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BioAssay: AID 624169

Luminescence-based cell-based primary high throughput screening assay to identify agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)

Name: Luminescence-based cell-based primary high throughput screening assay to identify agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A). ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 624169
Data Source: The Scripps Research Institute Molecular Screening Center (HTR2A_ACT_LUMI_1536_1X%ACT PRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-05-22

Data Table ( Complete ):           Active    All
BioActive Compounds: 2412
Depositor Specified Assays
624203Summary of the probe development efforts to identify agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)summary
624380Counterscreen for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A): Luminescence-based cell-based high throughput screening assay to identify agonists of the mu 1 opioid receptor (OPRM1)screening
624381Luminescence-based cell-based high throughput confirmation assay for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)screening
624499Counterscreen for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A): Luminescence-based cell-based high throughput dose response assay to identify agonists of the mu 1 opioid receptor (OPRM1)confirmatory
624503Luminescence-based cell-based high throughput dose response assay for agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)confirmatory
720642On Hold
720643On Hold
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Laura Bohn
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: DA025158-01A1
Grant Proposal PI: Laura Bohn
External Assay ID: HTR2A_ACT_LUMI_1536_1X%ACT PRUN

Name: Luminescence-based cell-based primary high throughput screening assay to identify agonists of the mouse 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A).


Serotonin (5-hydroxytryptamine; 5-HT) is an abundant neurotransmitter synthesized from the essential amino acid L-tryptophan and plays a significant role in appetite, platelet aggregation, pain perception, sleep, hormone secretion, sexual behavior, and thermoregulation. Serotonin mediates much of its actions by binding to and activating G protein-coupled receptors (GPCR) on the surface of neurons and other cells. One of these GPCRs, the serotonin 2A receptor (5-HT2AR), is a 7-transmembrane spanning GPCR in the frontal cortex which impacts on development, anxiety, depression, perception, and cognitive function [1, 2]. In neurons, the 5-HT2AR is a constitutively internalizing receptor and this trafficking is dependent upon interactions with the regulatory protein, Beta-arrestin2 [3]. Studies in cell-based assays reveal that serotonin requires Beta-arrestin2 to internalize the receptors while other agonists, such as DOI, 5-MeO-DMT, and quipazine, internalize the receptor in the absence of Beta-arrestins [3, 4]. All 5-HT2AR agonists seem to recruit Beta-arrestin2 to 5-HT2AR in cultured cells. However, while serotonin requires Beta-arrestins to traffic receptors, other agonists such as DOI, 5-MeO-DMT, and quipazine can internalize the receptor in the absence of Beta-arrestins [3, 4]. It remains to be determined if the specific 5-HT2AR-Beta-arrestin interactions and trafficking events have physiological significance. It is conceivable that the regulation of 5-HT2AR in vivo may impact neurological sensitivity to serotonin and the responsiveness to pharmacological agents and drugs of abuse. We predict that drugs that disrupt the serotonin 2A receptor-Beta-arrestin interaction might provide a means to alter the sensitivity of the receptor to the levels of serotonin present in the brain[4]. These findings may inspire the development of drugs that could be clinically useful for treating depression, schizophrenia, and drug addiction [5-7].


