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Chattopadhyay A, editor. Serotonin Receptors in Neurobiology. Boca Raton (FL): CRC Press/Taylor & Francis; 2007.

Authors

G-protein-coupled receptors or GPCRs have been a major area of study in the past decades. This is not surprising as this group, comprising a thousand-odd proteins, constitutes the largest group of signal transducers across the cell membrane and is also implicated in a vast number of diseases [1]. All major neuromodulators and some fast neurotransmitters act through either a single GPCR or a family of GPCRs, collectively termed a neuromodulator receptor family (such as the serotonin receptor family). This dependence of proper brain function on GPCR signaling is reflected in the fact that most psychiatric diseases implicate one or many malfunctioning GPCRs in their pathophysiology. Consequently, drugs aimed at treating these disorders are primarily targeted to GPCRs.

The serotonin 2A receptor (5-HT_2A) has been implicated in mental disorders with complex etiologies that are still not clearly understood, in processes such as learning and memory, and also in neurogenesis. There are a large number of drugs targeted to this receptor. Though the receptor has been studied largely in relation to its multiple functions in the CNS, high levels of receptor expression in other areas such as the intestine, platelets and endothelial cells suggest that it could play crucial roles in other aspects of physiology. Research shows that some GPCRs, including the 5-HT_2A receptor, exhibit critical differences in aspects of functional regulation from those seen in conventionally studied model GPCRs such as the β-2-adrenergic receptor. The receptor also couples to a number of intracellular signaling cascades, making it an important receptor to study. The 5-HT_2A receptor could well serve as an important alternate paradigm in the study of GPCR function.

The 5-HT_2A receptor was initially identified by hybridization using conserved elements of the cloned 5-HT_2C receptor, followed by functional expression [2,3]. It bears strong similarity in primary sequence to the two other members of its subfamily, i.e., 5-HT_2B and 5-HT_2C. All members of the 5-HT₂ receptor subfamily primarily couple to PLC on activation. As with most GPCRs, 5-HT_2A functional regulation also involves desensitization and resensitization—regulatory processes that help prevent overstimulation and allow recuperation of signaling competence, respectively. Internalization and recycling of the receptor represent two processes that regulate this desensitization and resensitization. The receptor contains numerous recognition motifs for interacting partners to dock and facilitate receptor signaling, desensitization or trafficking. In some GPCRs, these processes are mediated by posttranslational modifications such as phosphorylation of one or more amino acid residues of the receptor. What makes these processes an area of consistent interest in 5-HT_2A research is that drugs, psychotropic or therapeutic, modify many aspects of receptor functionality. Conversely, it has been suggested that the pathophysiology of psychiatric disorders is based on malfunctions in one or more aspects of GPCR function.

The importance of the role played by the 5-HT_2A receptor in mediating CNS function can be seen by its considerable expression in various regions of the CNS and its wide-ranging effects.

LOCALIZATION OF 5-HT_2A MRNA AND RECEPTOR PROTEIN IN THE BRAIN

Initial localization studies of the 5-HT₂ class of receptors were based on radioactive ligand binding in tissues. These studies did not fully distinguish between various subtypes of 5-HT₂ receptors because sequence information was not available to design subtype-specific probes or generate subtype-specific antibodies. With the cloning and identification of 5-HT₂ receptor subtypes, initially from the rat and later other species (Figure 6.1), it became possible to determine subtype-specific localization of transcripts within the CNS. This could then be correlated with radioactive ligand binding in the same location. Development of subtype-specific antibodies has provided a detailed description of receptor localization as well as changes in levels in various experimental conditions.

FIGURE 6.1

Comparison of amino acid composition of the human, rat and mouse 5-HT_2A receptors. The amino acid sequence of the human 5-HT_2A receptor is shown with differences in amino acid residues of the rat and mouse receptors shown above and below the human sequence,(more...)

