Erika Cecon

Jean-Luc Guillaume

Ralf Jockers
email: ralf.jockers@inserm.fr

Functional investigation of melatonin receptors activation by homogenous cAMP assay

Keywords: Melatonin receptors, cAMP, adenylyl cyclase, TR-FRET

Cyclic adenosine monophosphate (cAMP) is an important ubiquitous second messenger and one of the major pathways transducing the activation of G protein-coupled receptors (GPCRs). Quantifying intracellular levels of cAMP in an accurate and high-throughput manner is, therefore, of high interest to access functional responses of GPCRs. The neurohormone melatonin is selectively recognized by two GPCRs in mammals, named MT1 and MT2. Both have an inhibitory action on intracellular cAMP levels. Here, we describe a homogeneous high-throughput-compatible methodology routinely used in our laboratory to measure cAMP levels following activation of melatonin receptors.

Introduction

Modulation of the levels of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is one of the main signaling pathways used by G protein-coupled receptors (GPCRs) to transduce extracellular signals (hormones, peptides, neurotransmitters, light) into a cellular response. When a ligand binds to its receptor at the cellular membrane, the active conformation of the receptor is stabilized and triggers GDP/GTP exchange on the Ga subunit of heterotrimeric G proteins, followed by dissociation of the Ga-GTP and Gß/g subunits (1, 2). The activated Ga subunit interacts with and regulates the activity of effectors, among them the adenylyl cyclase (AC) enzyme, which synthetize cAMP from ATP. Ga subunits are classified into four families: Gαi, Gαs, Gα12/13, and Gαq. AC enzyme is the main effector of Gαi and Gαs families, resulting thus in modulation of the intracellular levels of cAMP.

Agonist-induced activation of Gs-coupled receptors positively regulates AC activity leading to an increase in cAMP production, whereas activation of Gi-coupled receptors inhibits AC, lowering cAMP levels.

Both MT1 and MT2 melatonin receptors are mainly coupled to the Gai family of G proteins (3, 4, 5) and the melatonin-induced decrease of intracellular cAMP levels is one of the bestcharacterized cellular response to melatonin. Accordingly, measuring cAMP levels upon MT1 and MT2 activation is indeed a mandatory step in the characterization and validation of new ligands for melatonin receptors (3, 6). Detection of melatonin-induced decrease in cAMP is also a validated approach to testify for the presence of functional melatonin receptors endogenously expressed in tissues, primary cells or cell lines (7, 8, 9), which are often expressed only in few cells and at very low levels (10). Melatonin signaling through cAMP pathway mediates several of the main physiological actions of melatonin, including synchronization of endogenous rhythmicity (11), regulation of mitochondrial activity (6,[START_REF] Suofu | Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release[END_REF] and metabolic function (13, 14).

Several methods have been developed to measure cellular cAMP levels. The first cAMP quantification assays were based on radioimmuno assays (RIA), which relies on the competition of endogenous cAMP with radioactively-labelled cAMP to binding to cAMP antibodies (15). Over the past two decades, the development of fluorescent-based technologies significantly broadened the choices to measure cAMP levels (16, 17). Among the most sensitive commercially available assays for cAMP measurement is the one based on the homogenous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) (Cisbio Bioassays) (18). The assay is based on the energy transfer between energy donor and energy acceptor fluorophores, with donor fluorophores belonging to the lanthanide family.

These fluorophores display extended lifetime emission properties, allowing to measure FRET signal with a time delay where the background from non-specific transient fluorescence is already decayed, resulting thus in a high signal-to-noise ratio [START_REF] Bazin | Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions[END_REF][START_REF] Degorce | HTRF: A technology tailored for drug discovery -a review of theoretical aspects and recent applications[END_REF]. Upon excitation of the donor fluorophore, positive detection of acceptor emission indicates that energy donor and acceptor were in close proximity to each other (less than 10 nm) and successful FRET occurred. In the case of the cAMP assay, it relies on the use of a cryptate-labelled antibody recognizing cAMP (donor) and d2-labelled cAMP (acceptor). The d2-cAMP competes with endogenous cAMP for binding to the antibody and, thus, the TR-FRET is inversely proportional to the level of endogenous cAMP. A standard curve allows to infer the real cAMP concentration in the samples. Here we describe the detailed protocol to detect melatonin receptor-mediated decrease on cAMP levels using the TR-FRET cAMP assay.

