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

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Chapter 6Serotonin 2A (5-HT2A) Receptor Function: Ligand-Dependent Mechanisms and Pathways

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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-HT2A) 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-HT2A 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-HT2A receptor could well serve as an important alternate paradigm in the study of GPCR function.

The 5-HT2A receptor was initially identified by hybridization using conserved elements of the cloned 5-HT2C 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-HT2B and 5-HT2C. All members of the 5-HT2 receptor subfamily primarily couple to PLC on activation. As with most GPCRs, 5-HT2A 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-HT2A 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-HT2A 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-HT2A 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-HT2A receptors.

FIGURE 6.1

Comparison of amino acid composition of the human, rat and mouse 5-HT2A receptors. The amino acid sequence of the human 5-HT2A 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-HT2A receptor mRNA expression in the rat CNS was based on northern blots of RNA extracted from various regions of the CNS [2]. Further studies using in 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-HT2A receptor mRNA was detected in the striatum and cerebellum. In addition, 5-HT2A mRNA expression has been found in the dorsal horn of the spinal cord [4,5].

5-HT2A 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-HT2A 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 5HT2A receptors. In the cortex, the 5-HT2A 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-HT2A 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-HT2A receptor immunoreactivity has also been shown in the developing cerebellum [8].

Implication in Diseases

Given the extensive localization of this receptor to brain areas that mediate cognitive functions and social interaction, it suggests that the 5-HT2A receptor might be involved in diseases in which these functions are impaired. Disorders in which the 5-HT2A receptor seems to be involved range from schizophrenia, depression, obsessive compulsive disorder (OCD), and attention deficit–hyperactivity disorder (ADHD), to eating disorders such as anorexia nervosa, to autism spectrum disorders. Evidence to support such connections varies from genetic screens to binding and protein expression data and some molecular data.

Schizophrenia: 5-HT2A is thought to play a significant role in this disorder. This is partly due to the fact that most typical and almost all atypical antipsychotic drugs used in therapy, bind to 5-HT2A receptors, as do many hallucinogens, which mimic schizophrenia-like symptoms, bind to this receptor [9–11]. Another line of investigation has led to the hypothesis that 5-HT2A, by modulating dopamine release in the striatum and cortex causes the motor and cognitive defects seen in schizophrenia [12]. Adding to these observations are postmortem studies that show altered 5-HT2A receptor expression in areas like the Brodmann area 9 of the cortex in schizophrenic patients [13] and increased expression in suicide cases diagnosed with schizoid symptoms. Genetic studies, however, have not yielded conclusive results; the promoter polymorphism 1438 bp upstream of the coding region involving a G/A change has been the focus of much debate, with some studies claiming a correlation to schizophrenia, increased aggression, etc., and others refuting the claim [14–16]. To date, no polymorphism in the 5-HT2A gene has been consistently associated with the disease or any of its symptoms across numerous studies and populations [17].

Depression/anxiety: It is known that long-term administration of tri-cyclic anti-depressants causes a decrease in the cortical density of 5-HT2A receptors [18]. PET binding studies have reported a decrease in 5-HT2A levels in the hippocampus and platelets in patients with major depression [19]. It is also unclear whether there is a correlation between 5-HT2A receptor polymorphisms and the propensity or onset of the disease [20–22]. Recently, studies using a transgenic mouse with little or no expression of the 5-HT2A receptor in the brain indicate that it is required for modulation of anxiety-related behavior in mice [23]. 5-HT2A receptor agonists have been reported to result in significant but differential regulation of BDNF mRNA levels in the hippocampus and neocortex. BDNF mRNA expression has been reported to affect neurogenesis in the dentate gyrus and has bearing on the depressive phenotype [24].

OCD/ADHD: Patients with obsessive compulsive disorders show increased binding to 5-HT2A receptors in brain areas like the caudate nuclei [25]. Studies monitoring correlations between polymorphisms in the receptor as well as its promoter and disease onset have not yielded results that can be generalized across different ethnic groups [26]. 5-HT2A receptor levels seem unaffected in patients suffering from attention deficit hyperactivity disorder (ADHD) [27], yet the receptor polymorphisms may be a modulating factor in ADHD.

