more than 50% of currently marketed pharmaceuticals. Therefore, significant emphasis has been placed on understanding molecular mechanisms governing GPCR function. GPCR are believed to form dimeric or oligomeric complexes. However, little is known about how dimer/oligomer biogenesis occurs and how it regulates GPCR function. The purpose of this proposal is to address fundamental questions regarding GPCR dimer/oligomer formation using the serotonin 5-HT2C receptor as a model system. The 5-HT2C receptor is widely distributed throughout the brain and is a target for drugs used to treat anxiety, depression, schizophrenia, and obesity. It is the goal of the proposed study to determine if native GPCRs, endogenously expressed in their natural physiological environment, function as monomers, dimers, tetramers or higher order complexes. This will be accomplished using state-of-the-art confocal microscopy combined with fluorescence correlation spectroscopy (FCS) and bimolecular fluorescence complementation that will allow direct visualization of 5-HT2C receptors and provide evidence for homodimerization of native receptors expressed in living primary cell cultures. Native 5-HT2C receptors endogenously expressed in primary choroid plexus epithelial cells and in primary hippocampal neurons will provide the model systems for FCS analysis. By measuring the fluctuations in fluorescence intensity of fluorescent molecules diffusing through a very small volume, FCS allows single molecule detection sensitivity and provides information about the diffusion properties as well as the number of fluorescent molecules traveling together within a protein complex. Fluorescent Fab fragments generated from a monoclonal antibody specific for an extracellular domain of the 5-HT2C receptor will be used to label 5-HT2C receptors in live primary cultures. FCS experiments will be performed to determine if 5-HT2C receptors, in their native environment are expressed as monomers, dimers, tetramers and/or higher order complexes. These studies will begin to elucidate the true physiological/structural characteristics of the 5-HT2C receptor. Additional clues as to the role of dimer/oligomer formation in GPCR function may be provided by FCS studies performed in the absence and presence of serotonin. These studies will be among the first to provide information about the dimeric/oligomeric nature of endogenously expressed GPCR in their intact, native cellular environment. An understanding of the overall GPCR architecture is crucial to understanding GPCR function in normal and pathological conditions and to the development of novel therapeutic agents. PUBLIC HEALTH RELEVANCE: G-protein coupled receptors (GPCR) are present in all cells in the human body, they play vital roles in cell communication and survival, and are targets for more than 50% of currently marketed pharmaceuticals. The studies in this proposal will begin to elucidate how GPCR come together to form higher order complexes (dimers/oligomers) and how this process regulates receptor function. Understanding the mechanisms involved in GPCR activation is crucial to understanding receptor function in normal and pathological conditions and to the development of novel therapeutic agents. |
| STRUCTURAL BASIS FOR CANNABINOID RECEPTOR 2 SIGNALING |
LADIAS, JOHN A.A. |
BETH ISRAEL DEACONESS MEDICAL CENTER |
$224,438 |
| This proposal focuses on the crystal structure determination of the human cannabinoid receptor hCB2, a G protein-coupled receptor (GPCR) that plays a central role in mediating the effects of cannabinoids and endocannabinoids in human physiology and pathology. An accurate structural understanding of the molecular mechanisms underlying GPCR activation and subsequent signal transmission to the cognate G proteins represents a major challenge for modern molecular biology and medicine. The proposed research aims at filling the knowledge gap on the structural basis of GPCR signaling by focusing on the crystallographic analysis of the hCB2 receptor in the active and inactive states. The specific aims are: 1) To determine the crystal structure of the hCB2 receptor in the active state. 2) To elucidate the structural basis for hCB2 receptor inactivation. To increase the stability and crystallizability of the hCB2 receptor, most of its intracellular loop 3 will be replaced with the T4 lysozyme (T4L) and the C-terminal cytoplasmic region of the receptor will be deleted. The optimized hCB2-T4L fusion protein will be expressed in insect cells and purified using affinity, ion exchange, and size exclusion chromatography. The purified receptor will be covalently complexed with a synthetic agonist or antagonist, and will be crystallized in lipidic mesophases. Complete diffraction data sets of the obtained crystals will be collected using synchrotron radiation and the structures will be determined using cutting-edge macromolecular crystallography methods. The successful completion of these studies will have a major impact on biomedical research and human health. The obtained structural knowledge will greatly facilitate the development of