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Overview of the method used to measure FRET selectively at the plasma membrane. (A) A series of z-stacks were acquired for a cell co-expressing YFP-tagged 5-HT1A receptor. Three-dimensional reconstruction and orthogonal plane analysis shows the receptor distribution in the x, y and z plane. The images show localization of the receptors in the plasma membrane as well as minor florescence from intercellular structures (most prominent in the y–z plane) that can lead to incorrect characterization of FRET in whole cell measurements . The cyan lines shown in both the x–z and y–z planes represent the confocal depth of the x-y image which is representative of those acquired for FRET analysis. Scale bar represents 20 μm. (B) Simultaneous acquisition of fluorescence emission in eight channels allows for the reconstruction of per pixel fluorescence emission spectra of a FRET sample co-expressing 5-HT1A-CFP and 5-HT1A- YFP receptors, designated as 'IM'. Linear unmixing allows us to determine the weighted contributions from CFP, YFP and the background signal that combine to fit the measured spectra, designated as 'CFP', 'YFP', 'BG', and 'Fit', respectively. The weights of the donor and acceptor spectra that provide the best fit, along with calibration parameters, are used in the analysis of FRET
Source publication
In the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Förster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between...
Contexts in source publication
Context 1
... we adapted the lux- FRET approach to the application at confocal microscopy, allowing for the analysis of receptor oligomerization with sub-cellular resolution. Figure 1A shows the 3-D recon- struction of a series of confocal slices through an N1E-115 cell co-expressing 5-HT1A-CFP and 5-HT1A-YFP receptor constructs at a 1:1 ratio. The center panel shows the confocal slice where fluorescence was detected specifically at the plasma membrane immediately adjacent to the coverslip on which the cell is growing. ...
Context 2
... emission was collected simultaneously over eight channels for each acquisition, allowing for reconstruction of emis- sion spectra on a per pixel basis. Figure 1B shows the measured emission intensities of a single pixel over the eight channels used in the image acquisition for a cell co- expressing 5-HT1A-CFP and 5-HT1A-YFP. This figure also shows the weighted contributions of CFP, YFP and background signal which combine to provide a fit to the acquired fluorescent spectra of the FRET sample. ...
Context 3
... of the wild-type 5-HT1A receptor measured by lux-FRET. (A) Application of the two excita- tion FRET method described in "Materials and methods" and outlined in Fig. 1, allows us to create images of Ef D , Ef A , x D , as well as FRET corrected total concentration T D . (B) Mean values of 10×10 μm regions of interest from cells expressing different ratios of 5-HT1A-CFP and 5-HT1A-YFP receptors allows to characterize the ap- parent FRET efficiency over the donor mole fraction. The data were collected ...
Citations
... Typical RET-based approaches for quantifying protein-protein interactions rely on plotting the average against the ratio of the donor to acceptor concentrations in the sample [28], the fraction of donors or acceptors [29][30][31], or the total concentration of donors and acceptors [28,32,33], and then fitting a model derived from the kinetic theory of RET [34] to the experimental data using the concentration of oligomers as a fitting parameter. Unfortunately, this method only works well if the number of protomers within an oligomeric complex and the geometry of the oligomer are a priory known (and fixed during the fitting process) [34], since otherwise an unlimited number of combinations between geometrical parameters and pairwise RET efficiencies fit the data equally well. ...
Resonance energy transfer (RET) and fluorescence fluctuation spectroscopies (FFS) are powerful fluorescence-based techniques for quantifying the self-association of membrane receptors within oligomeric complexes in living cells. However, RET spectrometry's ability to extract information on the detailed quaternary structure of oligomers sometimes rests on assumptions regarding the relative abundances of oligomers of different sizes, while FFS techniques may provide oligomer size information but not quaternary structure details, as they lack a probe for inter-molecular distances. In this report, we introduce a method which we termed "intensity fluctuations and resonance energy transfer" (iFRET), which combines analysis of donor and acceptor intensity fluctuations with RET efficiency determination. Because the three measured quantities each have a unique dependence on the acceptor mole fraction (XA), simultaneous global fitting of all three dramatically reduces ambiguity in the data fitting and choice of the most appropriate fitting model. We demonstrate the effectiveness of the method on simulated brightness and RET efficiency data incorporating mixtures of monomers, dimers, and tetramers and show that iFRET analysis provides a major improvement in both identifying the correct quaternary structure model and extracting the relative abundances of the monomers, dimers, and tetramers. Conceivably, the enhanced resolution of iFRET could potentially provide insight into the functional significance of receptor oligomerization in the presence and absence of cognate ligands.