In addition, the 5-HT2A receptor has also been implicated in other diseases with less established etiologies. PET scans of patients suffering from Anorexia nervosa reveal decreased 5-HT2A receptor binding in the hippocampus, amygdala, and the cingulate cortex [28,29]. Autism and Asperger’s syndrome have been associated with increased platelet serotonin levels and decreased 5-HT2A binding in cortical regions [30]. The receptor has also been implicated in the pathophysiology of diseases like bipolar disorders, Alzheimer’s disease, progressive multifocal leukoencephalopathy, obstructive sleep apnea syndrome, and thermal or inflammatory pain [31–37].

In recent years, a handful of studies have attempted to integrate increased understanding about signaling pathways modulated by the receptor as well as its interacting partners and its possible roles in cellular and physiologic processes. Such studies will no doubt help elucidate the role 5-HT2A receptors play in the etiology of mental and peripheral disorders. A recent report identified the role of platelet-derived serotonin in mediating liver regeneration via the 5-HT2A receptor [38]. A signaling cascade linking the 5-HT2A receptor to cellular proliferation has also been recently delineated [39]. The involvement in platelet aggregation of the 5-HT2A receptor expressed in platelets has been widely investigated. In addition, there appears to be a role that the drosophila 5-HT2 homolog plays in mediating adhesion in drosophila [40].

INTRACELLULAR SIGNALING CASCADES ACTIVATED BY THE 5-HT2A 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.

Activation Studies

The 5-HT2A receptor activates PLC through Gq and leads to an accumulation of IP3, di-acylglycerol (DAG) and activation of protein kinase C (PKC) [41]. Increase in cytoplasmic IP3 causes a release of calcium from intracellular endoplasmic reticulum stores—a characteristic activation signature of many GPCRs. This cascade has been the most studied and is perhaps the most important signal transduction pathway regulated by this receptor. Two assays associated with characterization of this pathway are discussed below. It has also been found that the 5-HT2A receptor activates other signal transduction cascades in a ligand-dependent manner. This ligand-specific functional activation of intracellular signaling cascades is discussed later.

Calcium Release Experiments

Calcium release has been monitored by loading cells with Ca2+ sensitive fluorescent dyes like Fura-2AM, fluo-3AM and rhod-2AM, to name a few. Descriptions of the large number and types of dyes available, labeling substrate molecules and calculating quanta of Ca2+ release, are usually available with the manufacturers. Briefly, a Ca2+ release experiment involves loading live cells with a Ca2+ sensor dye in the form of a cell-permeable neutral AM (acetoxy-methoxy) ester. The dye is usually in solution in DMSO and is added along with a detergent to facilitate its entry into cells. Cellular esterases cleave the dye ester to a charged form, thus sequestering the dye in the cytoplasm. The ligand under study is then applied to cells, and fluctuations in Ca2+ levels in the cells are measured. The dye alters its fluorescence intensity or causes a spectral shift in emission or excitation wavelength when bound to the released Ca2+. Experimental considerations are:

  • The choice of dye, i.e., its affinity to calcium and the changes expected.
  • The nonuniformity of dye loading (dyes may be charged species that get sequestered selectively in some organelles exhibiting a potential difference across their membrane like Rhod-2AM in mitochondria).
  • The type of the measurement desired, i.e., qualitative or quantitative. Ratiometric dyes are preferred for quantitative measurements as measurements with such dyes can be made independent of bleaching, uneven dye uptake, or leakage or variation in cell thickness.

Imaging in such experiments is also a crucial factor. As one has to contend with dye bleaching, accurate background corrections have to be obtained in images to ascertain precise amounts of fluorescence enhancement. In summary, once standardized, this technique has the potential to answer questions on signaling pathways activated by receptors, the time course of activation, ligand-specific variation in efficacy of receptor activation, and intracellular Ca2+ fluctuations that might occur due to constitutive activity of the receptor [42–44]. It is also important to note that experimental observations are very sensitive to temperature. Other than temperature affecting the kinetics of release, heat stress also attenuates Ca2+ mobilization. This temperature-sensitive attenuation of Ca2+ release can be rescued by the expression of Hsp 70, possibly via a protease mechanism [45,46].

Studies have reported elevated platelet Ca2+ levels upon activation of 5-HT2A receptors in patients with bipolar disorders. Such studies should help in understanding receptor dysfunction in depression and could be incorporated in designing patient-specific drug regimens [47–53].