hCB2-selective agonists, partial agonists, and antagonists. Because of the central role of the hCB2 receptor in mediating endocannabinoid signaling, new treatments will be developed for a number of medical conditions, including inflammatory and neuropathic pain, hepatic fibrosis, atherosclerosis, and osteoporosis. PUBLIC HEALTH RELEVANCE: The human cannabinoid receptor hCB2 plays a central role in mediating the (endo)cannabinoid signaling in human physiology and pathology. The proposed research focuses on the elucidation of the mechanisms underlying hCB2 function at the atomic level using X-ray crystallography. Accurate knowledge of the three- dimensional structure of hCB2 will advance the research in the (endo)cannabinoid and GPCR fields, and will effectively facilitate the development of innovative therapeutics for serious human diseases, including drugs of abuse and HIV. |
| EFFICIENCY OF ENHANCED SAMPLING METHODS IN GPCR RESEARCH |
FILIZOLA, MARTA |
MOUNT SINAI SCHOOL OF MEDICINE OF NYU |
$209,095 |
| G protein-coupled receptors (GPCRs) are abundant membrane proteins of extreme pharmacological importance since they are the primary targets for about 30% of prescription drugs that are currently on the market, and are likely to be potential targets for new therapeutic agents. Despite a great deal of research activity in the GPCR field, the design of powerful drugs acting at these receptors has lagged behind due to the limited understanding of the ligand-induced conformational changes of these receptors associated with specific physiological functions. For years, conventional drug design at GPCRs has mainly focused on the inhibition of a single receptor at a usually well-defined ligand-binding site. The growing body of evidence that GPCRs form clinically relevant dimers/oligomers with implications in several disorders has recently added a new complexity to the field, making the understanding of the mechanisms and dynamics governing the interaction between receptor pairs and/or higher-order oligomers equally important for successful rational drug design. The wealth of new higher-resolution structural, biochemical, and biophysical information on GPCRs that has appeared in the recent literature, coupled to recent advancements in coarse-grained modeling and enhanced sampling algorithms, suggests new ways to efficiently explore the conformational space of GPCRs (both monomers and dimers/oligomers), and to generate novel testable hypotheses of molecular mechanisms underlying receptor function. In this grant application, we propose to conduct exploratory metadynamics-based computational studies of ideal membrane protein systems, i.e. ¨-adrenergic receptor, glycophorin A, and the transmembrane domain dimer of the amyloid precursor protein, to validate the efficiency of enhanced sampling methods in predicting ligand-specific activated states and/or dimerization-disrupting mutants that agree with experimental data. PUBLIC HEALTH RELEVANCE: The discovery of powerful therapeutic drugs acting at G-protein coupled receptors (GPCRs), the largest, most versatile, and most pharmaceutically important group of membrane proteins, has been impaired over the years by the limited understanding of the molecular mechanisms underlying their diverse functions. The overall goal of the work proposed in this grant application is to explore the extent to which advanced computational strategies using enhanced sampling methods can improve dynamic molecular models of GPCRs, and generate novel testable hypotheses of functional modulation to pursue rational drug design successfully. |
| MECHANISMS OF SODIUM TRANSPORT REGULATION IN CYSTIC FIBROSIS |
KELLEY, THOMAS J |
CASE WESTERN RESERVE UNIVERSITY |
$196,250 |
| Hyperabsorption of sodium across CF airway epithelium was identified over twenty years ago and is a key factor in the progression of airway disease. Much has been learned about mechanisms regulating the function of the epithelial sodium channel (ENaC), but why ENaC function is specifically increased in CF and how its regulation relates to CFTR function has remained largely elusive. Earlier data suggests that the presence of functional CFTR inverted the cAMP-PKA regulation of ENaC from positive regulation to negative regulation. We have recently reported that the loss of CFTR function triggers a feedback response in epithelial cells leading to increased cAMP signaling that results in cholesterol accumulation. We demonstrated in this work that the cAMP binding competitor Rp-cAMPS reverses cholesterol accumulation to wt distribution. An increase in the expression of arrestin-3, a protein involved in G-protein coupled receptor (GPCR) internalization and other signaling events, is the initiating factor in Rp-cAMPS-sensitive cholesterol accumulation in CF. Other work in our laboratory not yet published demonstrates the ability of Rp-cAMPS to reverse many other aspects of CF biology to wt profiles including markers of inflammation, signaling proteins we have previously reported on, and over 145 proteins in proteomic analysis. Given the broad impact of Rp-cAMPS in CF epithelial cells and