Tas1r3, encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac.

The ability to taste the sweetness of carbohydrate-rich foodstuffs has a critical role in the nutritional status of humans. Although several components of bitter transduction pathways have been identified, the receptors and other sweet transduction elements remain unknown. The Sac locus in mouse, mapped to the distal end of chromosome 4 (refs. 7-9), is the major determinant of differences between sweet-sensitive and -insensitive strains of mice in their responsiveness to saccharin, sucrose and other sweeteners. To identify the human Sac locus, we searched for candidate genes within a region of approximately one million base pairs of the sequenced human genome syntenous to the region of Sac in mouse. From this search, we identified a likely candidate: T1R3, a previously unknown G protein-coupled receptor (GPCR) and the only GPCR in this region. Mouse Tas1r3 (encoding T1r3) maps to within 20,000 bp of the marker closest to Sac (ref. 9) and, like human TAS1R3, is expressed selectively in taste receptor cells. By comparing the sequence of Tas1r3 from several independently derived strains of mice, we identified a specific polymorphism that assorts between taster and non-taster strains. According to models of its structure, T1r3 from non-tasters is predicted to have an extra amino-terminal glycosylation site that, if used, would interfere with dimerization.

Pineal opsin: a nonvisual opsin expressed in chick pineal.

Pineal opsin (P-opsin), an opsin from chick that is highly expressed in pineal but is not detectable in retina, was cloned by the polymerase chain reaction. It is likely that the P-opsin lineage diverged from the retinal opsins early in opsin evolution. The amino acid sequence of P-opsin is 42 to 46 percent identical to that of the retinal opsins. P-opsin is a seven-membrane spanning, G protein-linked receptor with a Schiff-base lysine in the seventh membrane span and a Schiff-base counterion in the third membrane span. The primary sequence of P-opsin suggests that it will be maximally sensitive to approximately 500-nanometer light and produce a slow and prolonged phototransduction response consistent with the nonvisual function of pineal photoreception.

Ggamma13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium.

Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The alpha subunit of gustducin (alpha-gustducin) is critical for transduction of responses to bitter or sweet compounds. We identified a G-protein gamma subunit (Ggamma13) that colocalized with alpha-gustducin in taste receptor cells. Of 19 alpha-gustducin/Ggamma13-positive taste receptor cells profiled, all expressed the G protein beta3 subunit (Gbeta3); approximately 80% also expressed Gbeta1. Gustducin heterotrimers (alpha-gustducin/Gbeta1/Ggamma13) were activated by taste cell membranes plus bitter denatonium. Antibodies against Ggamma13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducin heterotrimers transduce responses to bitter and sweet compounds via alpha-gustducin's regulation of phosphodiesterase (PDE) and Gbetagamma's activation of phospholipase C (PLC).

Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.

Extracellular matrix-associated protein Sc1 is not essential for mouse development.

Sc1 is an extracellular matrix-associated protein whose function is unknown. During early embryonic development, Sc1 is widely expressed, and from embryonic day 12 (E12), Sc1 is expressed primarily in the developing nervous system. This switch in Sc1 expression at E12 suggests an importance for nervous-system development. To gain insight into Sc1 function, we used gene targeting to inactivate mouse Sc1. The Sc1-null mice showed no obvious deficits in any organs. These mice were born at the expected ratios, were fertile, and had no obvious histological abnormalities, and their long-term survival did not differ from littermate controls. Therefore, the function of Sc1 during development is not critical or, in its absence, is subserved by another protein.

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.

The biochemistry and molecular biology of taste transduction.

The recent application of molecular biological techniques to taste cells has led to the identification and cloning of a number of proteins that are involved in signal transduction. Some of these are expressed only in taste cells, whereas others are expressed at higher levels in taste cells than in other, non-sensory lingual cells. Among these proteins are several G protein alpha subunits, of which alpha gustducin is particularly interesting because of its similarity to the alpha transducins expressed in the visual system, suggesting that there are similarities in taste and visual transduction mechanisms.