The initial report of 5-HT_2A receptor mRNA expression in the rat CNS was based on northern blots of RNA extracted from various regions of the CNS [2]. Further studies usingin situ hybridization indicated high expression levels in layers 1, 4, and 5a of the cerebral cortex, the entorhinal and the piriform cortex. The olfactory bulb and some brainstem areas like the hypoglossal, pontine, motor trigeminal, and facial nuclei, also showed expression. Intermediate expressing areas were the limbic system and basal ganglia. These studies did not detect transcripts in the cerebellum, thalamic nuclei and found low expression in the hippocampus. In humans, no 5-HT_2A receptor mRNA was detected in the striatum and cerebellum. In addition, 5-HT_2A mRNA expression has been found in the dorsal horn of the spinal cord [4,5].

5-HT_2A protein expression has been ascertained by a variety of immuno-histochemical techniques, coupled with light, fluorescence, and electron microscopy. In keeping with mRNA expression data, 5-HT_2A protein is highly expressed in all layers of the cortex with layer 5 having the highest concentration. Pyramidal neurons of the frontal, insular, orbital, parietal, entorhinal, cingulate, perirhinal, piriform, insular, and deep layer of the cingulated cortex express 5HT_2A receptors. In the cortex, the 5-HT_2A is also found on certain astrocytes—an observation that has implicated glia in playing a role in schizophrenia [6,7]. In the basal ganglia and forebrain, the 5-HT_2A has been immunologically localized to medium- and large-sized neurons in the lateral septal nuclei. In the hippocampus, the receptor is found in pyramidal cells in CA1-3 regions and granular cells of the dentate gyrus. 5-HT_2A receptor immunoreactivity has also been shown in the developing cerebellum [8].

INTRACELLULAR SIGNALING CASCADES ACTIVATED BY THE 5-HT_2A RECEPTOR

Signaling pathways are fundamental mechanisms that form the basis of cell physiology and the relation of a cell to its environment. These pathways interact and are interconnected in networks that regulate most cellular functions. Signaling pathways are the focus of current research in both experimental and theoretical studies.

INTRACELLULAR LOCALIZATION OF THE 5-HT_2A RECEPTOR

Although existing studies on receptor distribution in tissues using a variety of tissue-based techniques have allowed determination of steady-state levels and localization of the receptorin vivo, studies in cell culture have been most successful in addressing mechanistic details of receptor function and intracellular distribution. As the distribution of 5-HT_2A mRNA and radioligand binding indicate the 5-HT_2A receptor to be present in the mammalian cortex, the rat prefrontal cortex has been the focus of considerable interest in looking at subcellular localization of 5-HT_2A receptors. Localization of the receptor to the cortex also correlates with the effects of antagonists and inverse agonists on behavior and cognitive function. Light microscopy using antibodies shows the receptor to be localized to dendritic shafts of pyramidal and local circuit neurons. Electrophysiological studies predict presynaptic localization on glutamatergic cerebellar mossy fiber nerve terminals [86]; while neurochemical, immunoelectron microscopy and immunohistochemical studies suggest that 5-HT_2A may be presynaptic on dopaminergic axon terminals in the ventral tegmental area [87–90].

Ultrastructural immuno-electron microscopy also demonstrates that receptors are localized primarily to the dendrites of neurons in the rat prefrontal cortex. Moreover, 5-HT_2A receptors are expressed to a greater extent in apical dendrites than basilar dendrites [6].

Information on receptor targeting to various subcellular compartments on activation as well as inhibition of the receptor is available from bothin vivo studies as well as cell cultures. Although immunohistochemistry has been useful forin vivo experiments, modification of the receptor with tags has been useful in cell culture studies. Somato-dendritic targeting of the receptor has been established from bothin vivo and in vitro studies [91]. To study receptor localization in cell culturesin vitro, receptors fluorescently tagged (GFP) at the C-terminus or FLAG-tagged at theN-terminus of the receptor have been used [43]. Preliminary observations showed that receptor insertion into the plasma membrane remains unaffected using such tags [91–93]. On the other hand,N-terminal GFP tags have not been successful in obtaining receptor useful for experimental studies as plasma membrane targeting seems to be affected (unpublished data). Mutational analyses have also revealed the importance of an aspartate residue (Asp155), critical for targeting the receptor to the membrane [94].

