International Union of Basic and Clinical Pharmacology. CX. Classification of Receptors for 5-hydroxytryptamine; Pharmacology and Function.

5-HT receptors expressed throughout the human body are targets for established therapeutics and various drugs in development. Their diversity of structure and function reflects the important role 5-HT receptors play in physiologic and pathophysiological processes. The present review offers a framework for the official receptor nomenclature and a detailed understanding of each of the 14 5-HT receptor subtypes, their roles in the systems of the body, and, where appropriate, the (potential) utility of therapeutics targeting these receptors. SIGNIFICANCE STATEMENT: This review provides a comprehensive account of the classification and function of 5-hydroxytryptamine receptors, including how they are targeted for therapeutic benefit.

Beta2-adrenergic receptor homodimers: Role of transmembrane domain 1 and helix 8 in dimerization and cell surface expression.

Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this remains a highly controversial area of research and mechanisms governing homodimer formation are poorly understood. Crystal structures revealing homodimers have been reported for many different GPCR. For adrenergic receptors, a potential dimer interface involving transmembrane domain 1 (TMD1) and helix 8 (H8) was identified in crystal structures of the beta1-adrenergic (ß1-AR) and ß2-AR. The purpose of this study was to investigate a potential role for TMD1 and H8 in dimerization and plasma membrane expression of functional ß2-AR. Charged residues at the base of TMD1 and in the distal portion of H8 were replaced, singly and in combination, with non-polar residues or residues of opposite charge. Wild type and mutant ß2-AR, tagged with YFP and expressed in HEK293 cells, were evaluated for plasma membrane expression and function. Homodimer formation was evaluated using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and fluorescence correlation spectroscopy. Amino acid substitutions at the base of TMD1 and in the distal portion of H8 disrupted homodimer formation and caused receptors to be retained in the endoplasmic reticulum. Mutations in the proximal region of H8 did not disrupt dimerization but did interfere with plasma membrane expression. This study provides biophysical evidence linking a potential TMD1/H8 interface with ER export and the expression of functional ß2-AR on the plasma membrane. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Native serotonin 5-HT2C receptors are expressed as homodimers on the apical surface of choroid plexus epithelial cells.

G protein-coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti-5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10(-9) cm(2)/s and were expressed at 32 receptors/µm(2) on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit.

Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor.

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle.

The issue of G protein-coupled receptor (GPCR) oligomer status has not been resolved. Although many studies have provided evidence in favor of receptor-receptor interactions, there is no consensus as to the exact oligomer size of class A GPCRs. Previous studies have reported monomers, dimers, tetramers, and higher-order oligomers. In the present study, this issue was examined using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH) analysis, a sensitive method for monitoring diffusion and oligomer size of plasma membrane proteins. Six different class A GPCRs were selected from the serotonin (5-HT2A), adrenergic (α1b-AR and ß2-AR), muscarinic (M1 and M2), and dopamine (D1) receptor families. Each GPCR was C-terminally labeled with green fluorescent protein (GFP) or yellow fluorescent protein (YFP) and expressed in human embryonic kidney 293 cells. FCS provided plasma membrane diffusion coefficients on the order of 7.5 × 10(-9) cm(2)/s. PCH molecular brightness analysis was used to determine the GPCR oligomer size. Known monomeric (CD-86) and dimeric (CD-28) receptors with GFP and YFP tags were used as controls to determine the molecular brightness of monomers and dimers. PCH analysis of fluorescence-tagged GPCRs revealed molecular brightness values that were twice the monomeric controls and similar to the dimeric controls. Reduced χ(2) analyses of the PCH data best fit a model for a homogeneous population of homodimers, without tetramers or higher-order oligomers. The homodimer configuration was unaltered by agonist treatment and was stable over a 10-fold range of receptor expression level. The results of this study demonstrate that biogenic amine receptors freely diffusing within the plasma membrane are predominantly homodimers.

Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers.

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 µm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.

Functional significance of serotonin receptor dimerization.

The original model of G-protein activation by a single G-protein-coupled receptor (GPCR) is giving way to a new model, wherein two protomers of a GPCR dimer interact with a single G-protein. This article will review the evidence suggesting that 5-HT receptors form dimers/oligomers and will compare the findings with the results obtained from the studies with other biogenic amine receptors. Topics to be covered include the origin or biogenesis of dimer formation, potential dimer interface(s), and oligomer size (dimer vs. tetramer or higher order). The functional significance will be discussed in terms of G-protein activation following ligand binding to one or two protomers in a dimeric structure, the formation of heterodimers, and the development of bivalent ligands.