Mechanisms of taste transduction.

Taste cells use a wide variety of mechanisms for transduction. Ionic stimuli, such as salts and acids, interact directly with ion channels to depolarize taste cells. More complex stimuli, such as sugars and amino acids, utilize apically located receptors for transduction. Recent molecular biological results suggest that the metabotropic glutamate receptor mGluR4 may function in glutamate taste transduction. New biochemical studies have identified a bitter-responsive receptor that activates gustducin. Unexpected results with knockout mice suggest that gustducin may be directly involved in both bitter and sweet transduction. Electrophysiological experiments indicate that both inositol trisphosphate and cyclic nucleotides function in both bitter and sweet transduction events.

The molecular physiology of taste transduction.

Taste receptor cells use a variety of mechanisms to transduce chemical information into cellular signals. Seven-transmembrane-helix receptors initiate signaling cascades by coupling to G proteins, effector enzymes, second messengers and ion channels. Apical ion channels pass ions, leading to depolarizing and/or hyperpolarizing responses. New insights into the mechanisms of taste sensation have been gained from molecular cloning of the transduction elements, biochemical elucidation of the transduction pathways, and electrophysiological analysis of the function of taste cell ion channels.

Directing gene expression to gustducin-positive taste receptor cells.

We have demonstrated that an 8.4 kb segment (GUS(8.4)) from the upstream region of the mouse alpha-gustducin gene acts as a fully functional promoter to target lacZ transgene expression to the gustducin-positive subset of taste receptor cells (TRCs). The GUS(8. 4) promoter drove TRC expression of the beta-galactosidase marker at high levels and in a developmentally appropriate pattern. The gustducin minimal 1.4 kb promoter (GUS(1.4)) by itself was insufficient to specify TRC expression. We also identified an upstream enhancer from the distal portion of the murine gustducin gene that, in combination with the minimal promoter, specified TRC expression of transgenes. Expression of the lacZ transgene from the GUS(8.4) promoter and of endogenous gustducin was coordinately lost after nerve section and simultaneously recovered after reinnervation, confirming the functionality of this promoter. Transgenic expression of rat alpha-gustducin restored responsiveness of gustducin null mice to both bitter and sweet compounds, demonstrating the utility of the gustducin promoter.

Isolation of a clone which induces expression of the gene encoding the human tumor necrosis factor receptor.

Tumor necrosis factor (TNF) is a cytokine with pleiotropic effects upon cell growth, inflammation and immunologic responsiveness. High-affinity TNF receptors (TNFRs) of 55 and 75 kDa are found in many cell types. Using an Epstein-Barr virus (EBV)-based mammalian expression library, we have isolated a clone from human lymphoblastoid transfectants that induces overexpression of the TNFR-encoding gene (TNFR). Transfectants overproducing the TNFR were isolated by multiple rounds of sorting on a fluorescence-activated cell sorter using fluorescent TNF ligand binding as the selection procedure. Among the sorted transfectants were cells producing approx. 150,000 receptors per cell (Kd of approx. 1 nM). These cells have multiple copies of the TNFR gene present as extrachromosomal plasmids. These cells also overproduced the mRNA for TNFR. Low-Mr EBV episomes were isolated from these overproducing cells and used to transform Escherichia coli. One of the colonies isolated contained a plasmid encoding a portion of the noncoding region of the TNFR gene. Transfection of human lymphoblastoid cells with this DNA gave rise to high-level production of TNFR. Fluorescent TNF bound to these transfectants is fully and specifically displaced by an excess of TNF. The rescued clone contains approx. 10 kb of human genomic DNA including the 3'-untranslated region of TNFR and several Alu sequences; apparently during the selection procedure in human cells, recombination occurred to rescue a portion of the TNFR gene. Transient transfection was used to narrow down the region responsible for TNFR induction to 5.2 kb. The mechanism by which this clone induces TNFR expression has not been determined.

Ultrastructural localization of gustducin immunoreactivity in microvilli of type II taste cells in the rat.

