Question

1. Olfaction occurs when volatile compounds bind to specific odorant receptors. In mammals, each olfactory receptor neuron in the olfactory nasal epithelium expresses a single type of odorant receptor. These odorant receptors constitute a large multigene family (>1000 members) of related proteins. Binding of odorant induces a signaling cascade that is mediated via a G protein, G_aolf. Recent studies suggest that there are a small number of olfactory sensory neurons in the nasal epithelium that express members of the trace-amine associated receptor (TAAR) family, chemoreceptors that are G-protein coupled receptors (GPCRs) but are unrelated to classical odorant receptors (not necessary for this pset, but for further background, you can see Liberles and Buck, 2006, Nature 442:645-650). The mouse genome encodes 15 TAAR genes while the human genome encodes 6.

1. In order to examine the expression pattern of different TAARs in the olfactory nasal epithelium, researchers localized TAAR RNA by in situ hybridization in pairwise combinations. All possible pairwise combinations of the 15 mouse TAARs were examined. A typical example of the results obtained is shown in the top set of panels in the figure below in which TAAR6 and TAAR7 have been localized with fluorescent probes in the nasal epithelium of mouse. The TAAR6 probe was labeled with a green fluor, the TAAR7 probe with a red fluor. The lower setoff panels shows localization of mouse odorant receptor 28 (MOR28; green), a classical odorant receptor, and TAAR6 (red). Each stained patch in the images is the staining pattern of an individual olfactory neuron. The “merge” panels show the two other images superimposed. What do these data suggest about expression patterns of the TAARs?

Taar6 Taar7 Merge MOR28 Taar6 Merge

b. A number of cell lines that produce neither classical odorant receptors nor TAARs have each been transfected with the gene encoding a different TAAR. The cells have also been cotransfected with a gene encoding secreted alkaline phosphatase (SEAP) under control of a cAMP-responsive element. The cells are then exposed to various amines, as shown in the following figure, and SEAP activity in the medium is determined. The figure shows data for some representative TAARs (m=mouse, h=human). What do these data reveal about TAARs? What does the SEAP activity assay reveal about the signaling pathway utilized by chemoreception involving TAARs?

c. in a third set of studies, SEAP activity was measured in cells expressing mouse TAAR5 (mTAAR5) following exposure of the cells to diluted urine derived from two strains of mice or from humans, as indicated on the graphs below. Mice reach puberty at about one month of age. What do these data suggest may be a biological function for the TAAR5 neurons in mice? What additional studies would you conduct to support your hypothesis?

Answer 1

a. The main objective of RNA in-situ hybridization is to localize and assess the level of gene expression. As observed from the given figure:

Observation 1: In the top panel, TAAR6 and TAAR7 genes are expressed almost in equal amounts in the olfactory neurons, though in small amounts.

Observation 2: In the lower panel, the gene expression level for MOR28 is much higher than TAAR6.

CONCLUSION: though the TAAR(trace amine-associated receptors) are coexpressing in the olfactory neurons in the comparable amounts. but, when compared with the MOR28, an odorant receptor, it can be predicted that the odorant receptors have high expression in the olfactory neurons as compared to the TAARs.   

b. The data given above shows the SEAP activity of each TAAR for different amino acid ligands.

• comparing the TAARs of mouse and human: hTAAR1(human) responds to B, C, G, H (refer the diagrams)
• mTAAR3 responds to G and H while  mTAAR4 of mouse responds to the B amine group.
• Looking all together, mTAAR3 and mTAAR4 of the mouse show a certain amount of analogy with that of hTAAR1 in humans as they react to the same amine groups.

c. with the exposure to urine, the expression of mTAAR5 has been affected only in the male mice strain s while there is no change observed in the mTAAR5 expression level in humans.

anther study observed that the male mice that shows the change, their age ranges from the 25 to 46 days.