Chemosensory Receptors And The Basis Of Specificity
Steven D Munger, Associate Professor
Anatomy And Neurobiologyuniversity Of Maryland Baltimore
Grant 5R01DC005786-07 from National Institute On Deafness And Other Communication Disorders, IRG: IFCN
Abstract: Mammals use several chemosensory systems to detect and encode their chemical environment. How these systems discriminate relevant chemical cues is a major unresolved question. We hypothesize that differences in the stimulus selectivity of different populations of chemosensory cells largely reflects differences in the ligand selectivity and sensitivity of the chemosensory receptors (CRs) expressed therein. Difficulties in obtaining large amounts of receptor protein suitable for biochemical or structural analysis, as well as the small number of CRs for which ligands are known, has hampered efforts to characterize the basis of ligand specificity. One group of CRs, the T1R taste receptors, offers unique advantages that will permit the first systematic analysis of how CR structure/function relationships impact the ability of a chemosensory cell population to detect and discriminate physiologically relevant ligands. We will take advantage of the demonstrated sensitivity of T 1Rs for sweet-tasting ligands, and an extracellular N-terminal ligand-binding domain amenable to biochemical purification and structural characterization, to establish the role of different T1Rs in the detection of sweet tasting stimuli. Aim 1 The structure of the T1R ligand-binding pockets, in the presence and absence of ligands, will be solved by a combination of circular dichroism spectrophotometry and X-ray crystallography of T1R N-terminal domains. Aim 2 To determine the specific contributions of ligand binding to taste function, targeted mutations will be introduced in the ligand-binding pocket of T1R N-terminal domains both in vitro and by gene targeting in mice. Changes in ligand binding kinetics will be measured using isothermal titration calorimetry, while the effects of T1R deletion or mutation on taste function will be assayed by brief-access behavioral tasks where the sensitivity of targeted mice to sweet stimuli will be determined. Together, these studies will provide the first in-depth structural and quantitative analyses of the interactions between chemosensory receptors and their ligands, and will offer important new insights into how individual taste receptors contribute to the detection and discrimination of food cues critical for health and survival
Keywords: chemoreceptor, ligand, protein structure function, sensory discrimination, taste cue, gene mutation, histochemistry /cytochemistry, intermolecular interaction, receptor binding, receptor expression X ray crystallography, behavior test, calorimetry, circular dichroism, gene targeting, genetically modified animal, laboratory mouse, protein purification
Project start date: 2002-09-20
Project end date: 2009-08-31
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Chemosensory Receptors And The Basis Of Specificity
Steven D Munger, Associate Professor
University Of Maryland Baltimore 660 W Redwood St, Rm 021 Baltimore, Md 21201
Grant 5R01DC005786-06 from National Institute On Deafness And Other Communication Disorders, IRG: IFCN
Abstract: Mammals use several chemosensory systems to detect and encode their chemical environment. How these systems discriminate relevant chemical cues is a major unresolved question. We hypothesize that differences in the stimulus selectivity of different populations of chemosensory cells largely reflects differences in the ligand selectivity and sensitivity of the chemosensory receptors (CRs) expressed therein. Difficulties in obtaining large amounts of receptor protein suitable for biochemical or structural analysis, as well as the small number of CRs for which ligands are known, has hampered efforts to characterize the basis of ligand specificity. One group of CRs, the T1R taste receptors, offers unique advantages that will permit the first systematic analysis of how CR structure/function relationships impact the ability of a chemosensory cell population to detect and discriminate physiologically relevant ligands. We will take advantage of the demonstrated sensitivity of T 1Rs for sweet-tasting ligands, and an extracellular N-terminal ligand-binding domain amenable to biochemical purification and structural characterization, to establish the role of different T1Rs in the detection of sweet tasting stimuli. Aim 1 The structure of the T1R ligand-binding pockets, in the presence and absence of ligands, will be solved by a combination of circular dichroism spectrophotometry and X-ray crystallography of T1R N-terminal domains. Aim 2 To determine the specific contributions of ligand binding to taste function, targeted mutations will be introduced in the ligand-binding pocket of T1R N-terminal domains both in vitro and by gene targeting in mice. Changes in ligand binding kinetics will be measured using isothermal titration calorimetry, while the effects of T1R deletion or mutation on taste function will be assayed by brief-access behavioral tasks where the sensitivity of targeted mice to sweet stimuli will be determined. Together, these studies will provide the first in-depth structural and quantitative analyses of the interactions between chemosensory receptors and their ligands, and will offer important new insights into how individual taste receptors contribute to the detection and discrimination of food cues critical for health and survival.