mouse models on CF cell biology, we were interested in determining if Rp-cAMPS could influence amiloride-sensitive transepithelial potential difference (TEPD) in CF mice. Preliminary data demonstrate that treatment of Cftr -/- mice for 12 days with 5 mg/kg/day Rp-cAMPS via osmotic pump reduces amiloride sensitive transepithelial potential difference to wt levels. In cellular studies, statin treatment of primary renal collecting duct epithelial cells significantly reduces amiloride- sensitive baseline Isc suggesting sodium transport is being influenced. Previous data demonstrating the impact of Rp-cAMPS on cholesterol processing in CF and the influence of statin treatment on amiloride-sensitive transport in polarized cells lead to the postulation that cholesterol processing contributes to hyperabsorption of sodium across CF airway epithelium. The hypothesis of this study is that chronically increased cAMP signaling in CF contributes to hyperabsorption of sodium across CF airway epithelium through modulation of cholesterol trafficking. Mechanistically, the hypothesis that NPC1- driven control of membrane cholesterol content and subsequent modulation of Na+/K+ ATPase function mediate Rp-cAMPS sensitive regulation of sodium transport will be tested. We know that in vivo treatment with Rp-cAMPS is effective in reverting sodium transport in CF mice to wt levels. The goal of this study is to identify the mechanism of this action. PUBLIC HEALTH RELEVANCE: A fundamental issue in CF airway disease is the hyperabsorption of sodium, which influences fluid levels and mucociliary clearance. A mechanism explaining why absorption is increased specifically in CF has been lacking. This proposal hypothesizes that alterations in chronic cAMP signaling in CF airway epithelial cells increases sodium transport through modulating membrane cholesterol levels. |
| LIGAND ASSISTED PROTEIN STRUCTURE: A NOVEL METHOD FOR DEDUCING LIGAND BINDING ARC |
MERCIER, RICHARD W |
NORTHEASTERN UNIVERSITY |
$194,375 |
| Current knowledge as to the physiology of addiction designates the most influential target for drugs of abuse to be G-protein coupled receptors (GPCRs). GPCRs and the components associated with their signaling pathways are also important pharmaceutical targets. Yet, with very few exceptions, there is little direct structural information pertaining to these cell-surface receptors, and homology modeling has been widely used to gain provisional structural insight into most other GPCRs. The research proposed herein is directed at elucidating the structural components controlling the regulation and function of the human CB2 cannabinoid receptor (hCB2), a peripheral GPCR currently under intense investigation. Structural analyses of GPCRs by traditional methods such as x-ray crystallography have been hindered due to the arduous task of purifying adequate amounts of integral membrane protein in its intact native conformation. Consequently, I propose to pursue an alternative method in order to gain direct experimental information about GPCR tertiary structure by focusing primarily on the hCB2 receptor. For this purpose, specific cannabinergic ligands will be utilized (several as yet to be synthesized), as molecular probes directed to cysteine residues to identify and characterize hCB2's binding site(s) and to deduce receptor conformation(s) and structural features regarding activation and inactivation. In this strategy a reactive moiety will be strategically linked to a classical, high-affinity hCB2 cannabinoid agonist at distinct pharmacophoric positions. The reactive moiety will also be linked to a diarylpyrazole inverse-agonist congener. I propose to use these designer ligands as covalent affinity labels in tandem with expressed cysteine to serine or alanine site-directed hCB2 mutants to determine the ligands' site(s) of interaction and thereby deduce information regarding hCB2 receptor-ligand interaction (the cysteine knockouts will identify the region where the reactive moiety binds). Information gleaned from the empirical analyses of mutated and WT hCB2 challenged with the various cannabinergic ligands through binding and functional assays will be incorporated as constraints which may then be used to generate and refine three-dimensional computational models. Corroboration of experimental structural results may also be performed via mass spectroscopic analysis of covalently tagged, expressed receptor protein. PUBLIC HEALTH RELEVANCE: The strategies designed within this proposal will provide significant enhancement towards elucidating CB2 tertiary structure. A better understanding as to the regulation and function of GPCRs through ligand recognition and structural analysis will advance the scientific communities understanding of the molecular signals associated with drugs of abuse. This information will also aid in the design and synthesis of therapeutically relevant compounds. |
| SEX STEROID HORMONE REGULATION OF DRUG REINFORCEMENT |
LEBESGUE, DIANE |
ALBERT EINSTEIN COL OF MED YESHIVA UNIV |
$152,284 |
