A bi-functional IL-6-HaloTag® as a tool to measure the cell-surface expression of recombinant odorant receptors and to facilitate their activity quantification

Abstract

The functional cell surface expression of recombinant odorant receptors typically has been investigated by expressing N-terminally extended, "tagged" receptors in test cell systems, using antibody-based immunocytochemistry or flow cytometry, and by measuring odorant/receptor-induced cAMP signaling, mostly by an odorant/receptor-induced and cAMP signaling-dependent transcriptional activation of a luciferase-based luminescence assay. In the present protocol, we explain a method to measure the cell-surface expression and signaling of recombinant odorant receptors carrying a bi-functional, N-terminal 'IL-6-HaloTag^®'. IL-6, being a secreted cytokine, facilitates functional cell surface expression of recombinant HaloTag^®-odorant receptors, and the HaloTag^® protein serves as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by antibody-independent, chemical live-cell staining and flow cytometry. Here, we describe how to measure the cell surface expression of recombinant IL-6-HaloTag^®-odorant receptors in HEK-293 cells or NxG 108CC15 cells, by live-cell staining and flow cytometry, and how to measure an odorant-induced activation of these receptors by the fast, real-time, luminescence-based GloSensor^® cAMP assay.

Keywords: De-orphaning; G protein-coupled receptor; GPCR; HEK-293; NxG 108CC15.

Figure 1.. Vector map of expression plasmid pFN210A, carrying the IL-6-HaloTag^®-OR coding regions.

NheI, EcoRI, NotI, recognition sites for restriction endonucleases. TEV (tobacco etch virus) protease recognition sequence. Numbers are base pairs (bp).

Figure 2.. Light-optical microscopy of NxG 108CC15 cells in culture.

Microscopic image was taken with a Zeiss Axiovert 25, Zeiss A-Plan 20x/0.3 Ph 1 Var1 and AxioCam MRm.

Figure 3.. Example layout of a 96-well plate showing the distributions of dilutions for concentration-response relations with two differently tagged ORs.

Decreasing concentrations from A-G are color intensity-coded.

Figure 4.. Graphical summary of the methods and examples of expected experimental outcome.

A. Transfection of cells with expression plasmids coding for a given OR, the chaperone RTP1S, the olfactory G protein alpha subunit, and the cAMP-activated luciferase (pGloSensor™-22F). B. Left panel, odorant stimulation of transiently transfected cells. Right panel, Binding of fluorescence-coupled ligand to the HaloTag^®. C. Quantification of odorant-induced, cAMP-dependent luminescence (left panel) by plate reader (Fig. 2A in [12]), and of HaloTag^®/Ligand-dependent fluorescence by flow cytometry (right panel). D. Example result of a concentration-response relation of sotolone on OR8D1, equipped with different N-terminal tags. Data are mean ± SD (n = 3–5), normalized to the maximum amplitude of OR8D1 carrying the N-terminal IL-6-HaloTag^®. Red colored curve indicates receptor with the N-terminal IL-6-HaloTag^®, blue, with Rho-tag-HaloTag^®, and black curve indicates the empty plasmid control (Mock). Curves were derived from fitting the logistic function to the data. Arrows indicate EC₅₀ values (see ref [19], Fig. 1E). E. Flow cytometry-derived fluorescence distribution of ~1000 NxG 108CC15 cells expressing IL-6-HaloTag^®-OR8D1, and labeled with cell membrane-impermeant HaloTag^® Ligand-Alexa488 (see ref [19] Fig. S3E).