However, it was discovered that GFP, by obscuring a potential PDZ binding domain at the C-terminus of the receptor, results in reduced targeting to the dendritic compartment in neurons. The 5-HT_2A receptor is the one of the first GPCRs showing that the PDZ-binding domain may play a critical role in dendritic targeting.

In using tags to study targeting of the receptor, the receptor was targeted correctly to the dendrites if the GFP was shifted 20 amino acids upstream of the C-terminus within the gene to allow the PDZ-binding domain to remain exposed. This provides a caveat that the tag has to be appropriately located such that all motifs are maintained intact for appropriate receptor functioning [91].

Studying the subcellular localization of the receptor and its targeting to different compartments suggests that there exist within the protein, signals for subcellular targeting and trafficking that are yet to be characterized.

Antibodies against the 5-HT_2A receptor are also widely used, with most studies using antibodies raised against anN-terminal portion of the receptor. Antibodies have been particularly useful for live staining cells expressing the receptor when the epitope is extracellular and the receptor is properly inserted into the cell membrane [93,95,96].

INTRACELLULAR TRAFFICKING OF THE 5-HT_2A RECEPTOR

GPCRs, in addition to initiating intracellular signal transduction cascades, also trigger cellular and molecular mechanisms leading to regulation of receptor signaling. Often, this is achieved by receptor phosphorylation, followed by internalization, wherein the receptor undergoes a series of structural and functional changes before being targeted to their final destination. This regulation of the receptor is brought about by interactions with regulatory molecules and trafficking to various subcellular compartments.

5-HT_2A receptors are internalizedin vitro andin vivo by several ligands including agonists as well as some antagonists [10,42–44]. 5-HT_2A endocytosis has also been subverted by the human JCV virus, which utilizes 5-HT_2A as a cellular receptor. Inhibiting receptor endocytosis reduces the risk of infection, arguing that some receptor antagonists that modulate receptor internalization, could be useful in treatment of progressive multifocal leukoencephalopathy caused by the JCV virus [33]. The biochemical mechanisms responsible for the regulation of 5-HT_2A intracellular trafficking are largely unknown.

Internalization in HEK293 cells expressing rat 5-HT_2A receptors begins within 2 min after adding 5-HT and is complete within 10 min [43]. Agonist-mediated internalization of the 5-HT_2A receptor is seen to occurvia clathrin-mediated endocytosis [97]. The process of internalization is also dynamin dependent. Dominant-negative mutant forms of clathrin and dynamin both inhibit receptor internalization. The receptor, on internalization is also seen to colocalize with other proteins such as the transferrin receptor (Figure 6.2), known to internalize using clathrin-dependent pathways [43].

FIGURE 6.2

Expression of 5-HT_2A tagged to EGFP in HEK 293 cells. (A) HEK 293 cells expressing EGFP-tagged 5-HT_2A receptor (5-HT_2A-EGFP) show cell surface-localized receptors after cycloheximide treatment. (B) Agonist-induced internalization of EGFP-tagged receptors.(more...)

Many — but not all — antagonists (including antipsychotic drugs) have been shown to bring about 5-HT_2A receptor internalization without receptor activation. Studies bothin vivo as well asin vitro have shown receptor redistribution in response to exposure to antagonists. Antagonist-mediated receptor internalization has also been shown to bevia clathrin-mediated endocytosis in a dynamin-dependent manner. Incidentally, antagonist-mediated internalization of the rat 5-HT_2A receptor, unlike serotonin-mediated internalization, is independent of protein kinase C (PKC) activation [98].

