Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-01-26
2004-11-09
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S007200, C435S198000, C435S325000, C435S320100, C435S252300, C435S069100, C536S023200, C530S350000
Reexamination Certificate
active
06815176
ABSTRACT:
FIELD OF THE INVENTION
The invention identifies nucleic acid and amino acid sequences of a sensory cell specific phospholipase C that are specifically expressed in taste cells, antibodies to such phospholipase C, methods of detecting such nucleic acids and proteins, and methods of screening for modulators of sensory cell specific phospholipase C.
BACKGROUND OF THE INVENTION
Taste transduction is one of the most sophisticated forms of chemotransduction in animals (see, e.g., Avenet & Lindemann,
J. Membrane Biol.
112:1-8 (1989); Margolskee,
BioEssays
15:645-650 (1993)). Gustatory signaling is found throughout the animal kingdom, from simple metazoans to the most complex of vertebrates; its main purpose is to provide a reliable signaling response to non-volatile ligands. Higher organisms have four basic types of taste modalities: salty, sour, sweet, and bitter. Each of these modalities is thought to be mediated by distinct signaling pathways leading to receptor cell depolarization, generation of a receptor or action potential, and the release of neurotransmitter and synaptic activity (see, e.g., Roper,
Ann. Rev. Neurosci.
12:329-353 (1989)).
Mammals are believed to have five basic taste modalities: sweet, bitter, sour, salty and unami (the taste of monosodium glutamate) (see, e.g., Kawamura & Kare,
Introduction to Umami: A Basic Taste
(1987); Kinnamon & Cummings,
Ann. Rev. Physiol.
54:715-731(1992); Lindemann,
Physiol. Rev.
76:718-766 (1996); Stewart et al.,
Am. J Physiol.
272:1-26 (1997)). Extensive psychophysical studies in humans have reported that different regions of the tongue display different gustatory preferences (see, e.g., Hoffmann,
Menchen. Arch. Path. Anat. Physiol.
62:516-530 (1875); Bradley et al.,
Anatomical Record
212: 246-249 (1985); Miller & Reedy,
Physiol. Behav.
47:1213-1219 (1990)). Also, numerous physiological studies in animals have shown that taste receptor cells may selectively respond to different tastants (see, e.g., Akabas et al.,
Science
242:1047-1050 (1988); Gilbertson et al.,
J. Gen. Physiol.
100:803-24 (1992); Bernhardt et al.,
J. Physiol.
490:325-336 (1996); Cummings et al.,
J. Neurophysiol.
75:1256-1263 (1996)).
In mammals, taste receptor cells are assembled into taste buds that are distributed into different papillae in the tongue epithelium. Circumvallate papillae, found at the very back of the tongue, contain hundreds (mice) to thousands (human) of taste buds and are particularly sensitive to bitter substances. Foliate papillae, localized to the posterior lateral edge of the tongue, contain dozens to hundreds of taste buds and are particularly sensitive to sour and bitter substances. Fungiform papillae containing a single or a few taste buds are at the front of the tongue and are thought to mediate much of the sweet taste modality.
Each taste bud, depending on the species, contain 50-150 cells, including precursor cells, support cells, and taste receptor cells (see, e.g., Lindemann,
Physiol. Rev.
76:718-766 (1996)). Receptor cells are innervated at their base by afferent nerve endings that transmit information to the taste centers of the cortex through synapses in the brain stem and thalamus. Elucidating the mechanisms of taste cell signaling and information processing are critical for understanding the function, regulation, and “perception” of the sense of taste.
Although much is known about the psychophysics and physiology of taste cell function, very little is known about the molecules and pathways that mediate these sensory signaling responses (reviewed by Gilbertson,
Current Opn. in Neurobiol.