Material

DNA constructs

1. cDNA constructs coding for human MT1, MT2 and empty vector.

-cAMP standard concentrate.

-Stimulation buffer (5X).

-Lysis and Detection Buffer.

-Plate sealing polyester films.

-HTRF-certified plate reader (see Note 5).

METHODS

Cell Culture

1.

Cultivate HEK293T cells (see Note 6) in DMEM medium supplemented with 10 % FBS and 1 % penicillin/streptomycin, in 10 cm cell culture dishes in a humidified atmosphere of 5 % CO2 at 37 °C. Cells should be grown to confluency and split in cell culture medium twice per week at a 1:10 ratio.

2.

On day 1, remove the cell culture medium from a confluent HEK293T cell culture dish, wash it with 5 mL of sterile PBS, add 1 mL trypsin/EDTA solution and incubate cells 2-3 min at 37 °C. Add 9 mL of complete DMEM medium and resuspend the cells. Pipette the medium with cells up-and-down using a 10 mL pipette to better detach and dissociate the cells.

3.

Count and distribute the cells into a 6-well plate at an approximative concentration of 0.3 x 10 6 cells / well. Incubate cells for 18 h -24 h at 37 °C in a 5 % CO2 incubator to allow cells to recover and attach to the plate.

Cell transfection

To obtain HEK293T cells expressing hMT1 or hMT2 receptors, cells are transiently transfected using the JetPei transfection reagent (see Note 7). 2.

Distribute the 200 µL of transfection solution per well, dropwise, over the cells that are to be transfected.

3.

Allow cells to incubate at 37 °C, in a 5 % CO2 incubator, for 48 h (see Note 9).

Activation of melatonin receptors and cAMP assay

On day 4 (i.e. 48 h post-transfection), cells are stimulated with the ligand and cAMP is measured immediately after.

1.

Prepare the working solutions of the ligand in 1x Stimulation buffer supplemented with 2 mM IBMX. Prepare 1 µM Forskolin solution in the IBMX-Stimulation buffer (see

Note 10).

2.

To perform a concentration-response curve of melatonin, final concentrations should be in the range from 10 -12 to 10 -6 M. From the stock solution of melatonin at 10 -2 M, prepare 2X concentrated working solutions of melatonin by serial dilutions in forskolin working solution.

3.

Remove the cell culture medium from the transfected cells and wash the wells with 1 mL of sterile PBS. Remove the PBS, add 500 µL of PBS and pipette up-and-down to detach the cells and collect them in a microcentrifuge tube.

4.

Count the cells and resuspend in stimulation buffer in order to obtain 1500 cells / µL (see Note 11).

Operate on ice

6.

In the wells of assay Positive and Negative Control add 5 µL of Stimulation Buffer.

7.

In the wells of the Standard curve, add 5 µL of each cAMP standard, prepared from the stock by serial dilution using a dilution factor of 4, according to the manufacturer instructions.

8.

In the wells of assay Positive and Negative Control, and of the Standard curve add 5 µL of Stimulation Buffer.

9.

In the wells containing cells, add 5 µL of Stimulation Buffer to Basal wells; 5 µL of Forskolin solution to Forskolin group; and 5 µL of each melatonin concentration solution to melatonin-stimulated group.

10.

Seal the plate and incubate at 37 °C for 30 min.

11.

Add 5 µL of cAMP-Cryptate working solution to all wells.

12. Add 5 µL of anti-cAMP-d2 working solution to all wells, except in the Assay negative control wells.

13.

Add 5 µL of Lysis buffer to Assay negative control wells.

14.

Seal the plate and incubate at room temperature (20 -22 °C) for 1 h, protected from light.

15.

Remove the plate seal and read the plate on an HTRF-compatible reader.

Data analysis

1.

Calculate the TR-FRET signal, which is the ratio of the acceptor (d2, measured at 665 nm) over the donor (cryptate, measured at 620 nm) signals for each well. For convenience, the ratio is multiplied by 10000 (see Note 12).

TR-FRET signal = Signal 665 nm x 10 4 Signal 620 nm 2.

Data is then normalized using Delta F (see Note 13):

Delta F = TR-FRET signal sample -TR-FRET signal background TR-FRET signal background where TR-FRET signal background corresponds to the assay negative control group.

3.

Next, calculate Delta F / Delta F max of all samples:

Delta F Delta F max
where Delta F max corresponds to the Standard 0.

4.