The rat 5-HT_2A receptor is unusual among GPCRs, in that it is internalized and desensitized in a β-arrestin-independent manner. Receptor internalization remains unaffected when dominant-negative mutants of different forms of β-arrestin (Arr-2 and Arr-3) are transfected into cells expressing the rat 5-HT_2A receptor [57]. Interestingly, though stimulation of the 5-HT_2A receptor brings about arrestin-independent internalization, arrestin is sorted into intracellular compartments, distinct from those containing the 5-HT_2A receptor. The molecular basis of arrestin-insensitivity of 5-HT_2A receptor internalization is not known.

Agonist-induced internalization of 5-HT_2A receptors is accompanied by differential sorting of Arr-2, Arr-3, and 5-HT_2A receptors into distinct plasma membrane and intracellular compartments. Agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles distinct from those with the 5-HT_2A receptor also implies novel roles for Arr-2 and Arr-3 [93]. In another interesting study, it was found that a constitutively active mutant form of arrestin-2 induces 5-HT_2A internalization independent of stimulation by a ligand. This effect was not seen if wild-type arrestin is overexpressed in the same cells. Additionally, it was shown that constitutively active arrestin-2 effected a decrease in efficacy of agonist-induced PI hydrolysis with a simultaneous increase in agonist potency. This was explained by postulating that arrestin binds to and stabilizes a receptor conformation that exhibits a higher affinity for agonists, hence allowing for significant changes in properties of receptor trafficking as well as signaling [99]. These novel observations of arrestin function make the 5-HT_2A receptor an important and interesting model system for the study of arrestin-GPCR interactions.

As discussed earlier, receptor activation and trafficking can be functionally selective in that different ligands can stimulate different biochemical pathways and signal transduction cascades. It was observed that agonists and antagonists display differential effects on binding to the receptor. Rat 5-HT_2A receptor activation by agonists, but not antagonists, induces greater Arr-3 than Arr-2 translocation to the plasma membrane [93].

Arrestin-independent internalization is thought to arise because it is believed that the 5-HT_2A receptor is not phosphorylated in response to stimulation by an agonist [100]. This would be unusual in GPCR endocytosis where receptor phosphorylation is a common phenomenon observed prior to internalization. One possible hypothesis is that ligand-specific phosphorylation or other such regulated posttranslational modifications may allow the 5-HT_2A receptor to participate in different pathways and traffic differentially in response to various agonists and antagonists.

5-HT_2A receptors have also been shown to recycle to the cell surface after activation and internalization by agonists. In HEK 293 cells expressing GFP tagged rat 5-HT_2A receptors, activating the receptor with 5-HT or dopamine, brings about internalization followed by receptor recycling, with the entire process taking approximately 2.5 h. These studies provided strong visual evidence that the receptors can recycle to the cell surface after being internalized in the normal course of activation, as has been reported for many other GPCRs. Moreover, the lack of discernible receptors within cells, in the absence of agonist, suggests that receptors are not normally internalized and recycled to any detectable extent unless stimulated. Activation of PKC, in the absence of an agonist also causes at least a portion of the receptors present on the cell surface to internalize and subsequently recycle to the cell surface. The kinetics of internalization and recycling are similar to those seen after activation by 5-HT or dopamine (Figure 6.3). Internalization and recycling could be important processes in desensitization and resensitization of receptor signaling and continue to be areas of intense study [43,44].

FIGURE 6.3

Functional selectivity in the 5-HT_2A receptor trafficking. Serotonin and dopamine bring about receptor internalization via clathrin and dynamin-dependent pathways. The receptor is more sensitive to dopamine-mediated internalization if serotonin is first(more...)

A similar study on trafficking of the 5-HT_2A receptor on exposure to antagonists is not yet published. It has been speculated that antagonist-internalized receptors are targeted to the lysosomal compartments for degradation, but this has not been proven. Differences in agonist- and antagonist-mediated internalization and recycling/degradation could explain some aspects of functional selectivity in receptor trafficking and signaling.