3:532-539 (1993)). Electrophysiological studies suggest that sour and salty tastants modulate taste cell function by direct entry of H
+
and Na
+
ions through specialized membrane channels on the apical surface of the cell. In the case of sour compounds, taste cell depolarization is hypothesized to result from H
+
blockage of K
+
channels (see, e.g., Kinnamon et al.,
PNAS USA
85: 7023-7027 (1988)) or activation of pH-sensitive channels (see, e.g., Gilbertson et al.,
J. Gen. Physiol.
100:803-24 (1992)); salt transduction may be partly mediated by the entry of Na
+
via amiloride-sensitive Na
+
channels (see, e.g., Heck et al.,
Science
223:403-405 (1984); Brand et al.,
Brain Res.
207-214 (1985); Avenet et al.,
Nature
331: 351-354 (1988)). Most of molecular components of the sour or salty pathways have not been identified.
Sweet, bitter, and unami transduction are believed to be mediated by G-protein-coupled receptor (GPCR) signaling pathways (see, e.g., Striem et al.,
Biochem. J.
260:121-126 (1989); Chaudhari et al.,
J. Neuros.
16:3817-3826 (1996); Wong et al.,
Nature
381: 796-800 (1996)). Confusingly, there are almost as many models of signaling pathways for sweet and bitter transduction as there are effector enzymes for GPCR cascades (e.g., G protein subunits, cGMP phosphodiesterase, phospholipase C, adenylate cyclase; see, e.g., Kinnamon & Margolskee,
Curr. Opin. Neurobiol.
6:506-513 (1996)). Identification of molecules involved in taste signaling is important given the numerous pharmacological and food industry applications for bitter antagonists, sweet agonists, and modulators of salty and sour taste.
The identification and isolation of taste receptors (including taste ion channels), and taste signaling molecules, such as G-protein subunits and enzymes involved in signal transduction, would allow for the pharmacological and genetic modulation of taste transduction pathways. For example, availability of receptor, ion channels, and other molecules involved in taste transduction would permit the screening for high affinity agonists, antagonists, inverse agonists, and modulators of taste cell activity. Such taste modulating compounds could then be used in the pharmaceutical and food industries to customize taste. In addition, such taste cell specific molecules can serve as invaluable tools in the generation of taste topographic maps that elucidate the relationship between the taste cells of the tongue and taste sensory neurons leading to taste centers in the brain.
SUMMARY OF THE INVENTION
The present invention demonstrates, for the first time, taste cell specific expression of nucleic acid encoding phospholipase C beta 2. Phospholipase C plays a central role in transmembrane signaling by catalyzing the hydrolysis of phosphatidylinositol 4,5-biphosphate and generating second messenger molecules involved in signal transduction in cells. The phospholipase Cs that are specifically expressed in taste cells are involved in the taste transduction pathway, and can thus be used to screen for modulators of taste. The compounds identified in these assays would then be used by the food and pharmaceutical industries to customize taste, e.g., as addition to food or medicine so that the food or medicine tastes different to the subject who ingests it. For example, bitter medicines can be made to taste less bitter, and sweet substance can be enhanced.
In one aspect, the present invention provides a method for identifying a compound that modulates sensory signaling in sensory cells, the method comprising the steps of: (i) contacting the compound with a sensory specific phospholipase C, the phospholipase C comprising greater than 70% amino acid sequence identity to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; and (ii) determining the functional effect of the compound upon the phospholipase C.
In one embodiment, the phospholipase C specifically binds to polyclonal antibodies generated against SEQ ID NO:2 or SEQ ID NO:4.
In another embodiment, the functional effect is a chemical effect.
In another embodiment, the functional effect is a physical effect.
In another embodiment, the functional effect is determined by measuring changes in intracellular cAMP, cGMP, IP
3
, DAG, or Ca
2+
.
In another embodiment, the phospholipase C is expressed in a cell. In another embodiment, the phospholipase C is expressed in a eukaryotic cell.
In another embodiment, the functional effect is
Adler Jon E.
Lindemeier Juergen
Zuker Charles S.
Prouty Rebecca E.
Ramirez Delia
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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