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

Podsumowanie

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Streszczenie

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors^1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.

Wprowadzenie

The mammalian olfactory system has the ability to respond to a vast number of odorous stimuli, allowing for the detection and discrimination of thousands of odorants. Olfactory receptors (ORs) are the molecular sensors expressed by the olfactory sensory neurons in the olfactory epithelium¹². Mammalian odor recognition occurs through differential activation of ORs by odorants, and the OR gene family is extensive, with roughly 1,000 murine and 400 human receptors^12-16. Previous functional analyses of ORs in olfactory neurons and in heterologous cells have shown that different odorants are recognized by unique, but overlapping ensembles of ORs^10,17-20. Matching ligands to ORs is critical for understanding the olfactory code and essential for building viable models of olfaction. Due to difficulties expressing ORs in heterologous systems as well as the large number of both odorants and ORs, this data has been largely absent from the field; indeed, fewer than 6% of human ORs have a published ligand^1-11. This protocol describes the use of a luciferase assay to characterize odorant/OR interactions. This assay enables the high-throughput characterization of ORs, a task that is essential to understanding odorant/OR interactions as well as developing a model of odor coding.

High-throughput studies of ORs face three major challenges. First, ORs expressed in heterologous cells were retained in the ER and subsequently degraded in the proteasome^21,22, preventing the ORs from interacting with odorants in the assay system^23-25. This problem was addressed by the discovery of accessory proteins that facilitate stable cell-surface expression of a broad range of ORs^19,26,27. Receptor-transporter-proteins 1 and 2 (RTP1 and 2) promote OR cell-surface expression and activation in response to odorant stimulation¹⁹. Based on this work, HEK293T cells were modified to stably express RTP1 long (RTP1L) and RTP2, receptor expression-enhancing protein 1, and G_αolf, resulting in the Hana3A cell line^19,27. In addition, the type 3 muscarinic acetylcholine receptor (M3-R) interacts with ORs at the cell surface and enhances activation in response to odorants²⁶. Co-transfection of an OR with RTP1S and M3-R into Hana3A cells results in the robust, consistent, and functional expression of a broad range of ORs at the cell surface²⁷. Second, mammalian OR repertoires are quite large. In humans, for example, the OR repertoire is an order of magnitude more numerous than the gustatory receptor repertoire, and 2 orders of magnitude more numerous than the visual receptor repertoire. Although cloning a single OR is a relatively straightforward protocol, significant up-front effort is required to generate a comprehensive library. Third, although we know that in vision, wavelength translates into color and in audition frequency translates into pitch, the organization of odors is poorly understood, making it difficult for researchers to interpolate from a representative sample of odorants. Although some progress has been made on this front^10,28, the map of the olfactory landscape remains incomplete. Screening tens of thousands of molecules against hundreds of ORs is a daunting task; high-throughput screens in this domain require carefully defined campaigns. The major remaining challenges are those of logistics and cost rather than problems inherent to the technique. Although heterologous screening has not been widely used to identify ligands by academic groups, a private company has used the same technique to identify ligands for 100 human ORs ²⁹. Unfortunately, these data remain proprietary.

The high-throughput luciferase assay outlined here has several advantages over alternative methods used to assess OR activation. Although the responses of native olfactory sensory neurons have been measured using electrophysiology and calcium imaging, these techniques have difficulty teasing apart which OR leads to a neuron's response due to the overlap in response properties for olfactory neurons. Although knocking-in a GFP-labeled receptor type^30,31, delivering specific receptors via adenovirus to murine olfactory neurons^32,33, or performing RT-PCR after recordings^17,24,33 can link recordings to single receptor types, these methods are low-throughput and not suitable for large-scale screens. Heterologous screening systems are more scalable, and two major forms are found in the literature: cAMP pathway reporters and inositol triphosphate (IP3) pathway reporters. Upon odor stimulation, ORs activate a G_αolf transduction signaling cascade that results in the production of cyclic AMP (cAMP)¹². By co-transfecting a firefly luciferase reporter gene under the control of a cAMP response element (CRE), luciferase production can be measured as a function of odor response, allowing for the quantification of OR activation. OR activation can also be linked to the IP3 pathway by co-expressing G-proteins such as G_α15/16 or a G_α15-olf chimera^24,25,34. We have chosen the assay presented here based on three factors: (1) the co-expression of RTP1 with Rho-tagged olfactory receptors improves the expression of olfactory receptors at the cell surface^19,27; (2) use of a cAMP-responsive reporter gene allows for the measurement of OR activation through the canonical second messenger pathway; and (3) the assay is well-suited to high-throughput screens.