1. Hsieh, C.L., A.M. Bowcock, L.A. Farrer, J.M. Hebert, K.N. Huang, L.L. Cavalli-Sforza, D. Julius, and U. Francke, The serotonin receptor subtype 2 locus HTR2 is on human chromosome 13 near genes for esterase D and retinoblastoma-1 and on mouse chromosome 14. Somat Cell Mol Genet, 1990. 16(6): p. 567-74.
2. Berger, M., J.A. Gray, and B.L. Roth, The expanded biology of serotonin. Annu Rev Med, 2009. 60: p. 355-66.
3. Schmid, C.L., K.M. Raehal, and L.M. Bohn, Agonist-directed signaling of the serotonin 2A receptor depends on beta-arrestin-2 interactions in vivo. Proc Natl Acad Sci U S A, 2008. 105(3): p. 1079-84.
4. Bohn, L.M. and C.L. Schmid, Serotonin receptor signaling and regulation via beta-arrestins. Crit Rev Biochem Mol Biol, 2010. 45(6): p. 555-66.
5. Allen, J.A., P.N. Yadav, and B.L. Roth, Insights into the regulation of 5-HT2A serotonin receptors by scaffolding proteins and kinases. Neuropharmacology, 2008. 55(6): p. 961-8.
6. Raehal, K.M. and L.M. Bohn, The role of beta-arrestin2 in the severity of antinociceptive tolerance and physical dependence induced by different opioid pain therapeutics. Neuropharmacology, 2011. 60(1): p. 58-65.
7. Bohn, L.M. and P.H. McDonald, Seeking Ligand Bias: Assessing GPCR Coupling to Beta-Arrestins for Drug Discovery. Drug Discov Today Technol, 2010. 7(1): p. e37-e42.


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Assay Overview:

The purpose of this assay is to identify compounds that activate Htr2a, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs CHO-K1 cells which express Htr2a fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that activate Htr2a will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate (GalactonStarSubstratetrade mark) which yields a chemiluminescent signal, resulting in increased well luminescence. Compounds are tested in singlicate at a final nominal concentration of 7.6 uM.

Protocol Summary:

The CHO-K1 mHTR2A cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated Charcoal/Dextran-treated Fetal Bovine Serum, 800 ug/mL Geneticin, 300 ug/mL Hygromycin B, and 1X Penicillin-Streptomycin-Glutamine.

The day before the assay, 3 uL of cell plating media was dispensed into the first column of 1536 well microtiter plates and 1000 cells in 3 uL of cell plating media were seeded into the remaining wells. Plates were centrifuged and then incubated at 37 C, 5% CO2, and 95 % RH for 24 hours. Next, 23 nL of test compound in DMSO, Serotonin (30 uM final concentration) in DMSO, or DMSO alone (0.76% final concentration) were dispensed to the appropriate wells. The plates were then incubated for 2.5 hours at 37 C, 5% CO2, and 95 % RH. The assay was started by adding 2 uL of PathHunter reagent (prepared according to the manufacturer's protocol) to all wells. Plates were centrifuged and after 1 hour of incubation at room temperature, well luminescence was read on a ViewLux microplate reader (Perkin Elmer, Turku, Finland).

The percent activation for each compound was calculated as follows:

%_Activation = ( ( RLU_Test_Compound - Median_RLU_Low_Control ) / ( Median_RLU_High_Control - Median_RLU_Low_Control ) ) * 100


High_Control is defined as wells containing cells, Serotonin and DMSO.
Test_Compound is defined as wells containing cells, test compounds and DMSO.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for the entire run, i.e. any compound that exhibited greater % activation than the entire screen's cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-7, and for inactive compounds 7-0.

List of Reagents:

PathHunter CHO-K1 mHTR2A Beta-Arrestin Cell Line (DiscoveRx, part 93-0567C2)
PathHunter Detection Kit (DiscoveRx, part 93-0001)
PathHunter Cell Plating 20 Reagent (DiscoveRx, part 93-0563R20B)
Serotonin/5-HT (Control Agonist, Sigma, part H9523)
Ham's F-12 Nutrient Mixture (Invitrogen, part 11765-054)
Charcoal/Dextran-treated Feta Bovine Serum (Hyclone, part SH3006803HI)
Penicillin-Streptomycin-Glutamine (100X) (Invitrogen, 10378-016)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin(R) Selective Antibiotic (Invitrogen, part 10131-027)
Detachin Cell Detachment Reagent (Genlantis, part T100100)
T-175 Flasks (Nunc, part 159910)
1536-well plates (Corning, part 7298)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activation at 7.6 uM (7.6μM**)Normalized percent activation of the primary screen at a compound concentration of 7.6 uM.Float%

** Test Concentration.
Additional Information
Grant Number: DA025158-01A1

Data Table (Concise)