| As a student, postdoctoral fellow and instructor, I have studied the molecular biology and signal transduction mechanisms of G protein coupled receptors (GPCRs). Over the past 4 years, I investigated the neurobiology of estrogen action in the control of female reproductive function and in neuroprotection following brain insult. My experience in these two fields stimulated my interest in understanding the molecular basis of sex differences in and hormonal control of addictive disorders and psychiatric diseases. Indeed, sex differences in drug abuse liability are well documented, and there is growing evidence that estrogens potentiate drug reinforcement. My recent observation that the newly discovered estrogen receptor GPR30, which is a GPCR, is specifically expressed in dopamine cells of the ventral tegmental area (VTA) that project to the nucleus accumbens prompted me to examine the possibility that this GPCR plays a role in the hormonal regulation of drug addiction. The study of drug reinforcement and the neurobiology of addiction is an entirely new orientation for my career, and the topic to which I will devote my scientific career. During the mentored award period, I will learn several new methods needed to support my development as a drug abuse researcher. I will receive training from Dr. Mary Kritzer, a highly skilled neuroanatomist who has made seminal observations on the expression of sex steroid receptors in brain regions involved in motivated behaviors, and Dr. Mark Wightman, who is world renowned for the development of methods for real time measurement of synaptic dopamine levels by fast scan cyclic voltammetry in awake animals. I will be assisted by my primary mentor Dr. Saleem Nicola to properly design and carry out cocaine self administration experiments. My co- mentor, Dr. Anne Etgen will continue to advise me on mechanisms of estrogen action in the brain. This will equip me to develop independent research projects on the role of sex steroid hormones in the brain that do not overlap with the research projects going on in the lab of either of my mentors. This training will take place at an institutional environment with outstanding facilities and a long history of collaborative interactions among the research faculty. These experiences will facilitate achievement of my long term career objective, to conduct research in my own laboratory as a faculty member at a university or academic medical center. My long-term research goal is to determine the molecular basis and neural circuits that underlie sex differences in drug abuse liability using a combination of behavioral, electrochemical, molecular and pharmacological techniques. The proposed research plan tests the hypothesis that estradiol ( E2) enhances cocaine reinforcement by elevating synaptic dopamine levels in the nucleus accumbens, and that GPR30 mediates these actions of E2. Clinical and preclinical evidence indicates that ovarian steroid hormones, particularly estrogens, modulate dopamine neurotransmission, and that this may be relevant to sex differences in drug reinforcement, with females being more vulnerable than males to drug addiction and relapse. It is well documented that estradiol and related estrogens have rapid actions in the brain that can be observed within seconds to minutes, suggesting that estrogen binding molecules expressed at the plasma membrane participate in mediating cellular responses to estradiol. GPR30, a GPCR which was recently shown to mediate estradiol activation of several cell signaling pathways in vitro, is a potential candidate for mediating rapid estradiol regulation of brain functions. My preliminary immunohistochemistry studies show that GPR30 is highly expressed in midbrain dopamine neurons. Surprisingly little is known about the influence of ovarian steroids on the mesolimbic drug reward circuits, especially the possibility that estradiol rapidly modulates synaptic dopamine availability in the nucleus accumbens. Therefore, the proposed specific aims test the hypothesis that estradiol acts via GPR30 to enhance cocaine reinforcement by elevating synaptic dopamine levels in the nucleus accumbens. The proposed studies will employ a combination of immunohistochemistry and tract tracing, in vivo administration of estradiol or a specific agonist for GPR30 (G1) and in vivo knockdown of GPR30 in the VTA to investigate the role of GPR30 in mediating estrogen regulation of cocaine reinforcement and dopamine release in the nucleus accumbens. Aim 1 will use double-label immunocytochemistry for GPR30 and tyrosine hydroxylase, a marker of dopamine neurons, combined with retrograde tract tracing to identify the neuroanatomical distribution of GPR30-expressing neurons in the cortico-mesolimbic dopamine system. Aim 2 will determine whether the GPR30 agonist G1 mimics the ability of estradiol to potentiate cocaine reinforcement as measured by facilitation of the acquisition of cocaine self administration and to increase motivation to take cocaine under a progressive ratio schedule. We will then assess E2/G1 modulation of cocaine reinforcement in animals subjected to GPR30 knockdown using in vivo RNA interference targeted to the