This high-throughput luciferase assay is applicable to a variety of studies valuable to the field of olfaction. First, a large number of ORs can be screened against a single odorant in order to determine the receptor activation pattern for a specific odorant. This type of study identified OR7D4 as the OR responsible for responding to the steroid odorant androstenone⁸. Conversely, one OR can be screened against a panel of odorants in order to determine the receptor response profile¹⁰. When candidate olfactory odorant/OR pairs are identified via these screens, interaction can be confirmed by conducting a dose response experiment examining the response of the OR to increasing concentrations of odorant. Dose response curves can also assess how genetic variation in an OR affects in vitro odorant response^8,9,11,35, and these studies can be extended to interspecific OR variation, allowing for the examination of receptor evolution across species and causal mutations in evolution^36,37, Finally, this assay can be used to screen for odor antagonists that are able to antagonize OR response to a particular odorant for a known odorant/receptor pair^38,39. In summary, this high-throughput luciferase assay is applicable to a range of studies that will help characterize OR activation patterns and provide a better understanding of odor coding in the olfactory system.

Protokół

1. Culture of Hana3A Cells

1. Prepare M10 media by supplementing minimum essential medium (MEM) with 10% (v/v) FBS.
2. Culture Maintenance
   1. Maintain cells in M10 media. NOTE: The expression vectors for RTP1L, RTP2, REEP1, and G_αolf confer puromycin resistance to Hana3A cells, but maintaining the cells with this antibiotic does not significantly affect assay results.
   2. Subculture at a ratio of 1:8 in 10 cm dishes every 2-3 days.
   3. Incubate at 37 °C with 5% CO₂.

2. Plating Cells for Transfection

1. Aspirate media from a 100% confluent 10 cm dish of Hana3A cells.
2. Wash cells by adding 10 ml PBS, swirling the dish, and aspirating the PBS.
3. Add 3 ml of 0.05% trypsin/EDTA and wait for cells to dissociate (about 1 min).
4. Inactivate trypsin by adding 5 ml M10 and break up cell clumps by triturating roughly 10x with a 10 ml pipette. Pipette carefully to avoid introducing air bubbles into the media.
5. For each 96-well plate, transfer 1 ml of cells into a 15 ml conical tube, centrifuge at 200 x g for 5 min, and aspirate the supernatant without disturbing the cell pellet.
6. Resuspend the cells in 6 ml M10 per 1 ml of cells transferred in Step 2.5.
7. Pipette 50 µl of cells to each well of a 96-well plate and incubate overnight at 37 °C with 5% CO₂.

3. Transfection of Olfactory Receptors

1. Preparation of Plasmid DNA
   1. Prepare plasmid DNA via an endotoxin-free protocol. NOTE: Use plasmid DNA preparation kits designated "endotoxin-free," or add a phenol-chloroform extraction step to the plasmid DNA preparation protocol.
   2. Dilute DNA to a concentration of 100 ng/µl in TE buffer.
2. Observe plated cells (Step 2.7) to ensure a proper confluency of approximately 30-50% per well and return to incubator. NOTE: While this confluency is not optimal for the lipid transfection reagent, a confluency of 30-50% at this step is optimal for measuring luciferase activity 24 hr after transfection.
3. Preparation of Transfection Mix
   1. Pipette RTP1S-pCI, M3-R-pCI, pCRE-luc, and pSV40-RL plasmids into MEM medium per the volumes detailed in Table 1 to make the Plasmid mix (volumes indicated are per 96-well plate).

      Plasmid mix
      	per well	per 96-well plate
      MEM	--	500 µl
      RTP1S-pCI	5 ng	480 ng
      M3-R-pCI	2.5 ng	240 ng
      pCRE-luc	10 ng	960 ng
      pSV40-RL	5 ng	480 ng

      
      Table 1. Plasmid mix components. Per well and per 96-well plate volumes of RTP1S-pCI, M3-R-pCI, pCRE-luc, and pSV40-RL, and MEM.
   2. For each 96-well plate, dilute 18 µl lipid transfection reagent in 450 µl MEM medium.
   3. Pipette Plasmid mix (from Step 3.3.1), rhodopsin-tagged olfactory receptor in pCI plasmid (Rho-OR-pCI), and lipid transfection mix (from Step 3.3.2) to make the Complex detailed in Table 2. Mix the solution by trituration and incubate at room temperature for 15 min. Stop the reaction by adding M10 according to Table 2. NOTE: this reaction is time-sensitive and should not be allowed to continue for more than 30 min. The well + 10% calculation is important to ensure sufficient volume for subsequent steps.

