The serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2cR) are seven-transmembrane, G protein-coupled receptors (GPCRs) that are expressed in numerous brain regions. The 5-HT2CR protein is expressed predominantly in the central nervous system while the 5-HT2AR is also prominent in peripheral tissues, such as platelets and smooth muscle cells of the gut and vasculature . Both receptors are involved in a wide range of physiological (e.g., temperature regulation, feeding) and psychological processes in mammals  and are implicated in psychological disorders (e.g., addiction, anxiety, depression, and learning and memory) [3–5]. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. The most commonly studied downstream signaling pathway of the 5-HT2AR and 5-HT2CR is the activation of phospholipase Cβ (PLCβ) via Gαq/11 proteins and the production of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), leading to increased Ca++ release from intracellular stores [6, 7]. However, both receptors also activate phospholipase A2 (PLA2), possibly through Gi/o or G12/13, resulting in increased arachidonic acid (AA) release and have also been shown to activate phospholipase D [8, 9] independent of PLC activation. The degree to which these downstream signaling pathways are recruited varies between the receptors, both in terms of the level of constitutive (agonist-independent) activation of each of the pathways  (particularly for the 5-HT2CR, where constitutive activity is highly dependent on the degree of editing) as well as ligand-directed signaling of agonists for the 5-HT2AR and 5-HT2CR . These differences in signal activation may be a key feature distinguishing the functional effects of these two receptors. Thus, an appreciation of the full spectrum of downstream signal activation is critical when elucidating the functional actions of these receptors as well as in screening and evaluation of novel ligands for these receptors.
Parallel assays to measure simultaneous changes in IP3 and AA levels have been used to great advantage in deciphering differences in selective ligand-directed signaling , inverse agonism , desensitization [13, 14], coupling specificity  and constitutive activity  between the 5-HT2AR and 5-HT2cR. However, additional rapid and quantitative assays to distinguish among further cellular responses in intact cells would broaden our appreciation of the multiplicity of signaling cascades likely to be initiated by serotonergic ligands. In the course of our studies to discover novel ligands for the 5-HT2AR and 5-HT2CR , we have developed quantitative live cell assays in parallel plates that involve minimal cell sample manipulations and improve measurements of signals leading to functional activity for cells expressing these two receptors. These assays measure two types of signaling evoked by ligand activation, changes in intracellular calcium (Ca
++) and phosphorylation changes in downstream kinases, in very similar cell preparations.
The assay to measure Ca
++ levels utilizes detection of increased fluorescence in the presence of ionized calcium by the fluorescent dye Calcium 4 (Molecular Devices, Sunnyvale, CA) and is performed in live, attached cells, typically in 96-well plates . Changes in Ca
++ have long been recognized as critical to cell function, and techniques for measuring such changes have been rapidly evolving since the initial introduction of intracellular calcium-sensitive fluorescent dyes. We also developed an in situ immunoassay to detect activation of one class of the downstream mitogen-activated protein kinases (MAPKs), the extracellular-regulated kinases (ERK1 and ERK2), to measure an additional signaling event resulting from ligand binding. Phosphorylation of ERK is an example of an important integrator of upstream signaling events for many GPCRs as well as other cellular receptors [19–21], including the 5-HT2AR [22–27] and the 5-HT2CR [28, 29]. At this pathway intersection many upstream signals are summed to subsequently coordinate actions leading to important cellular functions, such as cell survival/apoptosis , growth regulation [31, 32] and differentiation . Recently, activation of ERK was found to be inversely correlated with the degree of RNA editing of the 5-HT2CR; stimulation of the non-edited (as used in our studies) and partially edited isoforms caused greater levels of ERK activation than occurred with the fully edited version [34, 35].
This assay to quantify the phosphorylated protein (pERK) uses phospho-specific antibodies and also is performed on attached cells fixed immediately following ligand treatment in 96-well plates [36, 37]. ERK1/2 activation following 5-HT2R stimulation [28, 34, 38] has previously been demonstrated by measuring pERK with traditional methods, primarily densitometry of immunoblots. However, the plate immunoassay for pERK is a much more sensitive assay  and the 96-well format allows for far greater flexibility in assay design than traditional immunoblots. Many simultaneous experimental perturbations can be performed in the same cell preparation due to the rapid processing of high numbers of samples with these assays [36, 37]. The assay also provides in situ detection of ERK activation as opposed to ELISAs and immunoblots that are performed on cell homogenates.
We developed these assays to compare the signaling effects resulting from stimulation by different serotonergic ligands in Chinese hamster ovary (CHO-K1) cell lines expressing either human (h)5-HT2AR or h5-HT2CR. These lines were developed by Berg and Clarke  and extensive data have been accumulated using the same lines in several laboratories [11, 13–15, 17, 28, 34]. However, it is necessary to optimize each assay for cell type and receptor system because access to signaling response compartments is different in cells of different tissues and lineages. In addition, technical aspects of measuring antigens (such as antibody concentrations and incubation times) vary for each protein. Moreover, values for these optimized parameters offer windows into the biological behavior of cells expressing different receptor subtypes. For example, the effects of cell crowding (density) on the downregulation of membrane receptors were demonstrated with this approach  as were the influences of culturing in serum (which itself contains many ligands). The power of the assays developed herein is the ability to rapidly determine multiple and subtle effects (potency, efficacy, second messenger activation) of various ligands with minimal cell disruption or comparison artifacts to yield important information concerning differences in signaling pathways triggered by activation in the 5-HT2AR- and the 5-HT2CR-CHO cells. Thus, interactions of second messenger systems with converging downstream enzymatic endpoints can be investigated.