The center cannot hold: A Bayesian chronology for the collapse of Tiwanaku.

The timing of Tiwanaku's collapse remains contested. Here we present a generational-scale chronology of Tiwanaku using Bayesian models of 102 radiocarbon dates, including 45 unpublished dates. This chronology tracks four community practices: residing short- vs. long-term, constructing monuments, discarding decorated ceramics, and leaving human burials. Tiwanaku was founded around AD 100 and around AD 600, it became the region's principal destination for migrants. It grew into one of the Andes' first cities and became famous for its decorated ceramics, carved monoliths, and large monuments. Our Bayesian models show that monument building ended ~AD 720 (the median of the ending boundary). Around ~AD 910, burials in tombs ceased as violent deaths began, which we document for the first time in this paper. Ritualized murders are limited to the century leading up to ~AD 1020. Our clearest proxy for social networks breaking down is a precise estimate for the end of permanent residence, ~AD 1010 (970-1050, 95%). This major inflection point was followed by visitors who used the same ceramics until ~AD 1040. Temporary camps lasted until roughly ~AD 1050. These four events suggest a rapid, city-wide collapse at ~AD 1010-1050, lasting just ~20 years (0-70 years, 95%). These results suggest a cascading breakdown of community practices and social networks that were physically anchored at Tiwanaku, though visitors continued to leave informal burials for centuries. This generation-scale chronology suggests that collapse 1) took place well before reduced precipitation, hence this was not a drought-induced societal change and 2) a few resilient communities sustained some traditions at other sites, hence the chronology for the site of Tiwanaku cannot be transposed to all sites with similar material culture.

Nicotiana benthamiana as a Transient Expression Host to Produce Auxin Analogs.

Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce "new-to-nature" derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant Nicotiana benthamiana to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected N. benthamiana with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogs. Given the emerging importance of transient expression in N. benthamiana for industrial scale protein and product expression, this work provides insight into the capacity of N. benthamiana to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites.

Feasibility of Collecting Patient-Reported Outcomes for Inpatient Rehabilitation Quality Reporting.

OBJECTIVE: To evaluate rehabilitation inpatients' willingness and ability to complete patient-reported outcomes (PROs) and the burden of completion on patients and staff. DATA SOURCES/STUDY SETTING: Two inpatient rehabilitation facilities. STUDY DESIGN: Patients with neurological disorders were assigned randomly to receive a nominal monetary incentive during or 1 month after the stay. DATA COLLECTION: Patients responded using a tablet computer or paper. PRINCIPAL FINDINGS: Of the 1,055 admissions, 74 percent were eligible, and 51 percent of eligible patients completed the survey. Most answered without assistance. A majority completed the survey 1 month after discharge; incentive timing was unrelated to postdischarge completion. Half of the 285 follow-up respondents required at least two reminder calls. CONCLUSIONS: Collection of PROs from rehabilitation patients is feasible. Results inform policy makers regarding feasibility of PRO data in evaluating rehabilitation quality.

Serotonin 5-HT(2C) receptor homodimerization is not regulated by agonist or inverse agonist treatment.

Serotonin 5-HT(2C) receptors represent targets for therapeutics aimed at treating anxiety, depression, schizophrenia, and obesity. Previously, we demonstrated that 5-HT(2C) receptors function as homodimers. Herein, we investigated the effect of agonist and inverse agonist treatment on the homodimer status of two naturally occurring 5-HT(2C) receptor isoforms, one without basal activity (VGV) and one with constitutive activity (INI) with respect to Galpha(q) signaling. Cyan- and yellow-fluorescent proteins were used to monitor VGV and INI homodimer formation by western blot, and in living cells using bioluminescence and fluorescence resonance energy transfer (BRET and FRET). Western blots of solubilized membrane proteins revealed equal proportions of homodimeric receptor species from HEK293 cells transfected with either the VGV or INI isoform in the absence and presence of 5-HT. BRET ratios measured in HEK293 cells transfected with the VGV or INI isoform were the same and were not modulated by 5-HT. Similarly, FRET efficiencies were the same regardless of whether measured in cells expressing the VGV or INI isoform in the absence or presence of 5-HT or clozapine. The results indicate that serotonin 5-HT(2C) receptors form homodimers regardless of whether they are in an inactive or active conformation and are not regulated by drug treatment.

Stable expression of constitutively activated mutant h5HT6 and h5HT7 serotonin receptors: inverse agonist activity of antipsychotic drugs.