      Complex
      	per well	per well + 10%
      Plasmid mix	4.2 µl	4.58 µl
      Rho-OR-pCI	0.05 ng	0.06 ng
      Lipid transfection mix	4.2 µl	4.58 µl
      M10	41.7 µl	45.83 µl

      
      Table 2. Complex components. Per well and per well + 10% volumes of plasmid mix (Table 1), olfactory receptor plasmid (Rho-OR-pCI), and lipid transfection mix. M10 is added to quench the reaction following a 15 min incubation at room temperature.

4. Tap out the media on the cell plates.
5. Pipette 50 µl of complex to each well and incubate overnight at 37 °C with 5% CO₂.

4. Odor Stimulation

1. Observe the transfected cells to ensure a proper confluency of 50-80% per well and return to incubator. NOTE: If cells are less than 50% confluent, firefly luciferase and renilla luciferase readings may be too low for measurement of receptor activation. Consider discarding the plate.
2. Prepare 1 M stock solutions of each odor in DMSO.
3. Prepare odor stimulation solutions in CD293 medium.
   1. For screening experiments, dilute stock solution of odor to 100 µM. Also prepare a no-odor control (CD293 only) in order to control for OR background activation. NOTE: For screening experiments, each OR/odor pair is tested only once per experiment. Because some odor diffusion across wells is possible, it is recommended to stimulate with one odorant for each plate.
   2. For dose response experiments, prepare seven 10-fold serial dilutions of odor stock solution in triplicate starting at 1 mM for each receptor. Also prepare the same odor dilutions in triplicate for empty vector-transfected cells in order to control for odor background activation. NOTE: For dose response experiments, each odor concentration treatment should be conducted in triplicate.
4. Tap out the media on the cell plates.
5. Pipette 25 µl of odor stimulation solution to each well and incubate for 4 hr.

5. Measuring OR Activity via Luciferase Assay

1. Resuspend firefly luciferase substrate per the manufacturer’s instructions and store at -80 °C.
2. Thaw 1 ml of firefly luciferase substrate per 96-well plate.
3. Prepare fresh firefly luciferase reaction quencher and Renilla luciferase substrate reagent (5 µl luciferase quencher/Renilla luciferase substrate per 1 ml of buffer). NOTE: Approximately 1 ml of reagent is needed per 96-well plate.
4. Prepare the luminescent microplate reader. Open the microplate reader software. Within the system icon:
   1. Under the "Pre-heating" Tab, check the box for "ON" and set the temperature of the machine to 25 °C.
   2. Under the "Dispenser" tab, prime each dispenser with 1,000 µl of 70% ethanol followed by 1,000 µl of distilled water. NOTE: Use separate aliquots of alcohol and water for each dispenser. Ethanol is used to disinfect the dispensers, and water removes the residual ethanol.
   3. Prime each dispenser with 1,500 µl of air (remove dispensers from liquid). NOTE: priming with air ensures that luciferase substrates are not diluted with residual water.
   4. Prime dispenser 1 with 1,080 µl of firefly luciferase substrate (from Step 5.2). Prime dispenser 2 with Renilla luciferase substrate (from Step 5.3). NOTE: Be careful not to cross-contaminate the luciferase substrates. Priming with luciferase substrates fills the dead space in the reagent dispensers.
5. Set up the following protocol to read both firefly and Renilla luciferase luminescence. Within the software associated with the microplate reader, under the "File" menu, click on "New Task". Highlight "Protocols" and click on "Create New". In the next window, the circle next to "Standard Protocol" should be selected. Click "OK." Double click on "Procedure" on the left hand side of the screen.
   1. Dispense 10 µl of firefly luciferase substrate to all wells using dispenser 1. Under the "Actions" menu, click "Dispense". In the "Dispense Step" window, set: "Dispenser" to 1, "Priming" to none, "Dispense Volume" to 10 µl and "Rate" to 225 µl/sec. Click "OK".
   2. Shake the plate for 30 sec. Under the "Actions" menu, click "Shake". In the "Shake Step" window, set "Intensity" to Medium and "Duration" to 0:30 MM:SS. Click "OK".
   3. Read the luminescence of all wells for 0.5 sec per well. Under the "Actions" menu, click "Read". In the "Read Step" window, set: "Detection Method" to Luminescence, "Read Type" to Endpoint, "Integration Time" to 0:00:50 MM:SS:ss, "Filter Sets" to 1, "Emission" to Hole, "Optics Position" to Top, "Gain" to 135, and "Read Height" to 1.00 mm. Click "OK".
   4. Dispense 10 µl of Renilla luciferase substrate to all wells using dispenser 2. Set the conditions as in Step 5.6.1, except set "Dispenser" to 2.
   5. Shake plate for 30 sec. Set the conditions as in Step 5.6.2.
   6. Read luminescence of all wells for 0.5 sec per well. Set the conditions as in Step 5.6.3.
6. Remove the lid from the 96-well plate and place the plate in the microplate reader. Start the program set up in Step 5.5 to read plate luminescence.
7. Clean the reagent dispenser pumps. From the system icon under the "Dispenser" tab:
   1. Purge 1,000 µl of firefly luciferase substrate from the firefly luciferase dispenser into a recovery tube. NOTE: Firefly luciferase can be stored at -80 °C and reused.
   2. Prime each dispenser with 1,000 µl of distilled water, followed by 1,000 µl of 70% ethanol, and finally 1,500 µl of air (remove dispensers from liquid). NOTE: Water removes luciferase substrates from the reagent pumps, ethanol disinfects, and air dries any residual ethanol.