... Overexpression of the serotonin 5-HT 1A receptor in the heterologous cell system showed a statistically significant increase in the FRET signal (Fig. 4a) as compared to the controls conditions, which indicates close interaction, namely oligomerization or dimerization of 5-HT 1A R, and remains in agreement with previously published data [31,32]. We could not observe any changes in fluorescence signal after 5-HT 1A receptor agonist administration (Fig. 4b), while Kobe et al. reported that such agonist decreased FRET signal over a few minutes, however, they studied different compound we used the selective 5-HT 1A R agonist (R)-(+)-8-OH DPAT because of the possibility of the presence of other serotonin receptors on the HEK-293 cell line, such as 5-HT 7 or 5-HT 6 [31,33]. ...
Background
The serotonin 5-HT1A receptor (5-HT1AR) and metabotropic glutamate receptor 4 (mGlu4) have been implicated as sites of antipsychotic drug action. 5-HT1AR belongs to the A class of G protein-coupled receptors (GPCRs); mGlu4 is a representative of class C GPCRs. Both receptors preferentially couple with Gi protein to inhibit cAMP formation. The present work aimed to examine the possibility of mGlu4 and 5-HT1A receptor cross-talk, the phenomenon that could serve as a molecular basis of the interaction of these receptor ligands observed in behavioral studies.Methods
First, in vitro studies were performed to examine the pharmacological modulation of interaction of the mGlu4 and 5-HT1A receptors in the T-REx 293 cell line using SNAP- or HALO–tag and cAMP accumulation assay. Next, the colocalization of these two receptors was examined in some regions of the mouse brain by applying RNAScope dual fluorescence in situ hybridization, immunohistochemical labeling, and proximity ligation assay (PLA).ResultsThe ex vivo and in vitro results obtained in the present work suggest the existence of interactions between mGlu4 and 5-HT1A receptors. The changes were observed in cAMP accumulation assay and were dependent on expression and activation of mGlu4R in T-REx 293cell line. Moreover, the existence of spots with proximity expression of both receptors were showed by PLA, immunofluorescence labeling and RNAscope methods.Conclusion
The existence of interactions between mGlu4 and 5-HT1A receptors may represent another signaling pathway involved in the development and treatment psychiatric disorders such as schizophrenia or depression.
... The potential implications of oligomerization are far reaching, specially keeping in mind the role of GPCRs as major drug targets [14]. Evidence of GPCR dimers or higher-order oligomers has been reported in the last few years [1,11,24,35,60] and implicated in receptor trafficking, signaling and pharmacology. Oligomerization of GPCRs in live cell membranes has been studied extensively utilizing fluorescence resonance energy transfer (FRET) approaches such as hetero-FRET (FRET between two different fluorophores) and bioluminescence resonance energy transfer [26]. ...
... GPCRs can form homo-and hetero-oligomers ( Bulenger et al., 2005), whereas heterooligomers were found between partners of the same family ( Panetta and Greenwood, 2008;Woehler et al., 2008), as well as between different classes of GPCRs ( Trifilieff et al., 2011). ...
... Fluorescence labeled GPCRs in combination with Förster Resonance Energy Transfer (FRET) based biosensors for second messengers have enabled the determination of kinetic parameters for various steps of GPCR signaling. We have shown that serotonin receptors type 7 (5-HT7R) and type 1A (5-HT1AR) can form homodimers ( Kobe et al., 2008;Renner et al., 2012;Woehler et al., 2008), as well as heterodimers , both in vitro and in vivo. Both 5-HTRs regulate the intracellular level of the second messenger cyclic AMP, although in the opposite direction: the activation of Gs via the 5-HT7R results in increased production of cAMP ( Adham et al., 1998;Wirth et al., 2017), while Gi activation via the 5-HT1AR leads to a decrease in cAMP concentration (Albert et al., 1999;Kvachnina, 2005). ...