Studies reported so far have not looked in detail at the process of receptor internalization and the subcellular compartments that receptors localize to during trafficking. Data indicates that agonist-mediated internalization targets the receptor to a recycling endosome (Figure 6.2) [43,44]. The mechanistic details of 5-HT_2A recycling and pathways followed during these processes remain to be addressed. The role played by the cytoskeleton at all stages of receptor trafficking and molecular players such as Rab GTPases, which should be involved in these intracellular processes are yet to be identified. How biochemical mechanisms involved in 5-HT_2A recycling compare with those observed in other GPCRs also remain to be seen.

A number of studies on 5-HT_2A receptor internalization and trafficking have used fluorescently-tagged receptor constructs or fusion proteins wherein the receptor is tagged to an epitope for which an antibody is available. This has allowed for direct and occasionally real-time visualization of receptor trafficking in cell cultures andin vivo [43,44,93]. Similarly, intracellular transport of molecular players is also being studied for possible roles in functional interactions with the 5-HT_2A receptor. In such experiments the proteins studied are often fluorescently-tagged, whereas the receptor is visualized using antibodies.

While studying trafficking using fluorescently tagged receptors, it is often crucial to differentiate between receptors that have been newly synthesized and are being trafficked to the cell membrane and those that are being trafficked in response to, say, a stimulus. In order to do so, cells can be treated with protein synthesis inhibitors such as cycloheximide to ensure that all of the receptors get to the cell surface before the stimulus is provided (Figure 6.2). Though cycloheximide may not affect receptor signaling [101], it is seen that extended treatment of cells with protein synthesis inhibitors proves toxic. If so, alternative methods of reducing protein synthesis can also be employed such as serum starvation. In many cell lines, this also forces cells to enter the G₀ phase of the cell cycle. This may serve to reduce cell-cycle dependent variations, if any, in experimental observations.

Alternatively, an inducible system could be used to circumvent the necessity of inhibition of protein synthesis. Photoactivatable GFP (PAGFP) could be ideal in following receptor trafficking over time as PAGFP-tagged receptors, once photoactivated can be chased with no new contribution to the fluorescence from newly synthesized receptors [102]. Other inducible systems activated, for example, by tetracycline or pronesterone may be useful [103].

Biotinylation of surface receptors is a method routinely used in determination of receptor levels at the cell surface as well as in quantitative studies of receptor endocytosis and recycling. This method depends on specific exposed residues of the receptor being covalently modified with biotin. Streptavidin is subsequently used to quantify the levels of biotinylated receptor. It would be useful to remember that artifacts could arise from the biotinylation of specific residues if they change properties of the receptor and interfere with subsequent processes.

Receptor internalization can also be visualized by making use of a fluorescent ligand as receptor and ligand internalize together and remain associated for some time [104,105]. Unfortunately, no fluorescent ligands are available to visualize 5-HT_2A receptor trafficking. Antibody feeding experiments which bind to extracellular portions of the receptor partially evade this issue by allowing antibody-labeling of surface receptors in live cells after which the receptor-antibody complex can be chased to observe intracellular trafficking [43,44]. Imaging of the antibody-receptor complex can be carried only until the endosome, where low pH breaks apart this association. In another interesting development, quantum dots have been covalently attached to serotonin which may allow real-time imaging of receptor trafficking as long as receptor and ligand remain associated. As quantum dots are nontoxic and photostable they can be imaged using conventional fluorescence microscopy for an extended period [106,107].

Currently, information on 5-HT_2A receptor trafficking has been largely derived from cell cultures. Considering the obvious limitations on extending cell culture studies toin vivo situations it would be useful to generate transgenic mice expressing fluorescently tagged or epitope-tagged 5-HT_2A receptors. An important aspect of 5-HT_2A function that has arisen out of these studies is that a number of observations are cell-type specific. Therefore, choosing an appropriate cell type for study becomes all the more crucial. Observations from such anin vivo system could be correlated with data obtained from cell lines. This would also allow simultaneous study over several cell types to carefully document cell-type specific characteristics of receptor trafficking. If receptor expressionin vivo is also inducible it would add considerably more to our understanding of receptor trafficking lifetimes and region-specific behavior of the receptor. It would be also useful to generate knock-in transgenic mice expressing similar levels of modified receptor to that seen in the wild-type as well as use a model organism wherein the receptor is considerably over-expressed.

