Partners and International Organizations
(English)
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Prof. V. Torre, Prof. H. Breer, Prof. B. Kaupp, Dr. A. Menini
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Abstract
(English)
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The final third year of the project was dedicated to finishing the ongoing work on the heterologous expression system for of the olfactory receptor (OR) genes. Additionally, some studies were carried out on profiling the endogenous responses of rat olfactory neurons.
1) Expression of OR genes in established cell lines
We have demonstrated that setting up the functional expression system for olfactory receptor (OR) genes is hampered by inability of the receptors to reach the plasma membrane of the conventional non-neuronal cells. Using highly efficient Semliki Forest Virus (SFV) expression system, suitable for infecting a wide range of cell lines and primary cultures, we showed that although the ORs are produced in high amounts, they fail to be properly targeted to the cell surface and are retained in the endoplasmic reticulum. Additionally, in these cell lines the odorant receptors are not properly glycosylated. Unfortunately, even using the cell lines of neuronal or even olfactory epithelial origin failed to solve the receptor localisation problem, possibly due to the incomplete differentiation of these cells. Similarly, addition of non-olfactory amino acid sequences at the N-termini of the ORs (rhodopsin tag, N-terminus of b2-adrenergic receptor, cleaveable signal sequence of influenza hemagglutinin) did not render the receptors functional.
2) Expression of OR genes in the rat primary olfactory epithelium cultures
The rat I7 receptor was tagged with the Green Fluorescent Protein (GFP), and the fusion receptor protein expressed in the primary cultures of the olfactory epithelium (OE). Confocal microscopy was performed to assay co-localisation of the I7-GFP and the plasma membrane labelled with Concanavalin A-Texas Red. Evaluation of the data, including the scatter plot of red and green signals, demonstrated that GFP-tagged I7 co-localised with the plasma membrane of the cultured OE neurons. Similarly, when the mature rat OE neurons were infected with SFV-I7GFP, the receptor was localised on the plasma membrane and functional. Analysing the receptor glycosylation status in these cultures we showed an additional tunicamycin-sensitive band corresponding to the fully glycosylated receptor.
3) Functional expression of OR genes in mature OE neurons
The OE neurons infected with SFV-I7 were tested for responses to octanal, and the other I7 agonists, identified by S. Firestein (Zhao et al., Science (1998) 279, 237-242). The background response to octanal was small, never exceeding 1% of the responsive cells in a field. However, the number of neurons responding to octanal significantly increased in cultures infected with recombinant SFV-I7. Typically, at least 30% of all the cells in the field were responding to the I7 ligands octanal and nonanal. There was no response to buffer, or to C4 aldehyde butanal, which is in agreement with published data. The neurons infected with I7-GFP fusion gave responses to octanal, indicating that the insertion of GFP at the C terminus of an OR does not interfere with the receptor coupling to G proteins.
4) Endogenous receptor profiling in primary olfactory sensory neurons
A series of experiments was performed to monitor the response patterns of individual olfactory neurons which express the endogenous receptor(s). Uninfected OE neurons were exposed to pure sandalwood oil, and the rare responsive neurons identified by calcium imaging. They were subsequently probed with selected members of the sandalwood family of odorants from the Givaudan collection. The patterns of activation were quite similar in independent experiments, indicating the fairly narrow tuning of sandalwood receptor(s). Experiments are underway to isolate the responsive cells and clone the expressed receptor(s) using single-cell RT-PCR.
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