New methods of visualizing intracellular calcium dynamics are also being introduced and should lead to increased sensitivity in studying spatiotemporal regulation of Ca2+ in cells in response to receptor function [54].

5-HT2A activation has also been shown to effect Ca2+ signaling in cells by regulating voltage-gated Ca2+ channels. In acutely isolated prefrontal pyramidal cortical neurons, 5-HT2A receptors, via their classical pathway, activate calcineurin and inhibit activation of Cav1.2 L-type Ca2+ currents. This modulation and its blockade by atypical neuroleptics are postulated to have wide-ranging effects on synaptic integration and long-term gene expression in the deep-layer prefrontal pyramidal regions of the brain [55]. In contrast, in astrocytes, 5-HT2A stimulation opens voltage-independent Ca2+ channels, resembling depletion-operated calcium channels (DOCCs) [56].

IP3 Activation

Inositol 1,4,5-tris-phosphate accumulation by 5-HT2A receptor activation has also been used, not only to monitor and quantify receptor activation, but also to measure desensitization. IP3 assays routinely measure phosphoinositol hydrolysis using cells preincubated with radioactive myo-inositol or inositol, which acts as the source of labeled IP3. Following agonist or antagonist application, accumulation of total radiolabeled IP (inositol mono-phosphate, inositol bis-phosphate, and inositol tris-phosphate) is determined using ion exchange chromatography and scintillation counting. This assay not only provides quantitative estimates but also temporal data regarding receptor activation. The assay has yielded many insights into the mechanism of 5-HT2A desensitization and activation, as well as allowed identification of specific ligands against the 5-HT2A receptor [57–64]. In addition, many antagonists act as inverse agonists, inhibiting ligand-independent basal receptor activation. Decrease in basal levels of IP3 thereby provides a measure of antagonist efficacy.

5-HT2A Receptor Functional Selectivity

Many GPCRs display a phenomenon whereby different ligands can differentially activate signaling pathways via the same receptor. This differential activation arises independently of differences in the affinity of the ligand to the receptor. Ligands often display differences in efficacy and/or potency at one signaling pathway vs. another. A number of terms have been coined to describe this phenomenon, of which functional selectivity is gaining acceptance [65].

The 5-HT2A receptor is one of the first receptors to have been characterized as displaying the phenomenon of functional selectivity [66,67]. This was first hypothesized on the basis of the observation that hallucinogenic effects of drugs such as LSD do not correlate with their activation of the IP3/diacylglycerol pathway. The receptor has since been extensively studied and several conditions identified under which functional selectivity may be observed, and ligands that can bring about this selectivity have been characterized.

Stimulation of the 5-HT2A receptor leads to the production of at least three distinct biochemical signals, IP3/diacylglycerol, arachidonic acid (AA), 2-arachidonylglycerol (2-AG), and the relative activation of these pathways varies with the ligand used [68]. By measuring simultaneously at least two of these pathways using radioactively labeled IP3 and arachidonic acid (the signal transduction outputs of each of these pathways from the same cells) it has been seen that ligands vary in the efficacy with which they stimulate each of the different pathways.

More recently, identification of molecular players involved in the signaling cascade resulting in 5-HT2A receptor-induced arachidonic acid release, revealed that more than one signaling pathway is involved [69]. It raises the possibility that using a single readout such as arachidonic acid to characterize a biochemical pathway may not be sufficient. Different ligands may activate different pathways that finally impinge on the same molecule.

Numerous studies in different cell lines using antibodies to look at levels of molecular players have linked regulation of several signaling pathways to the 5-HT2A receptor. In addition to the three major pathways mentioned above, 5-HT2A stimulation can lead to a change in levels and/or activity of several molecular players including PLA2, PLD, ERK1/2, nitric oxide, calmodulin, CREB, Akt, Fos, TGF-β, EGFR, and JAK/STATs [39,70–79], depending on cell line used and the context of stimulation. Some, if not all, of these pathways will undoubtedly be involved in functional selectivity displayed by ligands targeting the receptor. This leads to a situation of ever-increasing complexity wherein accurate characterization of receptor signaling will require simultaneous measurements of a number of key molecular players involved in each of these pathways.