VTA. Aim 3 will employ a combination of in vivo infusions of E2 and G1, fast scan cyclic voltammetry measurement of dopamine release in the nucleus accumbens and in vivo RNA interference to knock down GPR30 in the VTA to test the hypothesis that activation of GPR30 modulates both tonic and phasic dopamine release specifically in the nucleus accumbens shell in response to cocaine. These studies will provide rigorous training for my future research in drug addiction. They will also provide insight into the general molecular mechanisms underlying presynaptic regulation of dopamine neurotransmission in the nucleus accumbens by estradiol and thus may also shed light on the basis of sex differences in drug abuse liability. PUBLIC HEALTH RELEVANCE: There is compelling evidence that women are more vulnerable than men to many different aspects of drug addiction. The proposed research will examine the neurobiological and molecular basis of the regulation of drug reinforcement by a major female sex steroid hormone, estradiol. |
| TYROSINE KINASES IN G PROTEIN MEDIATED SIGNALING |
LUTTRELL, LOUIS M |
MEDICAL UNIVERSITY OF SOUTH CAROLINA |
$150,899 |
| Over the past decade, the study of how G protein-coupled receptors (GPCRs) control cell growth, proliferation and differentiation has fundamentally changed our view of GPCR signal transduction. Far from the canonical model in which GPCRs function solely as activators of heterotrimeric G proteins, we now recognize that they are versatile signaling platforms that transmit both G protein-dependent and -independent signals. Our research originally focused on GPCR regulation of the ERK1/2 MAP kinase cascade. We established that GPCRs use several mechanistically distinct pathways to control ERK1/2 activity, including G protein- dependent signals transmitted by second messenger-dependent protein kinases and 'transactivated' EGF receptors, and novel G protein-independent signals that result from the -arrestin-dependent assembly of multiprotein 'signalsomes'. These results have defined two distinct GPCR signaling 'modes', and in some cases we have identified pathway-selective 'biased agonists' that dissociate them. Moreover, we have found that these pathways are not functionally redundant. Rather, the mechanism of activation determines the time course, spatial distribution, and ultimately the function of GPCR-regulated kinases. The central hypothesis of this proposal is that heterotrimeric G proteins and -arrestins serve as independent GPCR signal transducers that mediate distinct facets of the cellular response to GPCR stimulation. The proposal is organized into three Specific Aims, the first two focused on the structure and function of the GPCR-arrestin 'signalsome' and the third on how G protein-dependent and -arrestin-dependent signals are integrated to determine the cellular response. In each aim, we will focus on the angiotensin AT1A receptor, which utilizes both signaling mechanisms. Aims I and II employ transfected cell systems that allow us to use receptor and -arrestin mutants and rapid siRNA silencing of protein expression to maximum advantage. Experiments will determine the composition of the AT1AR--arrestin 'signalsome' and the structural features of the receptor and -arrestin that dictate signalsome composition and stability. We will employ advanced proteomic methodology to determine how G protein-independent signaling affects protein phosphorylation and determine how -arrestin signaling affects gene transcription. Aim III will concentrate on signaling by endogenous AT1A receptors in primary aortic vascular smooth muscle cells. We will employ pathway-selective agonists, pharmacologic inhibitors and shRNA expression silencing to study the cellular processes regulated by each type of signal in a physiologically relevant context. Experiments will determine the temporal, spatial and functional characteristics of the different types of signal, and how they are integrated to produce cellular changes associated with the development of atherosclerotic vascular disease. Completion of these studies will address a fundamental gap in our understanding of how GPCRs work and may provide insights into novel therapeutic applications of GPCR ligands with pathway-selective agonist or antagonist properties. PUBLIC HEALTH RELEVANCE: Contrary to the traditional view that G protein-coupled receptors (GPCRs) only signal by activating heterotrimeric G proteins, recent research has shown that they also transmit G protein-independent signals that are initiated by binding to adapter or scaffold proteins. This project focuses on the role of arrestins in angiotensin AT1A receptor signaling. Arrestins bind to activated GPCRs, 'uncoupling' them from G proteins while at the same time promoting the assembly of 'signalsomes' that affect protein phosphorylation and gene transcription. Our research will define the factors that control the assembly and function of the AT1A receptor- arrestin signalsome and determine how G protein-dependent and arrestin-dependent signals are integrated. These studies address fundamental gaps in our understanding of how GPCRs work and may provide insights into novel therapeutic applications of GPCR ligands with pathway-selective agonist or antagonist properties. |