Protein-protein interaction is often investigated using quantitative molecular microscopy with Förster resonant energy transfer (FRET). Here, we combined 'linear unmixing FRET' (lux-FRET) with the simultaneous application of a FRET-based biosensor for cAMP to investigate the oligomerization between the 5-HT7 receptor (5-HT7R, also known as HTR7) and the 5-HT1A receptor (5-HT1AR, also known as HTR1A) and its importance for cAMP signaling. We found that the 5-HT7R not only stimulates cAMP production, but also forms hetero-oligomers with 5-HT1AR, which blocks the inhibitory effect of the latter. 5-HT7R signaling, however, is not affected by this hetero-oligomerization. By modeling the kinetics of intracellular cAMP level changes in relation to the 5-HT7R:5-HT1AR stoichiometry, we were able to decipher the complex signaling characteristics of endogenous serotonin receptors in cultured hippocampal neurons. Our findings indicate that serotonergic signaling is not only modulated by the concentration of an individual receptor but also by its specific interaction with other receptors in endogenous systems. We conclude that the regulated ratio of serotonin receptors in immature and mature neurons may be critically involved in both the onset and response to treatments of psychiatric diseases, such as anxiety and depression.
... Recently, a novel molecular mechanism of the downregulation of 5-HT 1A receptor activity has been discovered. It was shown that 5-HT 1A receptor, as other G-coupled receptors, could form homodimer with another 5-HT 1A molecule as well as heterodimer with other kinds of 5-HT receptor molecule [72,73]. Importantly, heterodimerization of 5-HT 1A and 5-HT 7 receptors changed functional activity of 5-HT 1A receptors [74]. ...
Introduction: Selective serotonin reuptake inhibitors (SSRIs) are the most effective and most used antidepressant drugs. Acting by inhibiting serotonin (5-HT) transporter, SSRIs display a typical 3-4-week delay in their therapeutic effects, with nearly 40% of depressed patients remaining treatment-resistant. Recent evidence suggests complex interplay between 5-HT receptors and key proteins of 5-HT metabolism in molecular mechanisms of such delay and resistance to SSRIs.
Area covered: This paper concentrates on the interplay between 5-HT receptors in the delay of therapeutic effect of SSRIs, and the interaction between tryptophan hydroxylase 2 and 5-HT transporter in the SSRI resistance. Specifically, it discuss: (1) the data on the association between antidepressant drug efficacy and genetically defined characteristics of key proteins in the 5-HT signaling (TPH2, MAOA, SERT and 5-HT1A receptor), (2) the effect of dimerization of 5-HT7 and 5-HT1A receptors on the internalization and functioning of 5-HT1A presynaptic receptors, (3) the role of Tph2 deficiency in the resistance to SSRIs treatment. We shift the emphasis from individual proteins to their interactions in explaining antidepressant action of SSRI.
Expert opinion: These interactions should be considered when developing more effective antidepressant drugs as well as for predicting and improving the efficacy of antidepressant therapies.
... Recently, new mechanisms for 5-HT 1A receptor regulation associated with oligomerization and the interaction of the 5-HT receptors in addition to the previously revealed mechanisms were described. It was established that the 5-HT 1A receptor exists in the brain in several forms: as a 5-HT 1A monomer, 5-HT 1A /5-HT 1A homodimer or heterodimer with receptors of other type (Woehler et al., 2009;Kobe et al., 2012;Renner et al., 2012). New data on the role of dimerization in regulation of the 5-HT 1A receptor's functional activity were obtained in the study on the interaction between 5-HT 1A and 5-HT 7 receptors Popova and Naumenko, 2013;Naumenko et al., 2014). ...