In addition to these signaling pathways, receptor regulation also takes another form, i.e., internalization and recycling of the receptor. Interestingly, this pathway, too, is differentially regulated by different ligands.

Agonists such as 5-HT and those with partial efficacy on the same receptor, such as dopamine, bring about internalization of the rat 5-HT2A receptor, perhaps to different levels. A study performed in HEK293 cells using high resolution epifluorescence microscopy observed GFP-tagged receptors and their trafficking properties. Interestingly, it was observed that PKC activation is necessary for 5-HT-mediated internalization, whereas it is not required in dopamine-mediated internalization. Yet, both dopamine as well as 5-HT internalized receptors take the same amount of time to recycle to the cell surface. A further level of complexity is introduced when the receptor is primed toward dopamine-mediated internalization by low levels of 5-HT. Thus, two agonists (5-HT and dopamine) interact at the 5-HT2A receptor in a defined temporal sequence to bring about receptor internalization at concentrations lower than either of the two ligands could achieve individually [43,44].

Some antagonists (antipsychotic drugs) have also been reported to bring about an internalization of the receptor as well. The biochemical pathways stimulated by these ligands and the mechanisms involved in receptor internalization remain to be characterized [10,80,81].

Ligands differ in the extent to which they can stimulate signaling pathways as well as the biochemical pathways of receptor trafficking. It is conceivable that signaling pathways affected by the receptor will vary depending on its intracellular localization. This raises yet another issue in 5-HT2A signaling—it becomes necessary to consider subcellular localization at a time when the receptor is affecting a signaling pathway. The 5-HT2A receptor may, on internalization, enter a signaling endosome and, depending on the local environment in and around the endosome, affect signaling pathways in the cell. This has particular relevance in a polarized cell such as a neuron wherein the receptor may regulate different pathways in different subcellular locations.

Signaling could then be further affected by a number of factors including how long the receptor is retained in a milieu such as a signaling or recycling endosome. It is foreseeable that ligands, after bringing about receptor internalization, will generate signals that alter the time taken for receptor recycling. This could change the time that the receptor spends in a subcellular environment, adding another dimension to the signaling. Thus ligand-specific modifications to the receptor may introduce yet another factor in receptor regulation of signaling and biochemical pathways.

A detailed study characterizing functional selectivity, not only among signaling pathways but also among trafficking pathways, is essential, and structural features of the receptor that allow for differential stimulation of individual pathways require identification. Interacting partners of the receptor under different conditions when stimulated by agonists and antagonists might yield crucial information. Possible posttranslational modifications such as ligand-specific phosphorylation of the receptor might well have a role to play in functional specificity.

One possible mechanism whereby this functional selectivity is regulated could be by different structural conformations attained by the receptor. Studies have shown different receptor reserves for separate signaling pathways [68]. This further confounds the issue by bringing into focus changes in signaling brought about by altering levels of receptor expression and differences in conformation and/or interacting partners that might bring about alterations in receptor reserve.

In some conformations, receptors may activate effectors in the absence of a stimulating ligand. This receptor activity is decreased by inverse agonists and increased by agonists. This realization led to the development of multistate models of receptor function in which receptors can spontaneously form a variety of “active” conformations that regulate effector mechanisms in the absence of a ligand. Results from these studies have been interpreted as demonstration of multiple populations of receptor, each with a distinct conformation, with each conformation differing in affinity to different ligands and in its ability to stimulate signaling pathways. A naturally occurring single nucleotide polymorphism (SNP) has been identified that causes a histidine to tyrosine (H452Y) change and brings about a destabilization of the signaling conformation of the receptor [82]. The mechanism by which this mutation acts might give insights into the normal functioning of the receptor. Other point mutations in the receptor that change some aspect of receptor signaling and/or trafficking have been identified. For instance, a single amino acid mutation from cysteine to lysine (C322K) in the sixth transmembrane region makes the receptor constitutively active in that it results in continual stimulation of at least one signaling pathway, the IP3/diacylglycerol pathway [83]. In another exhaustive study, all serine and threonine residues in the protein were mutated in groups and the effects on signaling and desensitization of the receptor were characterized [84].

It is important to note that the activation of all these biochemical and signaling pathways is cell-type specific and care should be taken in choosing an appropriate system [57]. It could also be useful to identify commonalities of receptor signaling in different systems. At this point, comprehensive theoretical models of receptor signaling pathways might provide information at a system level that may not be arrived at experimentally.