| GERM CELL MIGRATION IN DROSOPHILA |
LEHMANN, RUTH E. |
NEW YORK UNIVERSITY SCHOOL OF MEDICINE |
$145,881 |
| Germ cells are essential for the maintenance of all sexually reproducing species. Many cellular and molecular aspects of germ cell behavior including their early development as primordial germ cells (PGCs) and their differentiation into sperm and egg are conserved throughout the animal kingdom. This proposal focuses on the analysis of PGC migration. In most organisms PGCs originate at one place of the early embryo and migrate through the embryos to reach the somatic part of the gonad, where they differentiate. This process is intricately regulated and failures in embryonic germ cell migration have been attributed to the origin of extragonadal germ cell tumors in humans. This proposal combines biochemical and genetic approaches with in vivo imaging analysis to understand how migratory cues are integrated overtime and space using Drosophila as a model. In vivo imaging will be used to follow the germ cell migratory path during their transepithelial migration through the posterior midgut and their subsequent homing towards the somatic gonad in wild type and mutants. The G protein coupled receptor (GPCR), Tre-1, will be analyzed with regard to its role in initiating the migratory program and in transepithelial migration. The molecular nature of attractant and repellant somatic guidance cues will be characterized by studying the function of the previously identified lipid phosphatase pathway that is controlled, by Wunen and Wunen2, two homologs of mammalian lipidphosphate phosphatase 3, and by analyzing the isoprenoid pathway, that is controlled by HMGCoA reductase. With a more detailed knowledge of the pathways involved, the interplay between GPCR and Wunen function in germ cells, and the integration of somatic signaling by the Wunen and HMGCoA reductase pathways in the soma will be addressed. The overall goals of this proposal are to connect the molecular network of germ cell migration with the cellular parameters of the developing embryo. Aspects of germ cell migratory behavior are shared with other solitary migrating cells, such as cell of the immune system and metastasizing cancer cells. Thus, the analysis of the genetically easily amenable fruit fly germ cell system is likely to reveal more general principles controlling cell migration. |
| MECHANISMS OF BRAIN-SPECIFIC ANGIOGENESIS INHIBITOR 1 (BAI1) IN NEURAL DEVELOPMEN |
DUMAN, JOSEPH G |
BAYLOR COLLEGE OF MEDICINE |
$124,846 |
| This proposal is for a K01 Mentored Research Scientist Development Award. The candidate is a postdoctoral associate with training in cell biology and biophysics. The goal of this proposal is to transition the candidate into molecular and developmental neuroscience. The candidate is working towards the establishment of a research lab studying the mechanisms of neuronal development with emphasis on signals and signal integration; these studies will be directed toward the understanding and treatment of mental disease. The research goal of this proposal is to elucidate the mechanisms by which brain angiogenesis inhibitor I (BAI1) mediates the formation of dendrites and dendritic spines. BAI1 is member of the B family of heteromeric G-protein coupled receptors (GPCRs) and possesses both an extensive extracellular segment containing thrombospondin repeats and a long cytoplasmic segment containing multiple potential signaling domains flanking the central GPCR moiety. The candidate's preliminary data demonstrate that (1) BAI1 is critical for the proper formation of dendrites and dendritic spines in hippocampal pyramidal neurons, (2) BAI1 interacts with Tiam1, an activator of the small GTPase Rac that links extracellular signals to dendrite and spine growth and development, and (3) BAI1 affects the actin cytoskeleton, at least partially through Rac. A variety of techniques will be used (1) to determine the domain(s) of BAI1 that is (are) required to promote the development of dendrites and dendritic spines, and (2) to measure the effects of BAI1 on dendritic and spine developmental pathways involving Rac. In particular, the candidate will investigate the role of BAI1 in modulating the activation of Rac through Tiam1 in response to brain-derived neurotrophic factor through its receptor TrkB. During this time, the candidate will participate in a variety of neuroscience teaching exercises, both formal and informal. The mentoring team includes both a well-known senior neuroscientist and an exciting young investigator. This training will address the candidate's long term goal of becoming a principal investigator by providing a high-quality environment in which to learn techniques, do research, and generate publications. In addition, the candidate will benefit from career development opportunities provided by Baylor College of Medicine and his senior co-mentor, be exposed to a large variety of scientific opportunities at the