Serotonin, a brain neurotransmitter, acting via 14 types of serotonin (5-HT) receptors regulates different forms of both normal and pathological behavior. All 5-HT receptors (except for 5-HT3 representing an ion channel (ionotropic receptor)) belong to a superfamily of metabotropic receptors coupled with G-proteins. Each receptoris characterized by a unique gene, spectrum of affinity to different agonists and antagonists, specific distribution in the brain, and a set of controlled functions. Among this diversity of 5-HT receptors, the evolutionarily most ancient 5-HT1A receptor is of a special interest since it plays a key role in autoregulating of the brain 5-HT system. This role of 5-HT1A receptors is caused by the peculiarities of their localization (pre- or postsynaptically on 5-HT neurons), depending on which they can exert the opposite effect on the functional activity of the 5-HT system. The review is devoted to the data of the literature and the results obtained by the authors about the factors regulating the expression and functional activity of 5-HT1A receptors and their influence on behavior. The structure of the 5-HT1A receptor gene was described; and the latest data on the posttranslational regulation of the activity of 5-HT1A receptors and interaction of 5-HT receptors were given. Special attention was paid to the role of the 5-HT1A receptor’s heterodimerization with the 5-HT7 serotonin receptor and the functional inactivation of the 5-HT1A receptor. The involvement of 5-HT1A receptors in regulation of aggressive behavior, catalepsy, anxiety, depression, and unique natural adaptation (hibernation) was demonstrated. Special attention was paid to the involvement of these receptors in regulation of (1)—fear-induced defensive aggression the base of thedomestication process; (2) intermale aggression resulting in the establishment of a dominant–subordinant relationship in the animal community and underlying human asocial behavior; and (3) the mechanisms of depression and the effect of clinically efficient antidepressants of the serotonin reuptake inhibitors group. The role of 5-HT1A/5-HT7 heterodimerization in the mechanism of the antidepressants effect was hypothesized.
... Nonetheless, when both receptors are co-expressed, the activation of one receptor in the 5-HT 1A R/µ-opioid heterodimer inhibits MAPK activation of the other receptor (Cussac et al., 2012). On the other hand, biochemical studies accomplished in neuroblastoma N1E-115 cells revealed that 5-HT 1A R forms dimers and homo-oligomers, being dimers the prevalent species at the plasma membrane (Kobe et al., 2008;Woehler et al., 2009). Moreover, kinetics of 5-HT 1A R dimer dissociation or association into high order homo-oligomers is not influenced by ligand binding (Kobe et al., 2008). ...
Serotonin (5-HT) is a neurotransmitter that plays an important role in neuronal plasticity. Variations in the levels of 5-HT at the synaptic cleft, expression or dysfunction of 5-HT receptors may alter brain development and predispose to various mental diseases. Here, we review the transduction pathways described in various cell types transfected with recombinant 5-HT1A receptor (5-HT1AR), specially contrasting with those findings obtained in neuronal cells. The 5-HT1AR is detected in early stages of neural development and is located in the soma, dendrites and spines of hippocampal neurons. The 5-HT1AR differs from other 5-HT receptors because it is coupled to different pathways, depending on the targeted cell. The signaling pathway associated with this receptor is determined by Gα isoforms and some cascades involve βγ signaling. The activity of 5-HT1AR usually promotes a reduction in neuronal excitability and firing, provokes a variation in cAMP and Ca2+, levels which may be linked to specific types of behavior and cognition. Furthermore, evidence indicates that 5-HT1AR induces neuritogesis and synapse formation, probably by modulation of the neuronal cytoskeleton through MAPK and phosphoinositide-3-kinase (PI3K)-Akt signaling pathways. Advances in understanding the actions of 5-HT1AR and its association with different signaling pathways in the central nervous system will reveal their pivotal role in health and disease.
... In the case of 5-HT 7 receptor, which can signal through Gs-or G12-proteins, palmitoylation may act as a molecular switch selecting the signaling pathway to be activated (Kvachnina, Dumuis et al. 2009). For 5-HT 1A receptors, in contrast, palmitoylation is irreversible and insensitive to agonist stimulation (Papoucheva, Dumuis et al. 2004, Kobe, Renner et al. 2008, Woehler, Wlodarczyk et al. 2009). ...