Gustducin is a transducin-like G protein (guanine nucleotide-binding protein) that is expressed in taste bud cells. Gustducin is believed to be involved in bitter and possibly sweet taste transduction. In the present study, we demonstrate that a subset of type II cells displays immunoreactivity to antisera directed against gustducin in taste buds of rat circumvallate papilla. Immunogold particles are present both in the microvilli and cytoplasm of the immunoreactive cells. Quantitative analysis of the data suggests that the number of colloidal gold particles (P<0.001) and nanogold particles (P<0.01) in the immunoreactive type II cells are much greater than in type I cells. There are also approximately 2.5 times (P<0.05) as many colloidal gold particles associated with the microvilli versus the cytoplasm in the immunoreactive type II cells. The ultrastructural distribution of gustducin immunoreactivity is consistent with its proposed role in the initial events of sensory transduction by gustatory receptor cells.

33P is preferable to 35S for labeling probes used in in situ hybridization.

We compared RNA probes labeled with 35S-UTP and 33P-UTP for use in in situ hybridizations. 33P-UTP was readily incorporated into in vitro transcribed RNA, producing 33P-labeled riboprobes of high specific activity. When the 33P- and 35S-labeled riboprobes were compared in in situ hybridizations using two different tissues, we found that the 33P-labeled riboprobes were less "sticky" than the 35S-labeled riboprobes, giving significantly less nonspecific background hybridization. Because of the low level of background stickiness, it was possible to use ten times more 33P-labeled riboprobe than 35S-labeled riboprobe without appreciably increasing background hybridization. Our findings indicate that, in most cases, 33P is the isotope of choice when labeling probes for in situ hybridizations.

Panning transfected cells for electrophysiological studies.

Panning was used to select co-transfected cells expressing plasmid-encoded ion channels. Adherent cells were cotransfected by the CaPO4 method with a plasmid encoding a cell surface marker (CD8) along with another plasmid encoding an ion channel. At 1-3 days post-transfection, the cells were suspended, treated with a biotinylated CD8-specific antibody and placed into streptavidin-coated bacterial petri dishes. After 2 h, these dishes were washed with a saline solution to remove cells that did not adhere to the streptavidin-coated dishes. By using molar ratios of > or = 8:1 of the ion channel encoding plasmid to the CD8 plasmid, we found that > or = 50% of the panned cells that adhered to coated dishes were positive for expression of the co-selected gene. Cells expressing plasmid-encoded channels (voltage-dependent sodium channels or cystic fibrosis transregulator chloride channels) were assayed using whole-cell recording, perforated-patch recording and single-channel recording. The method was tested on tsA201 and NIH3T3 cells, the latter of which transfected very poorly (usually < 4% efficiency) with our standard protocols. When the co-selected plasmid encoded the bacterial beta-galactosidase gene, it was possible to determine by histological assay the percentage of positively transfected cells (with and without panning). Panning in some cases increased the percentage of positively cotransfected cells by more than 20-fold. This technique is particularly useful when selecting co-transfected cells for electro-physiological recordings on individual cells.

Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction.

BACKGROUND: Taste receptor cells are responsible for transducing chemical stimuli into electrical signals that lead to the sense of taste. An important second messenger in taste transduction is IP3, which is involved in both bitter and sweet transduction pathways. Several components of the bitter transduction pathway have been identified, including the T2R/TRB taste receptors, phospholipase C beta2, and the G protein subunits alpha-gustducin, beta3, and gamma13. However, the identity of the IP3 receptor subtype in this pathway is not known. In the present study we used immunocytochemistry on rodent taste tissue to identify the IP3 receptors expressed in taste cells and to examine taste bud expression patterns for IP3R3. RESULTS: Antibodies against Type I, II, and III IP3 receptors were tested on sections of rat and mouse circumvallate papillae. Robust cytoplasmic labeling for the Type III IP3 receptor (IP3R3) was found in a large subset of taste cells in both species. In contrast, little or no immunoreactivity was seen with antibodies against the Type I or Type II IP3 receptors. To investigate the potential role of IP3R3 in bitter taste transduction, we used double-label immunocytochemistry to determine whether IP3R3 is expressed in the same subset of cells expressing other bitter signaling components. IP3R3 immunoreactive taste cells were also immunoreactive for PLCbeta2 and gamma13. Alpha-gustducin immunoreactivity was present in a subset of IP3R3, PLCbeta2, and gamma13 positive cells. CONCLUSIONS: IP3R3 is the dominant form of the IP3 receptor expressed in taste cells and our data suggest it plays an important role in bitter taste transduction.