RATIONALE: In order to determine the possible relationship between antipsychotic drug properties and inverse agonist activity at h5HT6 and h5HT7 receptors, constitutively activated forms of these receptors were created by site-specific mutagenesis. Typical and atypical antipsychotic drugs were assayed for their potencies as inverse agonists at these mutated receptors. OBJECTIVES: Stable cell lines expressing constitutively activated forms of the h5HT6 and h5HT7 receptors were created. Typical and atypical antipsychotic drugs demonstrating high to moderate affinities for the h5HT6 and h5HT7 receptors were assayed for their potencies in reversing the agonist-independent activity (inverse agonist activity). RESULTS: The E322R h5HT6 mutant and the S267K h5HT7 mutant displayed sufficiently robust agonist-independent activity when expressed in stable cell lines to allow the detailed, concentration-dependent, investigation of the inverse agonist activity of typical and atypical antipsychotic drugs. All the drugs tested displayed inverse agonist activity at both the activated h5HT6 and h5HT7 receptors. Native forms of these receptors did not display any constitutive activity. Interestingly, atypical antipsychotic drugs displayed potent inverse agonist activity, relative to typical antipsychotic drugs, at the h5HT7 receptor. LSD displayed neutral antagonist properties at the mutant h5HT7 receptor. CONCLUSIONS: Site-specific mutations in the third intracellular loop of the G(s)-coupled h5HT6 and h5HT7 receptors produce constitutive activation. Antipsychotic drugs display inverse agonist activity at the activated receptors. The inverse agonist mechanism-of-action of the atypical antipsychotic drugs at the h5HT7 receptors may be different from the typical antipsychotic drugs as these drugs displayed far higher potencies as inverse agonists at the h5HT7 receptor.

Constitutive activity of G-protein coupled receptors: emphasis on serotonin receptors.

Several lines of evidence indicate that G-protein coupled receptors (GPCR) may exist in a state that allows a tonic level of stimulation in vivo (constitutive activity). Several native forms of GPCR, when expressed in recombinant cell lines, display significant signal transduction stimulation in the absence of activating ligand. Many GPCR, including three serotonin receptors, display robust constitutive activation upon the mutation of a single amino acid, indicating mutations producing inappropriate constitutive activation may be etiological factors in diseases. If constitutive activity of GPCR is as common a phenomenon as some researchers suspect, this would suggest significant alterations in the classical model of ligand-receptor interactions. One of the most significant implications of constitutive activity for pharmacologists and medicinal chemists, is the possibility of developing drugs that lower the level of constitutive activity. Such compounds have been termed inverse agonists . These drugs, in theory, would have different physiological effects, and therefore possibly different therapeutic potential, than classical competitive receptor antagonists ( neutral antagonists ). Theoretical issues concerning constitutive activity in the GPCR family and some of the evidence supporting the existence of constitutive activity in the GPCR family is reviewed. Studies are presented demonstrating the procedures for producing and characterizing constitutive activated forms of serotonin receptors, including the demonstration of inverse agonist activity of drugs on these receptors.

Determining the oligomer number of native GPCR using florescence correlation spectroscopy and drug-induced inactivation-reactivation.

GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, there is surprisingly little information on GPCR dimer structure and function in primary cell culture or in vivo. We have used two novel approaches to develop methods to detect and study GPCR dimer function: FCS/PCH and "inactivation-reactivation". This review will focus on the data we have developed and our interpretations of those data. Using FCS/PCH 5-HT2C receptors have been detected directly and appear to exist as dimers, consistent with the inactivation-reactivation data on 5-HT7 and 5-HT2A receptors. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist ("inactivator") to one of the orthosteric sites of a homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR homodimers, indicating that GPCRs do exist and function as homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. This approach can be applied to heterodimers as well as homodimers and may aid in the development of novel drugs with heterodimer selectivity.

Fluorescence correlation spectroscopy and photon-counting histogram analysis of receptor-receptor interactions.

Fluorescence correlation spectroscopy (FCS) performed using a laser scanning confocal microscope is a technique with single-molecule sensitivity that is becoming more accessible to cell biologists. In this chapter, we describe the use of FCS for the analysis of diffusion coefficients and receptor-receptor interactions in live cells in culture. In particular, we describe a protocol to collect fluorescence fluctuation data from fluorescence-tagged receptors as they diffuse into an out of a small laser-illuminated observation volume using a commercially available system such as the Zeiss ConfoCor 3 or LSM-780 microscope. Autocorrelation analysis of the fluctuations in fluorescence intensity provides information about the diffusion time and number of fluorescent molecules in the observation volume. A photon-counting histogram can be used to examine the relationship between fluorescence intensity and the number of fluorescent molecules to estimate the average molecular brightness of the sample. Since molecular brightness is directly proportional to the number of fluorescent molecules, it can be used to monitor receptor-receptor interactions and to decode the number of receptor monomers present in an oligomeric complex.