Neurotransmitter receptors, the macromolecules specialized in decoding the chemical signals encrypted in the chemical signaling mechanism in the nervous system, occur either at the somatic cell surface of chemically excitable cells or at specialized subcellular structures, the synapses. Synapses have lipid compositions distinct from the rest of the cell membrane, suggesting that neurotransmitter receptors and their scaffolding and adaptor protein partners require specific lipid habitats for optimal operation. In this review we discuss some paradigmatic cases of neurotransmitter receptor-lipid interactions, highlighting the chemical nature of the intervening lipid species and providing examples of the receptor mechanisms affected by interaction with lipids. The focus is on the effects of cholesterol, glycerophospholipids and covalent fatty acid acylation on neurotransmitter receptors. We also briefly discuss the role of lipid phase states involving lateral heterogeneities of the host membrane known to modulate membrane transport, protein sorting and signaling. Modulation of neurotransmitter receptors by lipids occurs at multiple levels, affecting a wide span of activities including their trafficking, sorting, stability, residence lifetime at the cell surface, endocytosis, and recycling, among other important functional properties at the synapse.
... В последние годы к выявленным ранее механизмам регуляции 5-НТ 1А рецептора добавлены новые, связанные с олигомеризацией и взаимодействием 5-НТ рецепторов. Было установлено, что 5-НТ 1А рецептор существует в мозге в нескольких формах: как 5-НТ 1А мономер, 5-НТ 1А / 5-НТ 1А гомодимер и гетеродимер, связанный с другими рецепторами (Woehler et al., 2009;Kobe et al., 2012;. Новые данные о роли димеризации в регуляции функциональной активности 5-НТ 1А рецептора получены при изучении взаимодействия 5-НТ 1А и 5-НТ 7 рецепторов Naumenko et al., 2014). ...
Brain serotonin (5-HT) is known to be involved in the control of a wide range of physiological functions as well as of different kinds of behavior. Such polyfunctionality of 5-HT is mediated by numerous 5-HT receptors. Currently, 14 different 5-HT receptor subtypes expressed in the mammals have been identified. The 5-HT1А receptor is one of the most extensively characterised members of the serotonin receptor family. Increased interest to the 5-HT1А receptor is based on (1) a key role in the autoregulation of the brain serotonergic system due to the postsynaptic and presynaptic localization, (2) a great body of data demonstrating implication of 5-HT1А receptor in the control of various physiological functions (3) involvement of 5-HT1А receptors in the mechanisms of depression, anxiety and suicide. The review describes literature and original data on factors affecting the expression and functional activity of 5-HT1А receptors and the involvement of 5-HT1А receptors in the regulation of normal and pathological behavior. The structure of the 5-HT1А receptor gene is described and new data on the posttranslational regulation of 5-HT1А receptor functional activity are provided. A special focus was given to the interaction between 5-HT1А and 5-HT7 receptors followed by heterodimer formation and the role of heterodimerization in the functional inactivation of the 5-HT1А receptor. The implication of 5-HT1А receptors in the regulation of aggressive behavior, catalepsy, anxiety, depression and hibernation was shown. Special attention is focused on the involvement of 5-HT1А receptors in the regulation of 1) fear-induced aggression towards man – the basis of domestication, 2) intermale aggression underling asocial behavior in men, 3) depression and in the mechanism of antidepressant action. The described data extend the idea on the 5-HT1А receptor as a key player in the brain 5-HT system.
... Using a novel Fö rster resonance energy transfer (FRET) technique based on the spectral analysis, we have recently demonstrated that 5-HT 1A receptors can form homodimers at the plasma membrane [99,100]. We also showed that FRET efficiency measured for the 5-HT 1A receptor oligomers significantly decreased in response to agonist stimulation. ...
Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. Besides the important role of 5-HT receptors in the pathogenesis of depressive disorders and in their clinical medications, underlying mechanisms are far from being completely understood. This review focuses on possible cross talk between two serotonin receptors, 5-HT1A and the 5-HT7 . Although these receptors are highly co-expressed in brain regions implicated in depression, and most agonists developed for the 5-HT1A or 5-HT7 receptors have cross-reactivity, their functional interaction has not been yet established. It has been recently shown that 5-HT1A and 5-HT7 receptors form homo- and heterodimers both in vitro and in vivo. From the functional point of view, heterodimerization has been shown to play an important role in regulation of receptor-mediated signaling and internalization, suggesting the implication of heterodimerization in the development and maintenance of depression. Interaction between these receptors is also of clinical interest, because both receptors represent an important pharmacological target for the treatment of depression and anxiety.