Human taste cells express the G protein alpha-gustducin and neuron-specific enolase.

Expression of the alpha-subunit of the taste-specific G protein alpha-gustducin and the glycolytic enzyme neuron-specific enolase (NSE) was investigated immunohistochemically in human circumvallate and foliate taste papillae. Immunofluorescence for alpha-gustducin was observed in taste cells of both types of papillae and exhibited two patterns of immunofluorescence, plasmalemmal and cytosolic. The plasmalemmal pattern showed intense immunofluorescence localized to the apical region, and was exhibited by most immunoreactive taste cells. In contrast, the cytosolic pattern, observed in one or two immunoreactive cells in a taste bud per section, showed immunofluorescence distributed throughout the cytoplasm. A subpopulation of alpha-gustducin-immunoreactive taste receptor cells, most of which exhibited the cytosolic pattern, also expressed NSE. Optical sectioning, using confocal laser scanning microscopy, demonstrated the highest level of expression of alpha-gustducin in the apical microvillar region of the taste cells in close apposition to the taste pore. These studies indicate conservation of epitopes of alpha-gustducin in humans and rats, and suggest that this G protein is associated with taste transduction in both rats and humans. The patterns of expression of alpha-gustducin, and coexpression with NSE, may correlate with specialized subtypes or developmental stages of taste receptor cells.

SC1: a marker for astrocytes in the adult rodent brain is upregulated during reactive astrocytosis.

Astrocytes are the most abundant cell type in the mammalian central nervous system (CNS), and are involved in many processes critical for normal CNS maintenance and function. We have used double-label immunocytochemistry and in situ analysis to show that the SPARC (secreted protein acidic and rich in cysteine)-related protein SC1, co-localizes with the astrocyte marker glial fibrillary acidic protein (GFAP) in the adult rodent brain. Thus, SC1 is an astrocyte marker that may be used to investigate astrocyte heterogeneity and analyze glial cell lineages during neural development. Consistent with the presence of SC1 and GFAP in astrocytes, both proteins were markedly upregulated following reactive astrocytosis induced by focal mechanical trauma. Therefore, SC1 may play an important role in reactive astrocytosis subsequent to a wide variety of neural trauma, including neurodegenerative diseases and acute neural damage.

Molecular cloning of G proteins and phosphodiesterases from rat taste cells.

To identify and characterize those proteins involved in taste transduction, we cloned G proteins and phosphodiesterases from rat taste tissue. Using degenerate primers corresponding to conserved regions of G protein alpha subunits, the polymerase chain reaction was used to amplify and clone eight distinct cDNAs: alpha i-2, alpha i-3, alpha 12, alpha 14, a(s), alpha t-rod, alpha t-cone and alpha gustducin. alpha i-3, alpha 14, alpha s, and alpha t-rod are more highly expressed in taste tissue than in the surrounding nonsensory tissue. alpha gustducin is only expressed in taste cells. Rod transducin had previously been found only in the rod cells of the retina, where it converts light stimulation of rhodopsin into activation of cGMP phosphodiesterase. The primary sequence of alpha gustducin shows striking similarities to rod transducin in the receptor interaction domain and the phosphodiesterase activation site. We propose that gustducin and transducin regulate phosphodiesterase activity in taste cells and that this may promote bitter transduction and inhibit sweet transduction. Consistent with this proposal, we cloned two types of cAMP PDE from taste tissue: dnc-1 and PDE-3.