Plot the Delta F / Delta F max of the standard curve versus the cAMP concentration (a 0-to-100 % normalized curve), fit the curve using non-linear regression analysis, and the cAMP concentration in the samples can be obtained by interpolating the Delta F / Delta F max of the samples to the standard curve. An example of standard curve is shown in Figure 2.

5.

Plot the cAMP values versus melatonin concentration (see Note 14). An example of expected result is shown in Figure 3.

Notes

1.

Alternative transfection reagents can be used, and the manufacturer instructions should be followed.

2.

Alternative assays to measure cAMP can also be used. For example, we have also tested Surefire assays (PerkinElmer) which also work well.

3.

Several TR-FRET assays from Cisbio Bioassays are available, optimized for different purposes (detection of Gs signaling vs Gi signaling) based on different dynamic ranges. For melatonin-induced responses we use the assay optimized for Gi signaling.

4.

The stock solutions of the reagents from the cAMP kit should be prepared in advance and accordingly to the manufacturer instructions. Aliquots should be stored at -20 °C.

5.

Examples of HTRF-compatible plate readers are: TECAN F500 (TECAN) and EnVision (PerkinElmer).

6.

Other mammalian cell lines, such as HeLa or CHO cells, can also be used. In such cases, transfection reagents and efficiency should be checked and optimized for each cell type.

7.

This step is skipped if a stable cell line expressing melatonin receptors is used. We recommend using stable cell lines whenever possible, as we observed that using stable cell lines provide better amplitude in the cAMP assay.

8.

In our experience, low amounts of cDNA used for the transfection of melatonin receptors (between 1 to 10 ng per well) gives better amplitude in the Gi-cAMP response.

9.

Cells should not exceed 70 % confluency before transfecting to assure an optimal transfection rate. We found that an incubation time of 48 h (post-transfection) is optimal for the expression and plasma membrane translocation of membrane receptors.

10.

Forskolin activates the adenylyl cyclase enzyme, leading to increased intracellular levels of cAMP. It is used to increase the basal levels of cAMP prior to activation of Gicoupled receptors. The final concentration of forskolin used should be optimized depending on the cell type and density.

11.

Optimum cell density will depend on the cell type and should be optimized.

12.

The ratiometric data analysis significantly improves data quality by compensating eventual discrepancies in the amount of energy donor between the wells due to pipetting error or imprecisions.

13.

Delta F normalization allows inter-assay comparisons and pooling together data from assays run at different days or by different users.

14.

Forskolin-stimulates group is the control group (absence of melatonin). The basal group is not plotted but it should be compared to the forskolin-stimulate group to certify that cells responded properly to forskolin.

1 .

 1 On day 2, prepare the transfection solution containing the cDNAs for hMT1 and hMT2 in separated microcentrifuge tubes. For each transfection, prepare one microcentrifuge tube containing 100 µL 150 mM NaCl, 10 ng of MT1 or MT2 cDNA (see Note 8), and 990 ng of empty plasmid as carrier. In another tube, prepare the JetPei solution by adding 3 µL of JetPEI into 100 µL 150 mM NaCl. If multiple wells are to be transfected, multiply the volume by the number of transfections. Transfer 100 µL of the JetPEI mix to each tube containing the cDNA/NaCl mix and incubate for 15 min at room temperature (20 -22 °C).

for steps 5 to 9 .

 9 Distribute 5 µL of the cell solution per well in the 384well plate. Assay should be performed in triplicates and should contain the following cells (7 data points) An example of plate template is shown in Figure 1.

Figure 1 -

 1 Figure 1 -Example of a plate template in a 384-well plate for the cAMP assay. Posit. Ct. =

Figure 2 -

 2 Figure 2 -Example of a Standard curve result, plotting Delta F / Delta F max vs. cAMP

Figure 3 -

 3 Figure 3 -Example of expected result of the effect of melatonin decreasing cAMP levels in

Acknowledgement:

We thank the members of Jockers lab that helped to optimize the protocol over the years. This work was supported by grants from the Fondation Recherche Médicale (Equipe FRM 2019, EQU201903008055); The French National Research Agency (ANR) ANR-19-CE16-0025-01 (Mito-GPCR), ANR-21-CE18-0023-01 (alloGLP1R), Recherches Partenariales et Innovation Biomédicale 2012 "MED-HET-REC-2"; INSERM; CNRS; La Ligue Contre le Cancer N/Ref: RS19/75-127; France Alzheimer 2020 (dossier: 2042).