The discovery of a new antibody for BRIL-fused GPCR structure determination.

G-protein-coupled receptors (GPCRs)-the largest family of cell-surface membrane proteins-mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.

Influence of Cholesterol and Its Stereoisomers on Members of the Serotonin Receptor Family.

Despite the ubiquity of cholesterol within the cell membrane, the mechanism by which it influences embedded proteins remains elusive. Numerous G-protein coupled receptors exhibit dramatic responses to membrane cholesterol with regard to the ligand-binding affinity and functional properties, including the 5-HT receptor family. Here, we use over 25 µs of unbiased atomistic molecular dynamics simulations to identify cholesterol interaction sites in the 5-HT1B and 5-HT2B receptors and evaluate their impact on receptor structure. Susceptibility to membrane cholesterol is shown to be subtype dependent and determined by the quality of interactions between the extracellular loops. Charged residues are essential for maintaining the arrangement of the extracellular surface in 5-HT2B; in the absence of such interactions, the extracellular surface of the 5-HT1B is malleable, populating a number of distinct conformations. Elevated cholesterol density near transmembrane helix 4 is considered to be conducive to the conformation of extracellular loop 2. Occupation of this site is also shown to be stereospecific, illustrated by differential behavior of nat-cholesterol isomers, ent- and epi-cholesterol. In simulations containing the endogenous agonist, serotonin, cholesterol binding at transmembrane helix 4 biases bound serotonin molecules toward an unexpected binding mode in the extended binding pocket. The results highlight the capability of membrane cholesterol to influence the mobility of the extracellular surface in the 5-HT1 receptor family and manipulate the architecture of the extracellular ligand-binding pocket.

Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of Gs to four different GPCRs have been elucidated1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT1B receptor (5-HT1BR) bound to the agonist donitriptan and coupled to an engineered Go heterotrimer. In this complex, 5-HT1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the ß2-adrenoceptor (ß2AR) 3 or the adenosine A2A receptor (A2AR) 1 in complex with Gs. In contrast to the complexes with Gs, the gap between the receptor and the Gß-subunit in the Go-5-HT1BR complex precludes molecular contacts, and the interface between the Gα-subunit of Go and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the Go α-subunit. The molecular variations between the interfaces of Go and Gs in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

Assessment of the transmembrane domain structures in GPCR Dock 2013 models.

The community-wide blind prediction of G-protein coupled receptor (GPCR) structures and ligand docking has been conducted three times and the quality of the models was primarily assessed by the accuracy of ligand binding modes. The seven transmembrane (TM) helices of the receptors were taken as a whole; thus the model quality within the 7TM domains has not been evaluated. Here we evaluate the 7TM domain structures in the models submitted for the last round of prediction - GPCR Dock 2013. Applying the 7 × 7 RMSD matrix analysis described in our prior work, we show that the models vary widely in prediction accuracy of the 7TM structures, exhibiting diverse structural differences from the targets. For the prediction of the 5-hydroxytryptamine receptors, the top 7TM models are rather close to the targets, which however are not ranked top by ligand-docking. On the other hand, notable deviations of the TMs are found in in the previously identified top docking models that closely resemble other receptors. We further reveal reasons of success and failure in ligand docking for the models. This current assessment not only complements the previous assessment, but also provides important insights into the current status of GPCR modeling and ligand docking.

Sequential Co-immunoprecipitation and Immunoblot Approach to Determine Oligomerisation of G-Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) play a major role in psychiatric disorders and are the targets of several current therapeutic approaches in this field. A number of studies have now shown that GPCRs can assemble as high molecular weight homo- and hetero-oligomers, which could affect ligand binding, intracellular signalling or trafficking. This information could be critical in design of new drugs to treat neurological and psychiatric disorders. This chapter describes a sequential co-immunoprecipitation and immunoblot protocol for determining oligomerisation of the 5-hydroxytryptamine (HT)1A receptor with other GPCRs in co-transfected HEK-293 cells.

[THE THYROID STATUS OF RATS IMMUNIZED WITH PEPTIDES DERIVED FROM THE EXTRACELLULAR REGIONS OF THE TYPES 3 AND 4 MELANOCORTIN RECEPTORS AND THE 1B-SUBTYPE 5-HYDROXYTRYPTAMINE RECEPTOR].

