RESUMEN
The rod and cone transducins are specific G proteins originally thought to be present only in photoreceptor cells of the vertebrate retina. Transducins convert light stimulation of photoreceptor opsins into activation of cyclic GMP phosphodiesterase (reviewed in refs. 5-7). A transducin-like G protein, gustducin, has been identified and cloned from rat taste cells. We report here that rod transducin is also present in vertebrate taste cells, where it specifically activates a phosphodiesterase isolated from taste tissue. Furthermore, the bitter compound denatonium in the presence of taste-cell membranes activates transducin but not Gi. A peptide that competitively inhibits rhodopsin activation of transducin also blocks taste-cell membrane activation of transducin, arguing for the involvement of a seven-transmembrane-helix G-protein-coupled receptor. These results suggest that rod transducin transduces bitter taste by coupling taste receptor(s) to taste-cell phosphodiesterase. Phosphodieterase-mediated degradation of cyclic nucleotides may lead to taste-cell depolarization through the recently identified cyclic-nucleotide-suppressible conductance.
Asunto(s)
Hidrolasas Diéster Fosfóricas/metabolismo , Papilas Gustativas/metabolismo , Transducina/metabolismo , Animales , Bovinos , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Células Fotorreceptoras/metabolismo , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Compuestos de Amonio Cuaternario/farmacología , ARN Mensajero/metabolismo , Ratas , Rodopsina/metabolismo , Papilas Gustativas/efectos de los fármacos , Papilas Gustativas/enzimologíaRESUMEN
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.
Asunto(s)
Clonación Molecular , Proteínas de Unión al GTP/genética , Hidrolasas Diéster Fosfóricas/genética , Transmisión Sináptica/genética , Papilas Gustativas/fisiología , Gusto/genética , Secuencia de Aminoácidos/genética , Animales , ADN Complementario/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Transducción de Señal/genética , Transducina/genéticaRESUMEN
In vertebrate rod cells, the activated alpha-subunit of rod transducin interacts with the gamma (regulatory) subunits of phosphodiesterase to disinhibit the catalytic subunits. A 22-amino acid long region of rod transducin involved in phosphodiesterase activation has recently been identified. We have used peptides from this region of rod transducin and from several other G protein alpha-subunits to study the nature and specificity of the G protein alpha-effector interaction. Although peptides derived from rod transducin, cone transducin and gustducin are similar, only the rod peptide is capable of activating rod phosphodiesterase. Using substituted peptides we have identified five residues on one exposed face of rod transducin as important to phosphodiesterase activation. These results disagree with previous models which propose that loop regions of rod transducin interact with phosphodiesterase gamma.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Transducina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Células Fotorreceptoras Retinianas Bastones/enzimología , Homología de Secuencia de AminoácidoRESUMEN
In the vertebrate taste cell, heterotrimeric guanine nucleotide-binding proteins (G proteins) are involved in the transduction of both bitter and sweet taste stimulants. The bitter compound denatonium raises the intracellular Ca2+ concentration in rat taste cells, apparently via G protein-mediated increases in inositol trisphosphate. Sucrose causes a G protein-dependent generation of cAMP in rat taste bud membranes; elevation of cAMP levels leads to taste cell depolarization. To identify and characterize those proteins involved in the taste transduction process, we have cloned G protein alpha subunit (G alpha) cDNAs from rat taste cells. Using degenerate primers corresponding to conserved regions of G proteins, we used the polymerase chain reaction to amplify and clone taste cell G alpha cDNAs. Eight distinct G alpha cDNAs were isolated, cloned and sequenced from a taste cell library. Among these clones was alpha gustducin, a novel taste G alpha closely related to the transducins. In addition to alpha gustducin, we cloned rod and cone transducins from taste cells. This is the first identification of transducin expression outside photoreceptor cells. The primary sequence of alpha gustducin shows similarities to the transducins in the receptor interaction domain and the phosphodiesterase activation site. These sequence similarities suggest that gustducin and transducin regulate taste cell phosphodiesterase, probably in bitter taste transduction.
Asunto(s)
Transducina/genética , Animales , Clonación Molecular , ARN Mensajero/biosíntesis , Gusto/fisiologíaRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Herpesvirus Humano 4/genética , Receptores de Superficie Celular/genética , Factor de Necrosis Tumoral alfa/genética , Secuencia de Bases , Northern Blotting , Southern Blotting , Línea Celular Transformada , Clonación Molecular , Citometría de Flujo , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Receptores del Factor de Necrosis Tumoral , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
A new procedure using an asymmetrically tailed linker-primer plasmid has been developed to prepare extremely high complexity cDNA libraries. This procedure yields plasmid primed libraries with a final form equivalent to those made by the procedure of Okayama and Berg. However, the number of steps involved in library preparation is decreased. The form of the vector is such that one end of the linearized linker-primer plasmid has a 3' terminal extension of 40 deoxythymidylate residues (the dT end). The other end has a 3' terminal extension of 10 deoxycytidylate residues (the dC end). The dC end of the plasmid is blocked to further 3' extension by a 3' phosphate group. This configuration enables one to prime first strand cDNA synthesis at the dT end, tail the 3' end of the cDNA with deoxyguanylate residues without tailing the dC end (due to the 3' phosphate block). The plasmid primed cDNA can then be self-annealed and the 3' phosphate blocking group removed during the synthesis of double stranded cDNA. The efficiency of this procedure is significantly higher than other methods (including phage based libraries): linker-primer libraries have 15 to 900-fold higher complexity than libraries prepared by other methods. A cloning efficiency of 9 x 10(8) colonies per microgram of linker-primer DNA was achieved. This method should be useful for the cloning of cDNAs corresponding to extremely rare mRNAs.
Asunto(s)
Clonación Molecular/métodos , ADN/genética , Biblioteca de Genes , Vectores Genéticos/genética , Plásmidos/genética , Poli A/genética , Secuencia de Bases , Escherichia coli/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/genética , Transformación Bacteriana/genéticaRESUMEN
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.