Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Abstract

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein Gαolf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2 . Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3 . To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs. Protocol Procedure:The procedure comprises of three parts completed over a total time of 3 days: transferring cells, transfection and immunocytochemistry, one step carried out per day. Transfer and transfection of cells over the first two days must be carried out in sterile conditions in a laminar flow chamber. 2. Grow cells to a desired confluency in a 100mm culture plate, depending on the number of plates required to set up. It is important to determine the fraction of cells to be transferred to avoid overgrown or sparse cells: for instance, from a 100% confluent 100 mm dish, one may transfer about 3% to a 35 mm dish to obtain approximately 30% confluency in the latter. 3. Prior to transferring cells, in the sterile laminar flow chamber, coat 22 X 22 mm glass cover slips with sterile poly D lysine (1mg/ mL) and place one in each 35 mm cell culture dish, coated side up for plating cells (Figure 1). Allow the solution to dry for 5-10 minutes. During this interval, the UV source in the laminar chamber may be turned on to sterilize the cover slips, if desired. Poly D lysine helps heterologous cells stick to the cover slips. 4. To transfer cells, aspirate out the existing media in the 100mm dish, wash by carefully adding 8 mL sterile PBS, aspirate PBS and trypsinize the cells using 3 mL 0.05% Trypsin-EDTA; when cells detach...

Functional Evolution of Mammalian Odorant Receptors

“…To determine if individual differences in receptor activity are influenced by the amount of receptor at the cell surface, we assessed cell surface expression of each OR [36] , [37] . Using fluorescent immunocytochemistry in live cells, we measured the Cy3 signal intensity of each ortholog and paralog in our receptor set and compared them against the human counterpart.…”

“…Live-cell surface staining was done as previously described [25], [36], [37]. Briefly, Hana3A cells were seeded on poly-d-lysine coared glass coverslips in 35 mm dishes and transfected with 1000 ng OR, 250 ng RTP1S, and 50 ng of EGFP to control for transfection efficiency.…”

“…Hana3A cells were maintained in minimal essetial medium (Sigma) containing 10% fetal bovine serum (Sigma) (M10), 500 µg/ml peniciilin-streptomycin (Invitrogen) and 6 µg/ml amphotericin B (Sigma) [34] . Live-cell surface staining was done as previously described [25] , [36] , [37] . Briefly, Hana3A cells were seeded on poly-d-lysine coared glass coverslips in 35 mm dishes and transfected with 1000 ng OR, 250 ng RTP1S, and 50 ng of EGFP to control for transfection efficiency.…”

Ectopically expressed olfactory receptors OR51E1 and OR51E2 suppress proliferation and promote cell death in a prostate cancer cell line

“…Most GPCRs can be produced in a functionally active state in vitro using standard methods such as transient transfection of model cell lines. ORs, however, require the presence of special chaperones and/or modification with signal peptides and other sequences for receptor stability and trafficking to the surface ( 12 , 13 , 14 ). These discoveries facilitated expression of ORs in specially engineered cell lines via transient transfection, thereby allowing investigation of their function.…”

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

“…If previously uncharacterized receptors fail to respond to an odor, it may be due to lack of expression at the cell surface rather than a lack of interaction between odorant and receptor. Receptor cell surface expression can be examined via immunofluorescence before drawing conclusions from negative assay results 27,42 . Finally, due to low background luciferase activity in no-odor conditions, our assay is not designed to detect inhibitory responses.…”