RESUMEN
Taste represents a major form of sensory input in the animal kingdom. In mammals, taste perception begins with the recognition of tastant molecules by unknown membrane receptors localized on the apical surface of receptor cells of the tongue and palate epithelium. We report the cloning and characterization of two novel seven-transmembrane domain proteins expressed in topographically distinct subpopulations of taste receptor cells and taste buds. These proteins are specifically localized to the taste pore and are members of a new group of G protein-coupled receptors distantly related to putative mammalian pheromone receptors. We propose that these genes encode taste receptors.
Asunto(s)
Proteínas de Unión al GTP/genética , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/fisiología , Papilas Gustativas/química , Papilas Gustativas/fisiología , Animales , Anticuerpos , Expresión Génica/fisiología , Humanos , Hibridación in Situ , Mamíferos , Ratones , Datos de Secuencia Molecular , Neuronas Aferentes/química , Neuronas Aferentes/fisiología , ARN Mensajero/análisis , Conejos , Ratas , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/inmunología , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Transducina/análisisRESUMEN
Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many haptens are available as N-hydroxysuccinimide esters or isothiocyanates. Unfortunately, the hapten group may become covalently attached to or close to the combining site of antibodies, lectins, or other ligand-binding proteins during the process of haptenylation. Thus, the interaction of the corresponding protein with its ligand may become severely hampered. To overcome this restriction, we developed a novel procedure for the haptenylation of polyclonal antibodies that combines purification and haptenylation. Haptenylation during adsorption to the affinity matrix combines two advantages: the antigen binding site is protected and the labeling procedure becomes most convenient, as overlabeled proteins and unreacted haptens are easily removed by simple washing. Haptenylation during adsorption to the affinity matrix is a two-phase reaction, which requires different conditions to the conventional procedure. To obtain such optimal conditions, stabilities and reactivities of N-hydroxysuccinimide esters and isothiocyanate groups were investigated with a newly developed assay. Based on this information, antibodies against two recently described calcium-binding proteins, NCS-1 and NVP-3, were biotinylated or digoxigenylated. The haptenylated antibodies were successfully applied for biochemical determination and simultaneous immunoenzymatic double labeling of the two proteins.
Asunto(s)
Anticuerpos/inmunología , Proteínas de Unión al Calcio/inmunología , Cromatografía de Afinidad , Haptenos , Proteínas del Tejido Nervioso/inmunología , Neuropéptidos/inmunología , Receptores Sensibles al Calcio , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico , Absorción , Adsorción , Animales , Anticuerpos/aislamiento & purificación , Sitios de Unión de Anticuerpos , Biotina/análogos & derivados , Biotinilación , Química Encefálica , Señalización del Calcio , Digoxigenina , Ensayo de Inmunoadsorción Enzimática , Fluoresceína-5-Isotiocianato , Concentración de Iones de Hidrógeno , Inmunoglobulina G/inmunología , Neurocalcina , Proteínas Sensoras del Calcio Neuronal , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/inmunología , SuccinimidasRESUMEN
The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.