The activity of the hypothalamic-pituitary-thyroid (HPT) axis is controlled by the brain neurotransmitter systems, including the melanocortin signaling system. Pharmacological inhibition of type 4 melanocortin receptor (M4R) leads to disruption of the functioning of HPT axis and to reduction of the level of thyroid hormones. At the same time, the data on how prolonged inhibition of M4R affects this axis and on its role in regulation of M3R are absent. The relationship between the thyroid status and the activity of 1B-subtype 5-hydroxytryptamine receptor (5-HT1BR) is scarcely explored. The aim of this work to study the effects of chronic inhibition of M3R, M4R and 5-HT1BR induced by immunization of rats with BSA-conjugated peptide derived from the extracellular regions of these receptors on the thyroid status and the activity of thyroid stimulating hormone (TSH)-sensitive adenylyl cyclase signaling system (ACSS) in the thyroid glarid (TG) of the immunized animals. In rats immunized with the peptides K-[TSLHL WNRSSHGLHG11-25]-A of M4R, A[PTNPYCICTTAH269-280]-A of M3R and. [QAKAEE-EVSEC(Acm)-VVNTDH189-205]-A of 5-HT1BR levels of thyroid hormones such as fT4, tT4 and tT3 were significantly reduced. In rats immunized with M4R and M3R peptides, an increase of TSH was detected whereas in the animals immunized with 5-HT1BR peptide the level of TSH, on the contrary, was reduced. In the TG of rats immunized with M4R and M3R peptides, the stimulatory effects of hormones (TSH, PA-CAP-3 8) and GppNHp on adenylyl cyclase activity were attenuated, and the changes were most pronounced in the case M4R peptide immunization. After immunization with 5-HT1BR peptide the stimulatory effects of TSH, PACAP-38 and GppNHp were retained. Thus, the main cause of thyroid hormones deficit in rats immunized with M4R and M3R peptides was the decreased sensitivity of ACSS thyrocytes to TSH, whereas in rats iimunized with 5-HT1BR peptide the deficit of thyroid hormones was associated with decreased level of TSH. Our data on the negative impact of long-term immunization of rats with BSA-conjugated peptides derived from the extracellular regions of M4R, M3R.and 5-HT1BR on their thyroid status is a strong argument in favor of participation of these receptors and intracellular signaling pathways associated with them in the regulation of HPT axis.

The immunization with peptide 189-205, a derivative of serotonin receptor subtypes 1B, changes the sensetivity of adenylyl cyclase to hormones in the rat brain.

The aim of this work was to study the effect of multiple (during 12 months) immunization of rats with BSA-conjugated peptide 189-205 corresponding to the second extracellular loop of rat HT1BR on ACSS activity in the brain of immunized animals (group HT1BR) and its regulation by hormones.

Pharmacological stimulation of serotonin 5-HT1B receptors enhances increases in plasma active glucagon-like peptide-1 levels induced by dipeptidyl peptidase-4 inhibition independently of feeding in mice.

AIM: Glucagon-like peptide-1 (GLP-1), an incretin hormone, is released from intestinal L cells in response to nutrient ingestion. Dipeptidyl peptidase-4 (DPP-4) rapidly degrades the active form of GLP-1 to an inactive form in the bloodstream. The present study aimed to investigate the role of serotonin (5-HT)1B receptors in the regulation of plasma active GLP-1 levels and glucose tolerance under DPP-4 inhibition. METHODS: C57BL6J mice treated with or without alogliptin, a highly selective DPP-4 inhibitor, for 4 days were intraperitoneally injected with either saline, the 5-HT1B/2C receptor agonist meta-chlorophenylpiperazine (mCPP) at 2.5mg/kg and 5mg/kg or the selective 5-HT1B receptor agonist CP94253 at 2.5mg/kg and 5mg/kg, and food-deprived after treatment. An hour later, plasma active GLP-1 levels were determined. Also, a glucose tolerance test was done by injecting D-glucose (2g/kg) following the injection of saline or CP94253 (5mg/kg) in mice treated with alogliptin. RESULTS: Intraperitoneal injection of mCPP (2.5 and 5mg/kg) or CP94253 (2.5 and 5mg/kg) in mice treated with alogliptin for 4 days significantly increased plasma active GLP-1 levels compared with saline controls in mice that were food-deprived after the injections. While intraperitoneal injection of either mCPP or CP94253 alone had no significant effect on plasma active GLP-1 levels, the injection of CP94253 improved glucose tolerance in mice treated with alogliptin compared with saline. CONCLUSION: These findings suggest that pharmacological stimulation of 5-HT1B receptors enhances the increases in plasma active GLP-1 induced by DPP-4 inhibition independently of feeding and also improves glucose tolerance in mice.