Texas Medical Center, observe a laboratory being set up, and receive training in the ethical conduct of research. Both the training and research plans are facilitated by the availability of advanced imaging and other research technologies at Baylor, as well as the highly collaborative and excellent community of world-class researchers present there. PUBLIC HEALTH REVELANCE: Defects in dendrite and dendritic spine formation underlie many forms of mental disease, including autism, depression, and disorders associated with chronic stress. These defects also contribute to the pathologies of mental diseases, neurodegenerative diseases, and traumatic brain injuries. Determination of the signaling mechanisms that direct dendrite and spine formation should unveil new strategies for the treatment of these disorders. |
| STRUCTURAL STUDIES OF G PROTEIN-COUPLED RECEPTORS |
PALCZEWSKI, KRZYSZTOF NMI |
CASE WESTERN RESERVE UNIVERSITY |
$80,000 |
| G protein-coupled receptors (GPCRs) are important in biomedicine because they are the targets of about 50% of commercially available drugs. Only little is known about the structure and function of these receptors in any molecular or atomic detail. The only three-dimensional X-ray crystallographic model for a GPCR is that of ground-state rhodopsin in an inactive conformation. Rhodopsin, the light-sensing protein in retina, has been extensively studied as a prototypical GPCR. The integral membrane protein contains seven trans- membrane helices that provide a binding site for its 11-cis-retinal chromophore. Absorption of a photon triggers a change in the chromophore's conformation and then in the receptor's tertiary structure. This results in alterations on the receptor's cytoplasmic surface that permit binding of transducin (Gt), its cognate G- protein. This initiates further steps in the signal transduction process. Most GPCRs respond to molecular signals in the form of ligands. Binding of specific ligands by specific GPCRs results in a ligand-specific cellular response. The ligand binding site for most GPCRs coincides with the retinal pocket in rhodopsin. Binding of a ligand causes the same kinds of conformational changes as does absorption of a photon in rhodopsin, and the remaining molecular mechanisms for signal transduction are similar for all GPCRs. Our understanding of GPCR structure and function will be increased by components of this proposal. First, the oligomeric state of activated GPCRS will be addressed by experiments probing the quaternary structure of rhodopsin isolated under varying detergent condtions. Assessment of physiological function will be made for these preparations. The second part of the project will use single-molecule force microscopy to probe the interactions between the membrane and rhodopsin or the serotonin 5HT1AR receptor to understand the dynamics and stabilities of GPCRs. Crystallographic studies of activated rhodopsin make up the third part of the project. The last component of the project calls for further efforts in purifying the transducin/rhodopsin complex for biochemical and structural characterization. These projects all provide structural information for an important class of proteins, a class that provides extensive experimental and theoretical challenges. The importance of the protein family for human health makes this effort worthwhile. |
| G-PROTEIN COUPLED RECEPTORS AND AXON GUIDANCE IN THE DROSOPHILA EMBRYONIC NERVE C |
VANBERKUM, MARK F |
WAYNE STATE UNIVERSITY |
$73,519 |
| The development of axon pathways is a complex signal transduction process linking extracellular guidance cues to intracellular signals regulating axon outgrowth and steering. Guidance errors lead to structural birth defects in the nervous system that cause physical, mental, or behavioral impairment. The long-term goal of the investigator's laboratory is to understand how signaling events lead to the development of axon pathways and functional connectivity. Drosophila will be used as the model system as it allows molecular, genetic, and cellular tools to be applied to the in vivo study of axon guidance, while gene conservation makes the study relevant to human development. The goal of this application is to establish a role for G-protein coupled receptors (GPCRs) in axon pathway formation during development of the Drosophila embryonic nervous system. GPCRs are transmembrane receptors that activate trimeric G-proteins to regulate several key intracellular signaling pathways. GPCRs may directly function as guidance cue receptors, or modify second messenger levels to indirectly affect the signaling potential of other ligand-receptor systems operating at a guidance choice point. Based on vertebrate and Drosophila studies, it is hypothesized that GPCRs function in the developing Drosophila nerve cord to guide axons to their targets. Specific Aim 1 will test this hypothesis by systematically evaluate the role of GPCRs in axon pathway formation in the Drosophila embryo by genetically altering components of the canonical GPCR signaling pathway; and Specific Aim 2 will identify GPCRs that guide the developme |
