RESUMEN

The mammalian main olfactory pathway detects volatile chemicals using two families of G-protein-coupled receptors: a large repertoire of canonical odorant receptors and a much smaller set of trace amine-associated receptors (TAARs). The TAARs are evolutionarily conserved in vertebrates, including humans, suggesting an indispensible role in olfaction. However, little is known about the functional properties of TAARs when expressed in native olfactory sensory neurons. Here we describe experiments using gene targeting, electrophysiology, and optical imaging to study the response properties of TAAR-expressing sensory neurons and their associated glomeruli in mice. We show that olfactory sensory neurons that express a subset of the TAAR repertoire are preferentially responsive to amines. In addition, neurons expressing specific TAARs, TAAR3 or TAAR4, are highly sensitive and are also broadly tuned-responding to structurally diverse amines. Surprisingly, we find that TAAR4 is exquisitely sensitive, with apparent affinities for a preferred ligand, phenylethylamine, rivaling those seen with mammalian pheromone receptors. We provide evidence that this unprecedented sensitivity is mediated via receptor coupling to the canonical odorant transduction cascade. The data suggest that the TAARs are evolutionarily retained in the olfactory receptor repertoire to mediate high-sensitivity detection of a biologically relevant class of odorous stimuli.

Asunto(s)

Aminas/análisis , Receptores Acoplados a Proteínas G/fisiología , Aminas/metabolismo , Animales , Electrofisiología/métodos , Marcación de Gen , Humanos , Masculino , Ratones , Microscopía Fluorescente , Neuroimagen , Odorantes , Bulbo Olfatorio/fisiología , Mucosa Olfatoria/fisiología , Vías Olfatorias/fisiología , Neuronas Receptoras Olfatorias/fisiología , Técnicas de Placa-Clamp , Feromonas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología

RESUMEN

Olfactory stimuli are detected by over 1,000 odorant receptors in mice, with each receptor being mapped to specific glomeruli in the olfactory bulb. The trace amine-associated receptors (TAARs) are a small family of evolutionarily conserved olfactory receptors whose contribution to olfaction remains enigmatic. Here, we show that a majority of the TAARs are mapped to a discrete subset of glomeruli in the dorsal olfactory bulb of the mouse. This TAAR projection is distinct from the previously described class I and class II domains, and is formed by a sensory neuron population that is restricted to express TAAR genes prior to choice. We also show that the dorsal TAAR glomeruli are selectively activated by amines at low concentrations. Our data uncover a hard-wired, parallel input stream in the main olfactory pathway that is specialized for the detection of volatile amines.

Asunto(s)

Aminas/metabolismo , Vías Olfatorias/fisiología , Percepción Olfatoria/fisiología , Compuestos Orgánicos Volátiles/metabolismo , Animales , Femenino , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Familia de Multigenes/fisiología , Vías Olfatorias/metabolismo , Percepción Olfatoria/genética , Neuronas Receptoras Olfatorias/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Especificidad por Sustrato , Volatilización

RESUMEN

Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through target primed reverse transcription (TPRT). RLE-bearing elements have been used as a model system for investigating non-LTR retrotransposon integration. R2 elements target a specific site in the 28S rDNA gene. We previously demonstrated that the two major sub-classes of R2 (R2-A and R2-D) target the R2 insertion site in an opposing manner with regard to the pairing of known DNA binding domains and bound sequences-indicating that the A- and D-clades represent independently derived modes of targeting that site. Elements have been discovered that group phylogenetically with R2 but do not target the canonical R2 site. Here we extend our earlier studies to show that a separate R2-A clade element, which targets a site other than the canonical R2 site, does so by using the N-terminal zinc fingers and Myb motifs. We further extend our targeting studies beyond R2 clade elements by investigating the ability of the N-terminal zinc fingers from the nematode NeSL-1 element to target its integration site. Our data are consistent with the use of an N-terminal DNA binding domain as one of the major targeting determinants used by RLE-bearing non-LTR retrotransposons to secure a protein subunit near the insertion site. This N-terminal DNA binding domain can undergo modifications, allowing the element to target novel sites. The binding orientation of the N-terminal domain relative to the insertion site is quite variable.