Structure and function of serotonin G protein-coupled receptors.

Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein-coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling.

A dynamic view of molecular switch behavior at serotonin receptors: implications for functional selectivity.

Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT1B and 5-HT2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and ß-arrestin signaling at the 5-HT1B receptor but clearly favoring ß-arrestin over G protein coupling at the 5-HT2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT1B receptor, helix 6 movement in the 5-HT2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity.

Effect of peptides corresponding to extracellular domains of serotonin 1B/1D receptors and melanocortin 3 and 4 receptors on hormonal regulation of adenylate cyclase in rat brain.

The ligand-recognizing part of G protein-coupled receptors consists of their extracellular loops and N-terminal domain. Identification of these sites is essential for receptor mapping and for the development and testing of new hormone system regulators. The peptides corresponding by their structure to extracellular loop 2 of serotonin 1B/1D receptor (peptide 1), extracellular loop 3 of melanocortin 3 receptor (peptide 2), and N-terminal domain of melanocortin 4 (peptide 3) were synthesized by the solid-phase method. In synaptosomal membranes isolated from rat brain, peptide 1 (10(-5)-10(-4) M) attenuated the effects of 5-nonyloxytryptamine (selective agonist of serotonin 1B/1D receptor) and to a lesser extent serotonin and 5-methoxy-N,N-dimethyltryptamine acting on all the subtypes of serotonin receptor 1. Peptide 2 (10(-5)-10(-4) M) significantly reduced the adenylate cyclase-stimulating effect of γ-melanocyte-stimulating hormone (agonist of melanocortin receptor 3), but had no effect on the adenylate cyclase effect of THIQ (agonist melanocortin receptor 4). Peptide 3 reduced the adenylate cyclase-stimulating effects of THIQ and α-melanocyte-stimulating hormone (non-selective agonist of melanocortin receptors 3 and 4), but did not modulate the effect of γ-melanocyte-stimulating hormone. The effect of peptide 3 was weaker: it was observed at peptide 3 concentration of 10(-4) M. Peptides 1-3 did no change the adenylate cyclase-modulating effects of hormones acting through non-homologous receptors. Thus, the synthesized peptides specifically inhibited the regulatory effects of hormones acting through homologous receptors. This suggests that the corresponding extracellular domains are involved in ligand recognition and binding and determine functional activity of the receptor.

Structural basis for molecular recognition at serotonin receptors.

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Structural features for functional selectivity at serotonin receptors.

Drugs active at G protein-coupled receptors (GPCRs) can differentially modulate either canonical or noncanonical signaling pathways via a phenomenon known as functional selectivity or biased signaling. We report biochemical studies showing that the hallucinogen lysergic acid diethylamide, its precursor ergotamine (ERG), and related ergolines display strong functional selectivity for ß-arrestin signaling at the 5-HT2B 5-hydroxytryptamine (5-HT) receptor, whereas they are relatively unbiased at the 5-HT1B receptor. To investigate the structural basis for biased signaling, we determined the crystal structure of the human 5-HT2B receptor bound to ERG and compared it with the 5-HT1B/ERG structure. Given the relatively poor understanding of GPCR structure and function to date, insight into different GPCR signaling pathways is important to better understand both adverse and favorable therapeutic activities.

The function and structural influence of selective relaxed constraint at functional intracellular loop3 of 5-HT(1A) serotonin-1 receptor family.