In addition, most studies have used the rat 5-HT_2A receptor, and it is likely that important differences will arise when the human receptor is characterized. The few amino acid changes between the rat and human receptors may yield a whole new panoply of interacting proteins. Ligands will undoubtedly display completely altered efficacies and potencies at the rat and human receptors as is evident from ligand-binding studies with overexpressed receptors in cell lines [108]. Signaling and trafficking could turn out to be substantially different when using the human receptor as a model.

LONG-TERM REGULATION OF 5-HT_2A RECEPTOR

Long-term changes in 5-HT_2A receptor protein and mRNA expression have been the focus of many studies in the last decade. These time scales are of relevance as drugs that target the receptor are often administered for weeks before their effects are evident. Earlier studies looked at receptor protein levels whereas the advent of more sensitive techniques such as the ribonuclease protection assay (RPA) and reverse transcriptase polymerase chain reaction (RT-PCR) have allowed accurate estimation of receptor mRNA levels.

Long-term regulation of receptor levels in tissues has been monitored by radio-ligand binding assays. These assays initially involved the use of membrane fractions from tissue samples that express the receptor. Subsequently, cell lines that overexpress the receptor have been used. Receptor levels are determined by measuring binding of a range of concentrations of radioactive ligand to known amounts of the receptor in cell or tissue membranes. The membranes are washed free of excess ligand either by filtration or centrifugation and levels of the radioactive bound ligand determined. The concentration of radio-ligand that produces half-maximal occupancy represents K_d, the dissociation constant of the ligand to the receptor [109]. B_max, a measure of the maximum values of bound ligand, would represent the availability of receptors. Receptor up-regulation and down-regulation cause an increase and reduction in B_max, respectively, leaving K_d unaltered. The assay is performed under various conditions when looking at regulation of levels of receptor protein.

Designing the assay to have the ligand compete with an already-bound radio-labeled ligand of known affinity would allow for the measurement of relative affinity between various ligands for the receptor. Such competitive binding experiments are helpful in the determination of affinity of drugs to a receptor and also determine variation in affinities among mutant forms of the receptor or its homologues in different species.

Positron emission tomography (PET) is a useful and powerful method to studying receptor levelsin vivo, particularly in humans in real time. PET studies have also been carried out in rodent models. Radioligands that are often used in these studies are [F18] Altanserin and [C11] MDL 100907 labeled, which have been administered to patients that are either drug-naïve or have undergone treatment. Such studies have shown altered receptor densities in people with enhanced risk of schizophrenia [110], anorexia, and bulimia nervosa [111], and in untreated patients of obsessive compulsive disorders [25]. These methods have also been employed to examine the effectiveness of a certain treatment regime in altering receptor density in a disease [112,113].

mRNA levels of the 5-HT_2A receptor have been monitored byin situ hybridizations, ribonuclease protection assays, and real time RT-PCR.In situ hybridizations have been carried out on brain slices or cell cultures and involve hybridization of a radioactive or nonradioactively labeled antisense probe to the RNA species present in cells in the tissue or in culture. Although levels of mRNA can be estimated using autoradiography with radiolabeled probes, nonradioactivein situ hybridizations provide better cellular resolution though changes in transcript levels are not as easily determined [114,115]. Ribonuclease protection assays offer a quantitative and sensitive approach to determine levels of a specific transcript and have been successfully used to determine 5-HT_2A transcript levels. In this method, an antisense radiolabeled RNA probe specific to the transcript, is hybridized with RNA extracted from the tissue of interest and then treated with single-stranded specific ribonuclease T₁. The presence of the transcript and its hybridization to the probe results in the probe being protected from degradation by RNase T₁. Protected probe molecules are visualized and quantified after gel electrophoresis. This method allows quantification of minor variations in RNA levels, at the cost of spatial information. Such a study has yielded information on DOI-mediated long-term changes in 5-HT_2A mRNA levels in the prefrontal cortex [116].