Risperidone irreversibly binds to and inactivates the h5-HT7 serotonin receptor.

Risperidone displays a novel mechanism of antagonism of the h5-HT7 receptor. Pretreatment of the cells with 5 or 20 nM risperidone, followed by removal of the drug from the media, renders the 5-HT7 receptors unresponsive to 10 microM 5-HT for at least 24 h. Thus, risperidone seems to be producing a rapid, long-lasting inactivation of the h5-HT7 receptor. Whole-cell radioligand binding studies indicate that risperidone interacts in an irreversible or pseudo-irreversible manner with the h5-HT7 receptor, thus producing the inactivation. Internalization of the h5-HT7 receptor was not detected by monitoring green fluorescent protein-labeled fluorescent forms of the h5-HT7 receptor exposed to 20 nM risperidone. Ten other antagonists were tested for h5-HT7-inactivating properties, and only 9-OH-risperidone and methiothepin were found to demonstrate the same anomalous properties as risperidone. These results indicate that the h5-HT7 receptor may possess unique structural features that allow certain drugs to induce a conformation resulting in an irreversible interaction in the intact membrane environment. This may indicate that the h5-HT7 receptor is part of a subfamily of G-protein-coupled receptors (GPCRs) possessing this property or that many GPCRs have the potential to be irreversibly blocked, but only select drugs can induce this effect. At the very least, the possibility that highly prescribed drugs, such as risperidone, are irreversibly antagonizing GPCR function in vivo is noteworthy.

Serotonin 5-HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time visualization with confocal fluorescence resonance energy transfer.

Dimerization is a common property of G-protein-coupled receptors (GPCR). While the formation of GPCR dimers/oligomers has been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how and where GPCR dimerization occurs. The present study was performed to identify the precise cellular compartment in which class A GPCR dimer/oligomer biogenesis occurs. We addressed this issue using confocal microscopy and fluorescence resonance energy transfer (FRET) to monitor GPCR proximity within discrete intracellular compartments of intact living cells. Time-lapse confocal imaging was used to follow CFP- and YFP-tagged serotonin 5-HT2C receptors during biosynthesis in the endoplasmic reticulum (ER), trafficking through the Golgi apparatus and subsequent expression on the plasma membrane. Real-time monitoring of FRET between CFP- and YFP-tagged 5-HT2C receptors was performed by acceptor photobleaching within discrete regions of the ER, Golgi, and plasma membrane. The FRET signal was dependent on the ratio of CFP- to YFP-tagged 5-HT2C receptors expressed in each region and was independent of receptor expression level, as predicted for proteins in a non-random, clustered distribution. FRET efficiencies measured in the ER, Golgi, and plasma membrane were similar. These experiments provide direct evidence for homodimerization/oligomerization of class A GPCR in the ER and Golgi of intact living cells, and suggest that dimer/oligomer formation is a naturally occurring step in 5-HT2C receptor maturation and processing.

Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein.

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.

Creation, expression, and characterization of a constitutively active mutant of the human serotonin 5-HT6 receptor.

The serotonin 5-HT(6) receptor, a G-protein-coupled receptor, displays high affinity for antipsychotic, antidepressant, and psychotropic drugs. We created a constitutively active form of the human 5-HT(6) receptor in order to probe the molecular domains of receptor activation and to determine if inverse agonist activities of antipsychotic drugs contribute to their clinical profile. Previous studies from our laboratory support a critical role for the c-terminal region of the third intracellular loop (il3) in the activation of G(q)-coupled serotonin receptors. In the present study, PCR-based mutagenesis was used to mutate serine 267 (S6.34) in the c-terminal region of il3 to lysine (S267K). The native and S267K 5-HT(6) receptors were expressed in COS-7 cells to study the functional effects of the mutation. The S267K receptor shows 10-fold higher affinity for serotonin than the native receptor and demonstrates agonist-independent activity. Clozapine decreased the basal activity of the S267K receptor to vector control levels. Therefore, we can conclude that the S267K mutation renders the 5-HT(6) receptor constitutively active and that clozapine is an inverse agonist at the mutant 5-HT(6) receptor. These results indicate that the c-terminal region of il3 of the G(s)-coupled 5-HT(6) receptor is a key domain for G-protein coupling, similar to the G(q)-coupled 5-HT receptors. The inverse agonist action of clozapine indicates that drugs displaying competitive antagonist activity at native 5-HT(6) receptors may display inverse agonist activity at the constitutively activated form of the receptor.