Serotonin (5-HT) and its receptors have been involved in critical signal transduction mechanism and deregulation implicated in mood-related disorders. 5-HT activities are mediated through a family of transmembrane spanning serotonin receptors. Both within the family and species, 5-HT receptor protein sequence diversity and 7-transmembrane structural homogeneity have long been intriguing. In this study, we have analyzed the codon site constraint in 5-HT1 subclass receptors from 13 orthologous mammalian mRNA coding sequence. Further, the study was extended to computationally investigate the impact of non-synonymous sites with respect to function and structural significance through sequence homology algorithm and molecular dynamics simulation (MDS). Codon sites with significant posterior probability were observed in 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptor indicating variations in site constraint within the 5-HT1 sub-class genes. In 5-HT(1A) receptor, seven sites were detected at the functional intracellular loop(3) (ICL(3)) with higher substitution rate through Codeml program. Sequence homology algorithm identifies that these sites were functionally tolerant within the mammals representing a selectively relaxed constraint at this domain. On the other hand, the root mean square deviation (rmsd) values from MDS suggest differences in structural conformation of ICL(3) models among the species. Specifically, the human ICL(3) model fluctuation was comparatively more stable than other species. Hence, we argue that these sites may have varying influence in G-proteins coupling and activation of effectors systems through downstream interacting accessory proteins of cell among the species. However, further experimental studies are required to elucidate the precise role and the seeming difference of these sites in 5-HT receptors between species.

Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents.

Implication of 5-HT(2B) receptors in the serotonin syndrome.

The serotonin (5-HT) syndrome occurs in humans after antidepressant overdose or combination of drugs inducing a massive increase in extracellular 5-HT. Several 5-HT receptors are known to participate in this syndrome in humans and animal models. The 5-HT(2B) receptor has been proposed as a positive modulator of serotonergic activity, but whether it is involved in 5-HT syndrome has not yet been studied. We analyzed here, a putative role of 5-HT(2B) receptors in this disorder by forced swimming test (FST) and behavioral assessment in the open field. In FST, genetic (5-HT(2B)(-/-) mice) or pharmacological (antagonist RS127445 at 0.5 mg/kg) ablation of 5-HT(2B) receptors facilitated selective 5-HT reuptake inhibitors (SSRI)-induced increase of immobility time as well as expression of other symptoms related to 5-HT syndrome like hind limb abduction and Straub tail. Increase in immobility was also developed in FST by both wild type (WT) and 5-HT(2B)(-/-) mice after the administration of 5-HT(1A), 5-HT(2A) or 5-HT(2C) receptor agonists, 8-OH-DPAT (5 mg/kg), DOI (1 mg/kg), or WAY161503 (5 mg/kg), respectively. In contrast, the 5-HT(2B) receptor agonist BW723C86 (3 mg/kg) or 5-HT(1B) receptor agonist CGS12066A (2 mg/kg) decreased immobility time in both genotypes. The 5-HT syndrome induced by fluoxetine at high doses was blocked in WT and 5-HT(2B)(-/-) mice by administration of 5-HT(1A) and 5-HT(2C) receptor antagonists (WAY100635 0.5 mg/kg and SB242084 0.5 mg/kg) but not by the 5-HT(2A) receptor antagonist MDL100907 (1 mg/kg). By behavioral assessment, we confirmed that 5-HT(2B)(-/-) mice were more prone to develop 5-HT syndrome symptoms after administration of high dose of SSRIs or the 5-HT precursor 5-Hydroxytryptophan, 5-HTP, even if increases in 5-HT plasma levels were similar in both genotypes. This evidence suggests that the presence of 5-HT(2B) receptors hinders acute 5-HT toxicity once high levels of 5-HT are attained. Therefore, differential agonism/antagonism of 5-HT receptors should be considered in the search of therapeutic targets for treating this serious disorder.

[Peptides derived from the third cytoplasmatic loop of serotonin 1B receptor subtype selectively inhibit serotonin signal transduction via a homologous receptor].