There is a continuing need for a representation of signaling in dynamic compartments within a cell, with spatio-temporal information obtained from experiment. Such developments are essential for a quantitative understanding of how multiple functions of a cell are coordinated and regulated, and to evaluate the specifics of GPCR signaling.

All studies describing signaling pathways affected and their differential regulation by the 5-HT2A receptor have been carried out in cell culture. These studies are yet to be carried forward to studying signaling in an organism. For such studies perhaps drosophila or zebrafish may be useful initially as systems that allow easy genetic manipulation, and there exist a plethora of well-developed techniques available to study GPCR signaling and trafficking in these systems [85]. While using these organisms as model systems, in order to better understand the roles played by the receptor in mammals, it would also be important to look at both designed and naturally occurring mutants of this receptor and associated molecules.

Probing 5-HT2A Oligomerization

GPCRs oligomerization has profound implications on GPCR-ligand interactions, receptor functionality and on cross-talk between different signaling systems. GPCR Homodimerization can bring about cooperativity between associating receptors, leading to substantially altered functional interactions between receptor and one or more ligands [44]. Oligomerization of the 5-HT2A receptor has not been demonstrated to date, though it has been shown that 5-HT2C signaling is regulated by homodimerization [133]. The possibility of 5-HT2A multimerization needs to be addressed as characterization of the oligomerization state of the receptor would add to our understanding of 5-HT2A function.

Traditionally, association has been determined by coimmunoprecipitation of two populations of receptor tagged with the two different epitopes. However, coimmunoprecipitation may be unable to distinguish between direct receptor interactions and indirect associations within a complex [134].

Two techniques that are now commonly used that allow real-time identification of interacting partners in live cells are fluorescence resonance energy transfer (FRET) [135,136] and bioluminescence resonance energy transfer or BRET [136–138]. FRET and BRET studies have the added advantage of being able to study complexes in different subcellular locations. Some studies have utilized all three methods FRET, BRET and coimmunoprecipitation to validate their hypothesis [133].

If receptor multimerization is confirmed, it would be of interest to check if receptor multimerization is constitutive, ligand-induced, or even ligand-specific [139,140]. Furthermore, the stoichiometry of a receptor multimer can be determined using cross-linkers to covalently associate receptor assemblies whose molecular size is subsequently determined by gel electrophoresis [141]. TIRF microscopy could also identify stoichiometry of receptor complexes at the cell membrane by looking at bleaching characteristics of fluorescent receptors in these complexes.

Subsequently, residues and regions of the receptor involved in multimerization can be identified by sequential mutation of residues or deletion or portions of the receptor [133,142–146]. This has also been approached either through a variety of bioinformatics techniques [147–154].

INTRACELLULAR LOCALIZATION OF THE 5-HT2A 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 receptor in vivo, studies in cell culture have been most successful in addressing mechanistic details of receptor function and intracellular distribution. As the distribution of 5-HT2A mRNA and radioligand binding indicate the 5-HT2A 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-HT2A 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-HT2A 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-HT2A 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 both in vivo studies as well as cell cultures. Although immunohistochemistry has been useful for in 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 both in vivo and in vitro studies [91]. To study receptor localization in cell cultures in vitro, receptors fluorescently tagged (GFP) at the C-terminus or FLAG-tagged at the N-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-HT2A 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-HT2A receptor are also widely used, with most studies using antibodies raised against an N-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-HT2A 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-HT2A receptors are internalized in vitro and in vivo by several ligands including agonists as well as some antagonists [10,42–44]. 5-HT2A endocytosis has also been subverted by the human JCV virus, which utilizes 5-HT2A 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-HT2A intracellular trafficking are largely unknown.

Internalization in HEK293 cells expressing rat 5-HT2A receptors begins within 2 min after adding 5-HT and is complete within 10 min [43]. Agonist-mediated internalization of the 5-HT2A receptor is seen to occur via 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-HT2A tagged to EGFP in HEK 293 cells.