6. Data Analysis

1. Data Export
   1. In the microplate reader software, double click on "Report/Export Builders" on the left side of the screen.
   2. Click on the button "New Export to Excel" and click "OK".
   3. Highlight Export1 and click "Edit".
      1. Under "Content" check "System Description", "Procedure", "Plate Description", and "Plate Layout Matrix". Include "Raw Data" and "Calculated Data".
      2. Under "Workflow", check "Autoexecute on completion of the procedure". Under Export Mode, check "All plates in the same workbook" and "As a new worksheet".
      3. Under "File" choose file name format and file location, and click "OK".
      4. Close the "Report/Export Builders" window.
2. To obtain normalized luciferase values, divide the firefly luciferase luminescence reading for each well (Step 5.6.3) by the Renilla luminescence reading for each well (Step 5.6.6).

Wyniki

A primary screen tested 328 ORs against 26 odors at a concentration of 100 µM. This odor concentration has been demonstrated to effectively activate a large proportion of ORs with known ligands¹⁰. First, normalized luciferase activity was calculated by dividing the firefly luciferase reading by the Renilla luciferase reading. Next, baselined values were calculated by subtracting the normalized luciferase readings for the no odor control from the normalized luciferase readings for each well (

Dyskusje

Odorant identity is encoded by olfactory receptor activation patterns, but receptor activation patterns, including which receptors are activated and to what degree, are known for fewer than 6% of human olfactory receptors^1-11. Efforts to characterize olfactory receptors have been limited by their labor-intensive methods or applicability to only a subset of the olfactory receptor family^{17,23,24,33,34}. The Hana3A heterologous expression system supports the robust expression of the majority of tested o...

Materiały

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Hana3A cells	Avaiable from the Matsunami Laboratory upon request
RTP1S-pCI	Avaiable from the Matsunami Laboratory upon request
M3-R-pCI	Avaiable from the Matsunami Laboratory upon request
pCRE-luc	Agilent	219076	LUC
pSV40-RL	Promega	E2231	RL
Minimum Essential Media, Eagle	Sigma Aldrich	M4655	MEM
FBS	Life Technologies	16000-044	FBS
PBS (without Ca2+ and Mg2+)	Cellgro	21-040-CV	PBS
Trypsin (0.05% Trypsin EDTA)	Life Technologies	25300	Trypsin
CD293	Life Technologies	11913-019	CD293
96 well PDL white/clear plate	BD BioCoat	356693	plates
Lipid transfection reagent: Lipofectamine 2000	Life Technologies	11668-019	Lipofectamine
Firefly luciferase substrate, firefly luciferase quencher/Renilla luciferase substrate: Dual-Glo Assay	Promega	E2980	dual glo
Synergy S2	BioTek	SLAD	BioTek S2
Microplate reader software: Gen5 Data Analysis Software	BioTek	Gen5	Gen5
BIOSTACK	BioTek	BIOSTACK2WR	BioStack
Multiflo	BioTek	MFP	MultiFlo
300ul GripTips	Integra	4433	GripTips
12.5ul GripTips	Integra	4414	GripTips
300ul GripTips ViaFlo96	Integra	6433	XYZ tips
12.5ul GripTips 384 XYZ	Integra	6403	XYZ tips
384ViaFlo	Integra	6030	384ViaFlo
TE buffer	Macherey Nagel	740797.1
DMSO	Sigma Aldrich	D2650-100ML	DMSO
forskolin	Enzo Life Sciences	BML-CN100-0010	FOR