Abstract
The discovery of odorant receptors led to endeavors in matching them with their cognate ligands. Although it has been challenging to functionally express odorant receptors in heterologous cells, previous studies have linked efficient odorant receptor expression with N-terminal modifications and accessory proteins, including the receptor-transporting proteins (RTPs) and Ric8b. Here we have shown that a shorter form of RTP1, RTP1S, supports robust cell-surface and functional expression of representative odorant receptors. Using a combination of accessory proteins, including RTP1S, Ric8b, and G(alphaolf), a diverse set of untagged odorant receptors were successfully expressed heterologously due to the synergistic effects among the various accessory proteins. Furthermore, the addition of an N-terminal rhodopsin tag to the odorant receptors, along with the same set of accessory proteins, exhibits an additional level of synergism, inducing enhanced odorant receptor responses to odorants and thus defining a more efficient heterologous expression system. We then showed that the presence or absence of different N-terminal tags has little effect on the ligand specificity of odorant receptors, although the amount of receptor expressed can play a role in the ligand response profile. The accuracy of the odorant receptor heterologous expression system involving tagged odorant receptors and various accessory proteins promises success in high throughput de-orphaning of mammalian odorant receptors.
Highlights
15284 JOURNAL OF BIOLOGICAL CHEMISTRY sory neurons first successfully matched an odorant to a cloned Odorant receptors (ORs) [3]
Characterization of a Shorter and More Potent Form of RTP1— During the course of analyzing the function of a deletion series of RTP1 in OR expression,4 we found that one of the deletions, which started from the second methionine of the open reading frame, induced stronger expression of ORs when co-expressed in HEK293T cells
Western blot analysis using lysates from HEK293T cells expressing RTP1S or RTP1L as well as from the olfactory epithelium revealed that the RTP1 proteins expressed in the olfactory epithelium appeared to migrate to the same position as that of RTP1S, whereas no band corresponding to RTP1L was seen, suggesting that RTP1S is a major form of RTP1 produced in vivo (Fig. 1B)
Summary
Plasmid Construction—Rho-(N-MNGTEGPNFYVPFSNATGVVR-C), FLAG-(N-MDYKDDDDK-C), and HA-(N-MYPYDVPDYA-C) tags were subcloned into pCI mammalian expression vector (Promega). 0.8 g of OR DNA and 0.8 g total of all accessory proteins (RTP1S, RTP1L, Hsc70t, Ric8b, and/or G␣olf) or pCI were transfected per dish. The cells were washed in Hanks’ balanced buffer solution (Invitrogen) containing 15 mM NaN3 and 10 mM HEPES followed by incubation with Cy3-conjugated donkey anti-mouse IgG (Jackson Immunologicals) at 4 °C for 30 min and fixation in 1% paraformaldehyde at 4 °C and mounting in Mowiol. For each 96-well plate, 1 g of CRE-Luc, 1 g of pRL-SV40, 5 g of OR, and 1 g total of all accessory proteins (RTP1S, RTP1L, Hsc70t, Ric8b, and/or G␣olf) or pCI were transfected. HEK293T cells were seeded on glass-bottom plates with poly-D-lysine-coated coverslips (MatTek) and plasmid DNA of Olfr and either the accessory protein RTP1S, RTP1L, or pCI, and a G protein chimera, G15olf, was transfected with Lipofectamine 2000 and incubated for 24 h. Chemicals—All odorants were purchased from Sigma, except octanoic acid, which was purchased from Calbiochem
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