PCR based studies have also been used extensively in characterization of 5-HT_2A expression and regulation. From identifying expression of receptor transcripts in the dorsal root ganglion in humans [117], understanding the imprinting status of the gene in the human brain [118] to associating receptor polymorphisms in humans with a predisposition toward seasonal affective disorders [119]. Commonly used PCR-based techniques such as reverse-transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) have been used extensively to estimate receptor mRNA levels under various conditions. The two methods are based on mRNA extraction and subsequent generation of cDNA from samples using reverse transcriptase. In RT-PCR studies gene primers designed to amplify a portion of the cDNA containing the receptor sequence are used to determine the presence of mRNA of the gene of interest. RT-PCRs have been employed in examining agonist-induced receptor down-regulation in NIH 3T3 cells and Balb/c-3T3 cells [120,121], identifying neurons that respond to long-term treatment with clozapine by single-cell RT-PCR of rat cortical neurons [122] and mapping the effect ofN-methyl norsalsolinol on the expression of 5-HT_2A receptor transcripts in the caudate nucleus of rats [123]. qRT-PCR is a more accurate method and estimates the number of copies of a gene in the cDNA (and hence mRNA) present in a sample from the kinetics of amplification. qRT-PCR is a far more sensitive method and can be used in determining subtle changes in transcript status.

Microarray analyses have also been utilized by some groups to assess global changes in transcripts upon long-term stimulation or inhibition of the 5-HT_2A receptor. An interesting study coupled data from microarray experiments and qRT-PCRs to monitor differential regulation of the cortical transcriptome in response to hallucinogenic vs. nonhallucinogenic drug stimulation of the 5-HT_2A receptor using transgenic mice that expressed undetectable levels of the receptor in the cortex [124].

A striking observation that has arisen through studies on 5-HT_2A receptor levels is that chronic application of agonists like DOI, 5-HT and LSD, as well as antagonists like mianserin, ketanserin, and pipamperone, cause down-regulation of the receptor. The biochemical mechanisms behind this “paradoxical regulation” of the 5-HT_2A receptor are unknown. Agonist-induced long-term regulation has been examined in numerous studies.In vitro studies report variable results depending on the cell type used with up-regulation shown in MDCK cells, no difference observed in NIH 3T3 cells, and down-regulation in AtT-20 cells [125].In vivo studies, on the other hand, have consistently reported receptor down-regulation on chronic agonist application [116,126,127].

With the exception of SR 46349B, all currently available 5-HT_2A antagonists cause down-regulation of the receptor. It is postulated that antagonist-mediated 5-HT_2A receptor down-regulation is brought about by receptor internalization followed by lysosomal degradation of internalized receptors. It has also been suggested that this down-regulation may be the way that antipsychotics wield their therapeutic effect [60,80].

CONCLUSION

In this chapter we have attempted to describe the serotonin 2A (5-HT_2A) receptor as a model of GPCR function. We have tried to bring out complexities of signaling paradigms, interacting partners, and characteristics of the receptor in response to different agents/ligands. In keeping with the aim of the book, we have tried to give the reader an understanding of the techniques and methods used in the field to answer the questions being posed. Wherever possible, we have tried to discuss the pros and cons of the method over others that could have been used. In addition, we also suggest some avenues of future research to better understand the role played by the 5-HT_2A receptor in mediating CNS functioning, particularly those affected in diseases like schizophrenia, bipolar disorders, depression, and anxiety.

ACKNOWLEDGMENTS

We would like to acknowledge past and present members of our laboratory, particularly Samarjit Bhattacharyya for valuable discussions. This work was supported by the National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore. A.B. is a recipient of the Kanwal Rekhi Career Development Fellowship from the TIFR endowment fund.

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*

Authors have contributed equally.