Keywords: chemoreceptor, ligand, protein structure function, sensory discrimination, taste, cue, gene mutation, histochemistry /cytochemistry, intermolecular interaction, receptor binding, receptor expression, X ray crystallography, behavior test, calorimetry, circular dichroism, gene targeting, genetically modified animal, laboratory mouse, protein purification
Project start date: 2002-09-20
Project end date: 2009-08-31
5R01DC005786-06 (2007): $319362
5R01DC005786-05 (2006): $333917
5R01DC005786-04 (2005): $341629
5R01DC005786-03 (2004): $335737
5R01DC005786-02 (2003): $335674
Grants awarded to Steven D Munger
Transduction Mechanisms And CNS Targets Of GC-D Neurons
Steven D Munger, Associate Professor
University Of Maryland Baltimore 660 W Redwood St, Rm 021 Baltimore, Md 21201
Grant 5R01DC005633-05 from National Institute On Deafness And Other Communication Disorders, IRG: IFCN
Abstract: The sense of smell is the principal window on the rich and complex world of volatile chemicals. The main and accessory olfactory systems of mammals serve to detect and analyze odors and pheromones. However, it is becoming clear that the division of nasal chemosensory neurons into only these two groups is simplistic and does not reflect the functional diversity of receptor cell types in the nose. A novel group of chemosensory neurons, which target a unique population of glomeruli, the "necklace" glomeruli, in the posterior olfactory bulb, are found in several regions of the main olfactory epithelium. These neurons also exhibit a gene expression profile distinct from that of other chemosensory neurons. However, it is unclear if these receptor neurons and glomeruli function as a distinct chemosensory system. We have created a gene-targeted mouse that functionally disrupts the gene encoding the guanylyl cyclase, GC-D, a protein critical for the function of these cells. After targeting, this gene locus also encodes a histochemical reporter, tau-beta-galactosidase that permits the visualization of the neurons that express GC-D. We propose to use this mouse model to perform the first systematic analysis of the organization and functional characteristics of this poorly understood population of chemosensory neurons, as well as to characterize the functional role of these cells in chemosensory behavior. We will provide a systematic anatomical of these neurons and the necklace glomeruli and determine the developmental and anatomical expression patterns of GC-D in the olfactory system. We will also determine the chemosensory role of these neurons through behavioral and biochemical analyses of adult and neonatal animals. Finally, we will characterize the biochemical cascade involved in the transduction of specific chemosensory stimuli, including both odors and pheromones, in these cells. These results obtained in these studies will provide important insights into the mechanism by which these neurons and glomeruli detect and analyze sensory stimuli, the role of cGMP signaling in these processes, and will define the sensory role of this unique neuronal population.
Keywords: afferent nerve, biological signal transduction, chemoreceptor, guanylate cyclase, olfaction, calcium, cyclic GMP, innervation, ligand, maternal behavior, neural conduction, odor, olfactory lobe, pheromone, respiratory epithelium, behavior test, gene targeting, genetically modified animal, immunocytochemistry, in situ hybridization, laboratory mouse, morphometry, newborn animal, protein localization
Project start date: 2003-04-01
Project end date: 2009-03-31
5R01DC005633-05 (2007): $281609
5R01DC005633-04 (2006): $290021
5R01DC005633-03 (2005): $297000
5R01DC005633-02 (2004): $297000
1R01DC005633-01A1 (2003): $297000
CHARACTERIZING SPECIALIZED CHEMOSENSORY NEURONS
Steven D Munger, Associate Professor
Anatomy And Neurobiologyuniversity Of Maryland Baltimore
660 W Redwood St, Rm 021
baltimore, Md 21201
Grant 5R03DC004779-02 from National Institute On Deafness And Other Communication Disorders, IRG: ZDC1
Abstract: The proposed research is aimed at characterizing a specialized subset of neurons in the olfactory epithelium. These D neurons appear to be unique in expressing a novel signal transduction pathway (guanylyl cyclase, GC- D) among olfactory receptor cells. These neurons may also be involved in mediating suckling behavior in neonatal rodents. proposes to 1) isolate D neurons and characterize the signal transduction pathway and receptor molecules expressed in these cells; 2) show that GC-D is necessary for the normal expression of tyrosine hydroxylase (TH) in the olfactory bulbs, as an indicator of afferent activity; 3) examine the projection patterns of D neurons; and 4) show that GC-D neurons are necessary for nipple attachment and suckling in neonatal mice. These aims are related in an attempt to associate a specific subset of olfactory receptor cells, with unique signal transduction, to a particular set of neonatal behaviors. GC-D knockout/knockin mice will be generated to carry out the proposed experiments to identify D neurons, which make up less than 1% of olfactory receptor cells. A variety of molecular biological techniques will be used to screen isolated D neurons for the receptors and signal transduction components and use histochemical techniques for identifying projection patterns of D neurons and TH in the olfactory bulbs. In addition, GC-D transgenic mice will be used to examine the ability of neonates to attach to nipples and suckle