FIGURE 6.2

Expression of 5-HT2A tagged to EGFP in HEK 293 cells. (A) HEK 293 cells expressing EGFP-tagged 5-HT2A receptor (5-HT2A-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-HT2A receptor internalization without receptor activation. Studies both in vivo as well as in vitro have shown receptor redistribution in response to exposure to antagonists. Antagonist-mediated receptor internalization has also been shown to be via clathrin-mediated endocytosis in a dynamin-dependent manner. Incidentally, antagonist-mediated internalization of the rat 5-HT2A receptor, unlike serotonin-mediated internalization, is independent of protein kinase C (PKC) activation [98].

The rat 5-HT2A 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-HT2A receptor [57]. Interestingly, though stimulation of the 5-HT2A receptor brings about arrestin-independent internalization, arrestin is sorted into intracellular compartments, distinct from those containing the 5-HT2A receptor. The molecular basis of arrestin-insensitivity of 5-HT2A receptor internalization is not known.

Agonist-induced internalization of 5-HT2A receptors is accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2A 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-HT2A 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-HT2A 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-HT2A 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-HT2A 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-HT2A 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-HT2A receptor to participate in different pathways and traffic differentially in response to various agonists and antagonists.

5-HT2A 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-HT2A 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-HT2A receptor trafficking.

FIGURE 6.3

Functional selectivity in the 5-HT2A 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-HT2A 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-HT2A 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-HT2A recycling compare with those observed in other GPCRs also remain to be seen.

A number of studies on 5-HT2A 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 and in vivo [43,44,93]. Similarly, intracellular transport of molecular players is also being studied for possible roles in functional interactions with the 5-HT2A 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-HT2A 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-HT2A receptor trafficking has been largely derived from cell cultures. Considering the obvious limitations on extending cell culture studies to in vivo situations it would be useful to generate transgenic mice expressing fluorescently tagged or epitope-tagged 5-HT2A receptors. An important aspect of 5-HT2A 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 an in 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 expression in 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-HT2A 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.

TABLE 6.1

Proteins That Associate with the 5-HT2A Receptor

Interacting ProteinGeneRegion of the Receptor Involved in InteractionReference
Amyloid-b precursor protein intracellular domain associated protein-1aAIDA-1Ai3100
Eukaryotic translation initiation factor 3, subunit 5 eEIF3S5i3100
Neurotrophic tyrosine kinase, receptor, type 3 isoform c precursorNTRK3i3100
Melanoma-associated antigenMAAT1i3100
Paraoxonase 2PON2i3100
Microtubule associated protein 1AMAP-1Ai396,100
Ribosomal protein S6 kinase 2RSK2i3100
Nucleoside-diphosphate kinase 3NME3i3100
NADH dehydrogenase (ubiquinone) 1 b subcomplexNDUFB10i3100
Protein phosphatase 5, catalytic subunitPPP5Ci3100
Glutamine synthetaseGLULi3100
ADP ribosylation factor 1Arf1C-terminus128
Post synaptic density protein-95PSD-95C-terminus (PDZ-binding domain)92
Antioxidant protein-2AOP-2C-terminus (PDZ-binding domain)129
Activin receptor-interacting protein 1ARIP-1C-terminus (PDZ-binding domain)129
Synapse-associated protein 97SAP97C-terminus (PDZ-binding domain)129
MAGUK p55 subfamily member-3MPP-3C-terminus (PDZ-binding domain)129
Channel-interacting PDZ domain proteinCIPPC-terminus (PDZ-binding domain)129
Filamin cNot known130
Receptor for activated protein kinase CRACK1C-terminus130
CalmodulinCAMi2, C-terminus78
ArrestinARRB2i3131
Caveolin-1CAV-1Not known132

LONG-TERM REGULATION OF 5-HT2A RECEPTOR

Long-term changes in 5-HT2A 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 Kd, the dissociation constant of the ligand to the receptor [109]. Bmax, 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 Bmax, respectively, leaving Kd 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 levels in 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-HT2A receptor have been monitored by in 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, nonradioactive in 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-HT2A 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-HT2A mRNA levels in the prefrontal cortex [116].

PCR based studies have also been used extensively in characterization of 5-HT2A 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 of N-methyl norsalsolinol on the expression of 5-HT2A 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-HT2A 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-HT2A 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-HT2A 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-HT2A 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-HT2A antagonists cause down-regulation of the receptor. It is postulated that antagonist-mediated 5-HT2A 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-HT2A) 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-HT2A 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.

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