The third intracellular loops of hormonal receptors play the main role in the interaction of majority of the serpentine type receptors with heterotrimeric G-proteins. In recent years, it was shown that synthetic peptides corresponding to membrane-proximal regions of these loops could be selectively influenced with hormonal signal transduction via the receptors homologous to them and trigger signalling cascade in absence of the hormone. For the first time, we succeeded in synthesizing the peptides derived from C-terminal region of the third intracellular loop of the IB-subtype serotonin receptor and studied their influence on serotonin-sensitive adenylyl cyclase system in the rat brain. The peptides 300-316 and 306-316 (the numbers correspond to amino acid positions in the rat IB-subtype serotonin receptor) at micromolar concentrations in absence of hormone-stimulated GTP-binding of Gi,-proteins coupled with the IB-subtype serotonin receptors and inhibited forskolin-stimulated adenylyl cyclase activity. Using selective agonists and antagonists of serotonin receptors it was shown that the peptides 300-316 and 306--316 inhibited serotonin signal transduction via homologous to them receptor and weakly influenced other types of serotonin receptors. The peptide 300-316 is more active compared with its shorter analogue 306-316 in the selectivity and efficiency of action on adenylyl cyclase signalling system regulated via the IB-subtype serotonin receptors. These findings indicate that the regions 300-316 of the IB-subtype serotonin receptor are involved in interaction with Grproteins and consist of the main molecular determinants responsible for serotonin signal transduction to adenylyl cyclase.

The 5-HT1B receptor: a novel target for the pathophysiology of depression.

The serotonergic (5-HT) system has been widely implicated in the pathophysiology of Major Depressive Disorder (MDD). Although the 5-HT system is a popular target for drug therapy in MDD the role that serotonin plays in MDD is not clearly understood. An abundance of research suggests that several 5-HT receptor subtypes may be dysfunctional in patients with MDD including the 5-HT(1B) receptor. Evidence implicating 5-HT(1B) receptors in the pathophysiology of depression comes from a number of converging lines of research. Two common genetic polymorphisms of 5-HT(1B) receptors, G861C and C129T, have been implicated in affective disorders. Rats predisposed to learned helplessness have exhibited downregulation of 5-HT(1B) receptor messenger ribonucleic acid (mRNA) in dorsal raphe nucleus (DRN). Pharmacological studies have demonstrated augmentation of extracellular 5-HT levels and antidepressant effects following administration of selective serotonin reuptake inhibitors (SSRIs) in the absence or blockade of 5-HT(1B) receptors. 5-HT(1B) receptor agonists administered alone or with antidepressants have been shown to be effective in preclinical models of depression. Recent interest has focused on p11, an s100 EF-hand protein family protein which colocalizes with 5-HT(1B) receptors. P11 plays a central role in the modulation of 5-HT(1B) receptor function and is dysregulated in preclinical models of depression and postmortem MDD samples. A review of the literature provides strong evidence that 5-HT(1B) receptors and related factors such as p11 are involved in the pathophysiology of depression. The following explores possible factors which may render the 5-HT(1B) receptor dysfunctional, resulting in susceptibility to depression. Implications of using the 5-HT(1B) receptor as a biomarker for vulnerability to MDD are discussed.

Heterodimerization of hypothalamic G-protein-coupled receptors involved in weight regulation.

BACKGROUND: Melanocortin 3 and 4 receptors (MC3R and MC4R) are known to play an essential role in hypothalamic weight regulation. In addition to these two G-protein-coupled receptors (GPCRs), a huge number of other GPCRs are expressed in hypothalamic regions, and some of them are involved in weight regulation. So far, homodimerization was shown for a few of these receptors. Heterodimerization of unrelated receptors may have profound functional consequence but heterodimerization of GPCRs involved in weight regulation was not reported yet. METHODS: A selective number of hypothalamically expressed GPCRs were cloned into a eukaryotic expression vector. Cell surface expression was demonstrated by an ELISA approach. Subcellular distribution was investigated by confocal laser microscopy. A sandwich ELISA and fluorescence resonance energy transfer (FRET) were used to determine protein-protein interaction. RESULTS: Via sandwich ELISA and FRET approach we could demonstrate a robust interaction of the MC4R with GPR7, both of which are expressed in the hypothalamic nucleus paraventricularis. Moreover, we determined a strong interaction of MC3R with the growth hormone secretagogue receptor expressed in the nucleus arcuatus. CONCLUSION: Identification GPCR heterodimerization adds to the understanding of the complexity of weight regulation and may provide important information to develop therapeutic strategies to treat obesity.