Keywords: biological signal transduction, guanylate cyclase, neural information processing, neuron, olfaction, respiratory epithelium, sensory receptor cell adhesion molecule, oral behavior, receptor expression behavior test, immunocytochemistry, in situ hybridization, laboratory mouse
Project start date: 2000-08-01
Project end date: 2003-07-31
5R03DC004779-02 (2001): $74250
1R03DC004779-01 (2000): $74250
Chemosensory Receptors And The Basis Of Specificity
Steven D Munger, Associate Professor
Anatomy And Neurobiologyuniversity Of Maryland Baltimore
Grant 5R01DC005786-07 from National Institute On Deafness And Other Communication Disorders, IRG: IFCN
Abstract: Mammals use several chemosensory systems to detect and encode their chemical environment. How these systems discriminate relevant chemical cues is a major unresolved question. We hypothesize that differences in the stimulus selectivity of different populations of chemosensory cells largely reflects differences in the ligand selectivity and sensitivity of the chemosensory receptors (CRs) expressed therein. Difficulties in obtaining large amounts of receptor protein suitable for biochemical or structural analysis, as well as the small number of CRs for which ligands are known, has hampered efforts to characterize the basis of ligand specificity. One group of CRs, the T1R taste receptors, offers unique advantages that will permit the first systematic analysis of how CR structure/function relationships impact the ability of a chemosensory cell population to detect and discriminate physiologically relevant ligands. We will take advantage of the demonstrated sensitivity of T 1Rs for sweet-tasting ligands, and an extracellular N-terminal ligand-binding domain amenable to biochemical purification and structural characterization, to establish the role of different T1Rs in the detection of sweet tasting stimuli. Aim 1 The structure of the T1R ligand-binding pockets, in the presence and absence of ligands, will be solved by a combination of circular dichroism spectrophotometry and X-ray crystallography of T1R N-terminal domains. Aim 2 To determine the specific contributions of ligand binding to taste function, targeted mutations will be introduced in the ligand-binding pocket of T1R N-terminal domains both in vitro and by gene targeting in mice. Changes in ligand binding kinetics will be measured using isothermal titration calorimetry, while the effects of T1R deletion or mutation on taste function will be assayed by brief-access behavioral tasks where the sensitivity of targeted mice to sweet stimuli will be determined. Together, these studies will provide the first in-depth structural and quantitative analyses of the interactions between chemosensory receptors and their ligands, and will offer important new insights into how individual taste receptors contribute to the detection and discrimination of food cues critical for health and survival
Keywords: chemoreceptor, ligand, protein structure function, sensory discrimination, taste cue, gene mutation, histochemistry /cytochemistry, intermolecular interaction, receptor binding, receptor expression X ray crystallography, behavior test, calorimetry, circular dichroism, gene targeting, genetically modified animal, laboratory mouse, protein purification
Project start date: 2002-09-20
Project end date: 2009-08-31
1R01DC005786-01 (2002): $349013
Related Publications
The 15th International Symposium on Olfaction and Taste. Chem Senses. 2008 Oct; 33( 8): 735-8. No abstract available. PMID: 18849308 
Subsystem Organization of the Mammalian Sense of Smell. Annu Rev Physiol. 2008 Sep 22. [Epub ahead of print] PMID: 18808328 Modulation of taste sensitivity by GLP-1 signaling. J Neurochem. 2008 Jul; 106( 1): 455-63. Epub 2008 Jul 1. PMID: 18397368 
Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium. Proc Natl Acad Sci U S A. 2007 Sep 4; 104( 36): 14507-12. Epub 2007 Aug 27. PMID: 17724338 
Expression and purification of functional ligand-binding domains of T1R3 taste receptors. Chem Senses. 2006 Jul; 31( 6): 505-13. Epub 2006 Apr 18. PMID: 16621970 Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli. Curr Biol. 2005 Nov 8; 15( 21): 1948-52. PMID: 16271873 Inbred mouse strains C57BL/6J and DBA/2J vary in sensitivity to a subset of bitter stimuli. BMC Genet. 2005 Jun 20; 6: 36. PMID: 15967025 Haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taste sensitivity in inbred mice. BMC Genet. 2005 Jun 6; 6( 1): 32. PMID: 15938754 Taste sensitivities to PROP and PTC vary independently in mice. Chem Senses. 2003 Oct; 28( 8): 695-704. PMID: 14627538 Importance of the CNGA4 channel gene for odor discrimination and adaptation in behaving mice. Proc Natl Acad Sci U S A. 2003 Apr 1; 100( 7): 4299-304. Epub 2003 Mar 20. PMID: 12649326