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. 2006 Jun 13;103(24):9310-4.
doi: 10.1073/pnas.0600697103. Epub 2006 Jun 5.

Ric-8B promotes functional expression of odorant receptors

Luiz Eduardo C Von Dannecker  1 , Adriana F Mercadante, Bettina Malnic
Affiliations

Ric-8B promotes functional expression of odorant receptors

Luiz Eduardo C Von Dannecker et al. Proc Natl Acad Sci U S A. .

Abstract (VSports手机版)

Odorants are detected by a large family of odorant receptors (ORs) expressed in the nose. The information provided by the ORs is transmitted to specific regions of the brain, leading to odorant perception VSports手机版. The determination of the odorant specificities of the different ORs will contribute to the understanding of how odorants are discriminated by the olfactory system. However, to date only a few ORs have been linked to odorants they recognize, because ORs are poorly expressed on the cell surface of heterologous cells. Here we show that Ric-8B, a putative guanine nucleotide exchange factor for Galphaolf, promotes efficient heterologous expression of ORs. Our results also show that Ric-8B enhances accumulation of Galphaolf at the cell periphery, indicating that it promotes functional OR expression by improving the efficiency of OR coupling to Galphaolf. Expression systems containing Galphaolf and Ric-8B should contribute to the functional characterization of ORs. .

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

"V体育安卓版" Figures

Fig. 1.
Fig. 1.
Ric-8B promotes functional expression of mOR-EG. (A) Production of cAMP was measured in HEK293 T cells transfected as indicated with Gαolf, Ric-8B, RTP1, mOR-EG, or rhodopsin-tagged mOR-EG (RhoOR-EG) expression vectors. Activity was recorded in the absence (white bars) or presence (gray bars) of 300 μM eugenol. The data are expressed as means ± SD from three different experiments. cAMP accumulation is presented as femtomole per million cells. (∗∗, P < 0.007.) (B) Production of cAMP was measured as described above in cells transfected with Gαolf, Ric-8B, and mOR-EG expression vectors (gray bars) or with Gαolf, Ric-8B, and rhodopsin-tagged mOR-EG (RhoOR-EG) expression vectors (black bars). Activity was recorded in the absence (−) or presence (300 μM) of the indicated odorants. [∗∗, P < 0.009 compared with transfection of the same expression vectors but no addition of odorant (−)].
Fig. 2.
Fig. 2.
Ric-8B promotes functional expression of mOR-S6. (A) Production of cAMP was measured in HEK293 T cells transfected as indicated with Gαolf, Ric-8B, RTP1, and rhodopsin-tagged mOR-S6 (RhoS6) expression vectors. Activity was recorded in the absence (white bars) or presence (gray bars) of 300 μM nonanedioic acid. The data are expressed as means ± SD from three different experiments. cAMP accumulation is presented as femtomole per million cells. (∗, P = 0.0219.) (B) Production of cAMP was measured in cells expressing Gαolf, Ric-8B, RTP1, and rhodopsin-tagged mOR-S6 after stimulation with the indicated concentrations of nonanedioic acid. The data are expressed as the means from two different experiments.
Fig. 3.
Fig. 3.
Ric-8B promotes functional expression of mOR-I7. Production of cAMP was measured in HEK293 T cells transfected as indicated with Gαolf, Ric-8B, RTP1, and mOR-I7 expression vectors. Activity was recorded in the absence (white bars) or presence (gray bars) of 300 μM heptanal. The data are expressed as means ± SD from three different experiments. cAMP accumulation is presented as femtomole per million cells. (∗, P = 0.0288; ∗∗, P = 0.0035.)
Fig. 4.
Fig. 4.
Cellular localization of Gαolf and Ric-8B detected by immunofluorescence. (A–D) HEK293T cells transfected with Gαolf (A) or Gαolf and Ric-8B (B–D) expression vectors were permeabilized and immunostained by using anti-Gαolf (A and B) or anti-FLAG (C) and visualized on a fluorescence microscope. The merge between the images shown in B and C is shown in D. (E–G) Cells coexpressing Gαolf and Ric-8B and double-labeled with anti-Gαolf (E) and anti-FLAG (F) antibodies show that Gαolf and Ric-8B colocalize at the cell periphery (G, a merge of E and F). (H–J) Representative cells transfected with Gαolf alone (H) or with Gαolf and Ric-8B (I) stained with anti-Gαolf are shown. The percentages of cells with strong Gαolf fluorescent signal, like the ones shown in I (green cells), and with Ric-8B or Ric-8BΔ9 signal (red cells) in cells transfected with Gαolf alone, with Gαolf and Ric-8B, or with Gαolf and Ric-8BΔ9 were counted (700 cells from three independent experiments) and are shown in J. (∗∗∗, P < 0.0001.) Nuclei were visualized with Hoechst dye.
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